Location: Weed and Insect Biology Research
2023 Annual Report
Objectives
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.
Approach
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?
Progress Report
Objective 1: Research continues on the effects of low-temperature storage on pollinator post-storage quality. One important use of low-temperature pollinator storage is in the synchronization of emergence of the alfalfa leafcutting bee, Megachile rotundata, with the bloom of alfalfa grown for seed production. Seed producers use a degree day model to estimate when bloom will occur, and then move cold-stored alfalfa leafcutting bees to warmer temperatures for the 4 weeks of incubation required for emergence. During this period, unexpected weather changes can delay the crop bloom, which then requires a concurrent delay in bee emergence. While current protocol is to store the developing bees at low temperatures (4 to 10°C) to temporarily halt development, we have previously shown that exposing developing bees to this range of temperatures can induce sub-lethal effects such as deformed wings and a reduced ability to fly, that leads to a reduction in their ability to pollinate. However, we have now demonstrated that incubating bees at temperatures above their developmental threshold of ~18°C slows development and delays emergence without affecting bee quality. Since developmental rates vary with incubation temperature, we also developed an easy-to-use mathematical model that can accurately predict emergence based on the temperatures used. This tool will enable producers to greatly improve synchronization of the bee peak nesting activity with the bloom of alfalfa and other crops.
Work also continues on developing a more thorough understanding of the low-temperature physiology of the alfalfa leafcutting bee at the molecular level. While we have conducted numerous gene expression studies that have provided important insights into the response of this pollinator to low-temperature exposures, these studies have been correlative in nature, and don’t clearly establish the underlying molecular mechanisms. To address this problem, a gene knock-down technique using RNA interference is being developed. We have developed a reliable injection method, and preliminary experiments have demonstrated that the expression level of our initial target gene was significantly decreased for at least two days post injection. This new ability to decrease the expression of selected genes may greatly enhance our understanding of low-temperature physiology in this species.
We have also continued our studies on fluctuating thermal regimes (FTR), whereby the shelf-life of cold-stored M. rotundata can be significantly increased by subjecting the bees to a daily one-hour pulse of high temperature. While the current hypothesis prevalent in the literature is that the high temperature pulse helps to reestablish membrane integrity and ion homeostasis, we put forward a new hypothesis: that the warm temperature pulse could also be functioning as a zeitgeber (timekeeper) helping to synchronize an insect’s central and peripheral clocks. This could improve longevity since any impairment of a bee’s circadian mechanism could alter its ability to synchronize its daily activities and physiological processes to the environment. Our preliminary results support our hypothesis. Giving the bees a temperature pulse in the night significantly improved survival over that of bees receiving the pulse during the day. The result indicates that timing of the high-temperature pulse is an important factor in optimizing low-temperature storage protocols.
During this reporting period we continued our work on the physiology of two pest species, the Colorado potato beetle, Leptinotarsa decemlineata, and the sugar beet root maggot, Tetanops myopaeformis. Our efforts on the Colorado potato beetle have focused on the fact that determining the sex of adult beetles can be difficult for non-specialists. Drawing from previous datasets, we have isolated five sex-specific genes that can reliably identify the sex of an individual using either qPCR or simple agarose gel electrophoresis. Beside empowering more refined experimental design for future investigations, this technique will also enable the rescreening of older data sets to further our understanding of the role of sex on this beetle’s physiology. For the sugar beet root maggot, we conducted differential scanning calorimeter screening experiments to quantify the metabolic heat generated by this insect at a range of temperatures. We discovered that the maggots can maintain a stable heat capacity over a range of ecologically relevant overwintering temperatures, which was unexpected for an ectotherm. This novel result indicates that sugar beet root maggots are able to maintain a fairly consistent level of thermal energy needed to support overwintering metabolism, thereby permitting a degree of temperature independence.
Objective 2: Research continues on technologies to support the cryopreservation of insect germplasm. Although our main focus is on pollinators, we have also investigated the possibility that the honey bee sperm cryopreservation protocol developed during this research project could be used for other species. We focused on two lepidopteran species: the monarch butterfly, and the critically endangered Sacramento checkerspot butterfly (SCB). We were able to successfully collect viable sperm by dissecting males of both species, and post-cryopreservation assessments confirm that the procedure efficiently protects sperm from both species. Sperm samples from 3 SCBs and approximately 20 monarchs are currently in cryostorage at the USDA-ARS-Edward T. Schafer Agricultural Research Center (ETSARC) in Fargo, North Dakota.
We have also worked to improve the accessibility of honey bee germplasm cryopreservation by developing a non-activating medium for honey bee sperm. Normally, cryopreservation of germplasm requires ready access to liquid nitrogen which can substantially limit the feasibility of collecting and transporting germplasm. However, we have demonstrated that by using our non-activating medium, honey bee sperm can be stored at ambient temperatures for several weeks, and can even be transported at ambient temperatures without a decrease in viability. This new medium will greatly enhance the accessibility of honey bee sperm storage, such as enabling the collection of sperm from remote field sites that lack the facilities needed for liquid nitrogen.
