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?
Research continues on the effects of low-temperature storage on pollinator post-storage quality. Managed beneficial insects are commonly exposed to low temperatures to slow their development in order to match pollinator emergence to crop bloom or predatory insects with the presence of the pest they are to control. Our past research has demonstrated that the survival of the alfalfa leafcutting bee during low temperature storage could be significantly increased by implementing a daily one-hour pulse of high temperature (fluctuating thermal regime, FTR). Improving insect shelf-life with FTR is not exclusive to alfalfa leafcutting bees, and the current consensus within the published literature is that giving an insect a high temperature pulse during low-temperature storage enables physiological repair of damage incurred by low temperature exposure. We hypothesized that there should be an increase in metabolic heat corresponding to this physiological repair. This year, we used calorimetry on bees during the warm phase of FTR, and while we observed a small increase in metabolic heat during this period, we believe it is too small to account for the increase in survival. Additionally, to develop a more complete understanding of the metabolic changes induced by FTR, we examined how glycogen, trehalose, simple sugars and lipid levels changed over the course of a FTR exposure. We discovered a complex interaction between the age of the bee before the FTR exposure and the duration of the exposure. Our biochemical analysis supports our previous genetic results that indicated that FTR initiates dynamic gene expression processes. The applied implications of our results is that the physiology of an insect is changing over the course of storage and therefore we may need to change the storage conditions to optimize M. rotundata post-storage quality. One new hypothesis that we have developed is that low temperature exposure is leading to desynchronization of the various clocks within M. rotundata and that the pulse is serving as an environmental cue synchronizing the bees’ circadian clocks. This hypothesis is currently being tested, including the collection of samples to investigate the role of the clock genes during FTR storage. Secondly, we have continued our investigation into the sublethal effects of cold storage. While an insect may survive cold storage, it may be harmed by the process, leading to a variety of problems that would reduce their ability to pollinate crops. This year, we have demonstrated that two deformities can develop in bees stored at a constant temperature: wings that are not properly developed and mouth parts that do not work properly. Additionally, even when removing these individuals, cold stored bees are less likely to fly in a simple “drop test” than those not subjected to cold storage. Importantly, all of these sublethal effects are significantly reduced by the use of FTR. These results clearly demonstrate that pollination managers need to consider many parameters to ensure that high quality insects are released into the field. Finally, we have continued research on the abiotic stresses experienced during shipping bees for pollination services. Specifically, our studies have included atmospheric changes (decreases in oxygen and decreases in carbon dioxide), temperature extremes, and vibrations that can be experienced during shipping. These experiments are ongoing. Objective 2: We continued our work on refining cryopreservation techniques for germplasm cryo-storage and assessing the post-freezing quality of the germplasm. In our male germplasm storage studies, we investigated bee cryopreservation using material from different sources, including bee semen, whole testicular tissue, and spermatozoa derived from the seminal vesicle. While our research has focused on the European honey bee (Apis mellifera), we also continued to transfer this technology to the bumble bee (Bombus impatiens), and are investigating applying it to other high impact insect species as well. For honey bee embryonic cryostorage, we continue to use naturally oviposited embryos as source material. Previous studies have shown that honey bee embryos are highly susceptible to chemical solvents traditionally used during permeabilization, but this year, we have demonstrated that survival could be improved by using high molecular weight alkane mixtures, such as using heptane containing 5% dissolved nonane and devoid of trace isopropanol. The hatch percentage of these embryos was estimated in vitro and was noted to be comparable to untreated in vitro embryos. We have also made progress in penetrating embryos with cryoprotectants prior to freezing, including assessing embryos for their ability to vitrify (reaching very low temperatures without the formation of ice crystals). We also continue to improve our protocol to accommodate issues specific to honey bees, including the fact that they are highly susceptible to handling. We also continue to investigate the development of cryopreservation protocols for other insects of economic importance, such as the preservation of research strains of the western corn root worm, Diabrotica sp., in collaboration with ARS scientists in Brookings, South Dakota. Our third component of this objective is to rear larvae from cryopreserved embryos in vitro to assess the post-freezing embryonic quality and to ultimately produce a reproductively viable queen from a cryopreserved embryo. Much of our previous work has focused on determining how different larval diets affect whether a larva develops into a reproductively viable queen, a non-reproductive worker, or an intercaste, with traits of both types of bee. The larval diets tested this year resulted in significantly lower intercaste production. Additionally, since the hive environment includes constant grooming by the nurse bees, larval honey bees are susceptible to a wide range of complications, including sensitivity to handling, and a propensity for mold development. We have improved our handling protocols this year, leading to significant improvement in larval survival.
