Location: Weed and Insect Biology Research
2024 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: The ongoing research on how cold storage during development affects the quality of adult alfalfa leafcutting bees is important because cold storage is employed in order to synchronize bee emergence with optimal crop bloom when alfalfa is grown for seed production. Although most pollination managers currently store bees at temperatures below the developmental threshold of 18°C to temporarily pause development, our previous studies have shown that this can lead to sublethal effects such as deformities and decreased ability to fly in the resultant adults. We have characterized these effects at different temperature and duration combinations so that pollination managers can make more informed decisions. To do this, we have developed a standardized flight performance test which allows for simple and reproducible assessments of adult bee quality. We have also continued our research on incubating bees at temperatures above their developmental threshold, which appears to delay emergence without affecting bee quality. Although this leads to healthier adult bees, it also complicates the estimation of emergence since bees are still developing during storage. We continue to improve our mathematical model that can accurately predict emergence based on the thermal history of a bee, which will be helpful to producers using these warmer temperatures. Also, in response to stakeholder concerns, we are studying the effects of low temperatures during development in two important parasitic wasp species of alfalfa leafcutting bees to effectively manage their population.
Our work continues to progress understanding of low-temperature molecular physiology in multiple economically important insects. We have conducted a variety of gene expression studies to investigate the pollinator responses to low temperature stress and continue development of manipulative techniques such as RNA interference (RNAi) to move past lists of candidate genes towards functional characterizations of these candidates. A functional RNAi protocol for Megachile rotundata remains in development. We developed a reliable injection method for delivery of the RNAi constructs without substantive effects on insect survival post-injection, but preliminary experiments using multiple RNAi targets and carriers (e.g., liposomes) to facilitate cellular uptake of the constructs have yielded inconsistent results. We hypothesize that this inconsistency in RNAi response is due to either a lack of cellular uptake of the RNAi constructs and/or lack of a systemic response to the treatment leading to more localized RNAi response not detected by our current sampling methods. In continued efforts to develop a functional protocol, we are reevaluating our current methods in the Colorado potato beetle, Leptinotarsa decemlineata, which has a robust and systemic RNAi response. If successful, this comparative study will allow us to rule out any potential technical issues resulting from our methodology in M. rotundata and confirm that observed issues are biological and not methodological.
With respect to our studies on fluctuating thermal regimes (FTR), which when implemented significantly reduces cold-related injury during cold storage, there is literature evidence that a high-temperature pulse during FTR facilitates the reestablishment of physiological homeostasis, counteracting the effects of cold-related injury during storage. Our previous phenotypic and gene expression data support this hypothesis. In addition to counteracting cold injury, FTR also acts to synchronize adult emergence in M. rotundata, suggesting that FTR can affect circadian rhythm. During this reporting period, we tested the hypothesis that the periodic nature of the FTR warm pulse, which occurs at the same time each day, acts as a zeitgeber (timekeeper) to help synchronize insects’ central and peripheral clocks. Preliminary data on shifting the warm pulse to randomized times through the day, as well as different (but consistent) times of the day suggests that the timing of the temperature pulse is important for improving low temperature storage protocols. Additionally, we have sequenced the transcriptomes of pupal M. rotundata exposed to either constant low temperatures or FTR over a course of eight weeks to better understand how gene expression changes over time during these storage protocols. Our previous efforts have only focused on short sampling windows, so we anticipate that these data will provide a more nuanced description of FTR’s beneficial effects as well as new candidate biomarkers for stress in this species.
In this reporting period, we studied the overwintering physiology in multiple pest species, including the Colorado potato beetle (CPB) and the sugar beet root maggot (SBRM). Using sex-specific gene expression markers previously developed by our group, we have started investigating sex-specific differences in diapause regulation in CPB using RNA-seq. Our preliminary data suggests that while 40% of the variation in gene expression in our dataset is explained by sex, the transcriptomic signatures of diapause between sexes only differ by 6%. These results suggest that similar physiological mechanisms likely regulate diapause in both sexes, but we did observe variation in lipid metabolism, cell cycle regulation, and alternative splicing. Our group is also working to produce a chromosome-level genome assembly for the SBRM. During the previous reporting period, we generated a high quality contig assembly using the PacBio Sequel II and Hi-C scaffolding. Since we were unable to resolve the chromosome structure using these methods, we have started supplemental sequencing on sexed males and females to attempt to resolve this structural issue in the assembly.