We also developed a method for assessing the quality of cryopreservation media using differential scanning microcalorimetry (DCM). Stored sperm is usually a concentrated collection of a single cell type in suspended animation at liquid nitrogen. At higher temperatures these cells will emit unique thermal signatures that could be assessed using DCM. We adapted this technique to assess honey bee germplasm quality. The results indicate that the use of medium other than non-activating diluent leads to rapid loss of viable cells in as little as 48 hours even if stored at 14°C. Calorimetric assessment also shows that the primary cause of viability loss in honey bee germplasm that is stored under near-ambient conditions is microbial contamination. Microscopic assessments of such samples show the loss of nuclear material in the sperm cells. Microcalorimetry not only was efficient in determining the basal metabolic rate in the germplasm in storage, but also the heightened heat output from approximately 450 microbes per microliter of the germplasm. This non-invasive method of viability determination is not only very precise but also rapid.
Accomplishments
1. Development of a cryopreservation protocol for insect conservation. Insect populations are declining worldwide, and germplasm cryopreservation is a vital tool for conserving both economically and ecologically important species. Traditionally, germplasm cryopreservation requires the development of species-specific protocols and the use of readily available liquid nitrogen, which greatly inhibits the collection of germplasm from remote collection sites. ARS researchers in Fargo, North Dakota, have developed a medium for the cryopreservation of honey bee sperm that enables collection and transportation at ambient temperatures. The germplasm can be at ambient temperatures for up to 6 months before the samples need to be placed into liquid nitrogen for long-term storage. This novel cryopreservation protocol provides conservation organizations with a new tool for preserving the world’s insect biodiversity.
2. Cryopreservation of sperm from recently deceased insects. With the alarming worldwide decrease in population numbers of economically and ecologically important insect species, new conservation technologies are urgently needed. ARS researchers in Fargo, North Dakota, demonstrated that viable male insect germplasm can be collected up to 24 hours after the death of an individual, cryopreserved in liquid nitrogen, and later revived. Originally developed for the honey bee, this technology has been extended to a native bumble bee species and the monarch butterfly. This new technology offers conservation organizations a means to maximize the genetic potential of each male within a breeding colony.
3. Molecular markers for sex identification in Colorado potato beetles. Male and female insects can respond differentially to stress. Therefore, knowing the sex of an individual insect can be important when working to understand the complex processes underlying stress physiology. In the Colorado potato beetle, adults are difficult to sex for non-specialists. ARS researchers in Fargo, North Dakota, have identified five sex-specific genes that can separate male and female samples using standard molecular tools. While this work directly applies to future investigations and rescreening of older datasets in Colorado potato beetle, many insects have life stages that can’t be sexed, limiting our understanding of the role of sex on insect physiology. This novel technique may be adaptable to other species as well, which would provide researchers a new tool for understanding the role of sex of an insect on its physiology.
Review Publications
Torson, A.S., Roe, A.E., Doucet, D., Sinclair, B.J. 2023. Molecular signatures of diapause in the Asian longhorned beetle: gene expression. Current Research in Insect Science. https://doi.org/10.1016/j.cris.2023.100054.
Park, M.G., Delphia, C.M., Prince, C.M., Yocum, G.D., Rinehart, J.P., O'Neill, K.M. 2022. Effects of temperature and wildflower strips on survival and nutrition dynamics of Megachile rotundata under extended cold storage. Environmental Entomology. https://doi.org/10.1093/ee/nvac062.
Rajamohan, A., Prasifka, J.R., Rinehart, J.P. 2022. Vitrification of lepidopteran embryos - a simple protocol to cryopreserve the embryos of the sunflower moth, Homoeosoma electellum. Insects. 13(10). Article 959. https://doi.org/10.3390/insects13100959.
Pantzke, S., Ferguson, B., Rajamohan, A., Rinehart, J.P., Prischmann-Voldseth, D., Prasifka, J.R. 2023. Thermal biology and overwintering behavior of the red sunflower seed weevil (Coleoptera: Curculionidae). Environmental Entomology. https://doi.org/10.1093/ee/nvad041.
Campion, C., Rajamohan, A., Dillon, M. 2023. Sperm can't take the heat: Short-term temperature exposures compromise fertility of male bumble bees (Bombus impatiens). Journal of Insect Physiology. 146(2023). Article 104491. https://doi.org/10.1016/j.jinsphys.2023.104491.
Zhao, L., Wang, X., Liu, Z., Torson, A.S. 2022. Energy consumption and cold hardiness of diapausing fall webworm pupae. Insects. 13(9). Article 853. https://doi.org/10.3390/insects13090853.