1. Cryopreservation of semen from varroa resistant honey bee strain. The honey bee is an important component of the United States agricultural bioeconomy through its pollination services, honey, and wax production. Honey bee colonies have been declining significantly due to environmental factors, habitat loss and increasing prevalence of parasites and disease. For many years, the most serious pest of honey bees has been the varroa mite, and there is an urgent need for new control methods for this pest. Three strains of highly varroa resistant bee have recently been developed by ARS researchers in Baton Rouge, Louisiana. To safeguard this critical biological resource, ARS researchers in Fargo, North Dakota, have cryopreserved the sperm from these varroa resistant strains. These preserved stocks are now available to honey bee breeders in case of loss of the breeding stock of these strains.
2. Cryopreservation of malaria mosquito germplasm. In 2020 the World Health Organization reported 241 million cases of malaria resulting in approximately 627,000 deaths. To help combat this devastating disease, scientists have developed a number of genetically altered strains of the mosquito that transmit malaria. These strains are expensive to develop and to maintain, and can be lost due to due disease, equipment failure, or other calamities. Working with collaborators at the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, ARS researchers in Fargo, North Dakota, have developed a method to cryopreserve the mosquito sperm. The CDC is now working to employ this new technology to safeguard their colonies, thereby ensuring the availability of these strains for future researchers.
Cambron-Kopco, Yocum, G.D., Yeater, K.M., Greenlee, K. 2022. Timing of diapause initiation and overwintering conditions alter gene expression profiles in Megachile rotundata. Frontiers in Physiology. https://doi.org/10.3389/fphys.2022.844820.
Grula, C.C., Rinehart, J.P., Greenlee, K.J., Bowsher, J.H. 2021. Body size allometry impacts flight-related morphology and metabolic rates in the solitary bee Megachile rotundata. Journal of Insect Physiology. 133. Article 104275. https://doi.org/10.1016/j.jinsphys.2021.104275.
Melicher, D.M., Wilson, A.M., Yocum, G.D., Rinehart, J.P. 2021. Fluctuating thermal regimes extend longevity and maintain fecundity to increase colony shelf-life of Drosophila melanogaster. Journal of Thermal Biology. https://doi.org/10.1111/phen.12357.
Melicher, D.M., Bowsher, J.H., Rinehart, J.P. 2021. Fluctuating temperatures extend longevity in pupae and adult stages of the sepsid Themira biloba. Journal of Thermal Biology. 99. Article 102959. https://doi.org/10.1016/j.jtherbio.2021.102959.
Mahdi, O.S., Greenlee, K.J., Rose, E., Rinehart, J.P., Smith, D.J. 2021. The sporicidal activity of chlorine dioxide gas on Paenibacillus larvae spores. Journal of Apicultural Research. https://doi.org/10.1080/00218839.2021.1920761.
Debardlabon, K.M., Rajamohan, A., Rinehart, J.P. 2022. Vitrification of manually stage selected embryos of Drosophila melanogaster have significantly higher survival and adult emergence. Journal of Cryobiology. 105:83-87. https://doi.org/10.1016/j.cryobiol.2022.01.002.
Earls, K., Porter, M., Rinehart, J.P., Greenlee, K. 2021. Thermal history of alfalfa leafcutting bees affects nesting and diapause incidence. Journal of Experimental Biology. 224(22). https://doi.org/10.1242/jeb.243242.
Signor, S., Yocum, G.D., Bowsher, J. 2022. Life stage and the environment as effectors of transposable element activity in two bee species. Journal of Insect Physiology. 137. Article 104361. https://doi.org/10.1016/j.jinsphys.2022.104361.
Cambron, L.D., Yocum, G.D., Yeater, K.M., Greenlee, K. 2021. Overwintering conditions impact insulin pathway gene expression in diapausing Megachile rotundata. Comparative Biochemistry and Physiology. 256. Article 110937. https://doi.org/10.1016/j.cbpa.2021.110937.
Walter, R.M., Rinehart, J.P., Dillon, M.E., Greenlee, K.J. 2021. Size constrains oxygen delivery capacity within but not between bumble bee castes. Journal of Insect Physiology. 134. Article 104297. https://doi.org/10.1016/j.jinsphys.2021.104297.