Objective 2: The research specific to this objective encompasses three aspects of bee germplasm conservation and the testing of these technologies, for their applicability to other insects including bumble bees and lepidopterans. The Subobjectives 2a-2c delves on,
a. Developing cryopreservation protocols for male honey bee germplasm [Sub. Obj. 2a],
b. Standardization of bee embryo cryopreservation protocol [sub obj. 2b], and
c. Developing methods to rear the bee embryos and larva in vitro after the sub obj. 2b. [sub. Obj. 2c]
Male germplasm studies assessed the feasibility of using vitrification technique(s) to cryopreserve bee semen instead of the conventional controlled-freezing methods. In addition, a vitrification mixture without synthetic chemicals, such as dimethyl sulphoxide (DMSO), as cryoprotective agent was developed. The assessments of semen activity and nuclear stability in the vitrified and revived bee spermatozoa show that the seminal characteristics are comparable to the conventionally frozen samples. The methodology is currently awaiting the scheduled field assessments viz. insemination acceptance by the queen bee, brood formation, and brood quality characteristics.
Within the same Subobjective 2a, an aspect of importance for bee semen storage and transportation, namely, the long-term storage of semen at near ambient temperatures was assessed using calorimetry - a non-invasive method to evaluate the stability of the sperm cells. We noticed that the primary factor that caused damages to the sperm cell is the presence of bacteria. The semen obtained with conventional media is contaminated by the extender and the differential scanning calorimetry (DSC) data suggests that the semen at 14°C has significantly limited viability after 30 days of storage. Previously we have shown that the bee semen washed with an extender that was developed by the USDA-ARS researchers (FAV extender medium) could allow semen storage for 6 months or more.
As a quality control component of the above research, studies have been initiated to assess the seminal proteome that is responsible for sperm cell viability. Samples representing fresh semen, semen with traditional extenders, semen washed and extended with FAV extender medium, and semen stored over a period of 7, 14, 30, 45 and 60 days at 14°C have been collected and are being prepared for proteomic analysis.
Subobjectives 2b and 2c are dedicated to standardization of the cryopreservation protocol designed for the honey bee embryos by the USDA-ARS researchers and fine-tuning the post revival procedures to obtain an adult bee under in vitro conditions. The standardization steps address critical requisites to bringing the technology to the field. Towards this agenda, USDA researchers have fine-tuned the embryo collection and incubation conditions that make high throughput cryopreservation a feasibility for honey bee embryos. This is especially important when dealing with an organism with an extremely environmentally sensitive embryonic stage. Significant progress has been made with respect to developing the cryopreserved / treated embryos to larval stages with in vitro rearing techniques as a part of the research collaborative research done in the Subobjective 2c. Additionally embryonic and larval samples have been collected for proteomic assessments. Proteomic assessments of the embryos and larva are slated for the upcoming/future portions of the project plan. Proteomic research is intended to decipher the reasons for the embryo and larval acceptance in the hive, especially after intensive treatments such as in the cryopreservation procedures.
In addition, testing of the applicability of these techniques to other agriculturally important species such as the bumble bees were carried out during this study period. These studies have resulted in a stable vitrification procedure for bumble bee semen as well as the seminal plasma. The male germplasm that has been cryopreserved from various species are scheduled to be assessed for quality and once ascertained, part of the germplasm will be transferred to the National Animal Germplasm Repository in Fort Collins, Colorado.
Accomplishments
1. Nest size affects bee size in solitary bees. Solitary, cavity nesting bees, such as the alfalfa leafcutting bee are an underappreciated but critically important pollinator in agriculturally dominated ecosystems. While much is known about the management of this insect, how the size of nesting cavities affects the size and quality of the bees has received less attention. USDA-ARS researchers in collaboration with North Dakota State University conducted a study to compare bees reared in a variety of nest sizes with those reared in the industry standard cavities that are 7mm in diameter. The results show that adult bee body size varies directly with the diameter of the nest. This is important because in other species, characteristics such as foraging distance and the relative size of pollen loads that can be carried are affected by the size of the bee. Hence, using different sized nests can help develop a bee optimized for a specific application.
2. Length of the telomere caps at the ends of the chromosomes is longer after diapause in two solitary bee species. As organisms get older, they exhibit external, physical signs of aging, but aging also occurs at the molecular level. However, the cellular and molecular mechanisms that contribute to aging are poorly understood. Measuring the length of the telomeres is one way to detect organismal aging. Telomeres are repetitive sequences of non-coding DNA located at the end of chromosomes, which protect coding DNA during replication. As the organism ages, telomere length decreases. This pattern occurs in many vertebrates, but there are very limited studies on the telomere dynamics in insects. USDA-ARS researchers in Fargo, North Dakota, have determined how telomere length changes throughout the lifespan and in response to stress in two agriculturally important solitary bee species, the blue orchard bee (Osmia lignaria) and the leaf cutting bee (Megachile rotundata). The results show a contrasting pattern to what generally occurs in vertebrates and other insects. Telomere length in solitary bees increases in later life stages after emergence from a dormant state. Thus, the USDA researchers have demonstrated that aging dynamics in diapausing solitary bees differ from other animals and insects.
3. Genome sequencing and transcriptomic characterization of dormancy in an orchard pollinator. The blue orchard bee, Osmia lignaria, is an economically important orchard pollinator in the United States. Many insect species in temperate environments enter a state of dormancy to endure harsh environmental conditions. Most insects go dormant during winter, but O. lignaria goes dormant twice during its lifecycle: once as a larva to avoid hot temperatures during summer and once as adults to overwinter. Insects employ two main types of dormancies – one that is a programmed part of the lifecycle and one that occurs directly in response to stress. These two different types of dormancies are managed differently in commercially reared insect species. However, it was unclear whether O. lignaria’ s larval dormancy is a programmed part of the lifecycle or if it simply occurs in response to stressful environmental conditions. Therefore, ARS researchers in Fargo, North Dakota, in collaboration with North Dakota State University and the Ecological Forestry Applications Research Centre in Spain measured metabolic rates and gene expression to compare adult and larval dormancy. Researchers found that both dormancies are characterized by temperature-independent decreases in metabolic rate, consistent with a programmed dormancy, but they exhibit very different physiology. Developing a better understanding of this prepupal dormancy will be critical for the continued development of optimized blue orchard bee management protocols for pollination services.
4. Cryopreservation of monarch butterfly sperm bundles. Importance of butterflies and moths to the agriculture and the ecosystem is not trivial. However, to better protect them, it important to understand their reproductive physiology and develop techniques to better preserve their germplasm, namely, the eggs/embryos and the sperm cells. Most of the male butterflies and moths transfer the sperm cells that are required to fertilize the eggs in the female butterfly in the form of bundles of fertilization capable sperm cells while the fertilization incapable sperm are transferred as free cells. ARS researchers have developed a method that could be used to cryopreserve and revive the sperm cell bundles of the monarch butterfly paving way to a full-fledged germplasm conservation program for the lepidopterans in general. This study was published in June 2024 in the Scientific Reports. The USDA-ARS researchers have extended this study to other highly endangered species in consultation with the U.S.Fish and Wildlife Service and the University of New Mexico researchers.
Review Publications
Grula, C.C., Richard, Z.S., Walsh, D.B. 2024. Bee diversity and abundance in perennial irrigated crops and adjacent habitats in central Washington state. Florida Entomologist. https://doi.org/10.1515/flaent-2024-0007.
Rinehart, J., Grula, C.C., Rinehart, J.P., Bowsher, J. 2023. Nesting cavity diameter has implications for management of the alfalfa leafcutting bee (Hymenoptera: Megachilidae). Physiological Entomology. https://doi.org/10.1093/jee/toad207.
Pantzke, S., Rajamohan, A., Ferguson, B., Rinehart, J.P., Prischmann-Voldseth, D.A., Prasifka, J.R. 2024. Development of a degree-day model for adult emergence of the red sunflower seed weevil, Smicronyx fulvus LeConte (Coleoptera: Curculionidae). Journal of Kansas Entomological Society. 97(1):26-38.
Warcup, K., Torson, A.S., Yocum, G.D. 2023. Identification of sex-specific markers based on gene expression in adult Colorado potato beetles. Journal of Insect Science. 23(4):1-6. https://doi.org/10.1093/jisesa/iead071.
Grula, C.C., Rinehart, J.D., Anacleto, A., Kittison, J.D., Heidinger, B.J., Greenlee, K.J., Rinehart, J.P., Bowsher, J.H. 2024. Telomere length increases following quiescence in two solitary bee species. Scientific Reports. 14. Article 11208. https://doi.org/10.1038/s41598-024-61613-2.
Bennett, M.M., Debardlabon, K.M., Rinehart, J.P., Yocum, G.D., Greenlee, K.J. 2023. Effects of developmental state on low-temperature physiology of the alfalfa leafcutting bee, Megachile rotundata. Bulletin of Entomological Research. https://doi.org/10.1017/S0007485321001103.