Source: UNIVERSITY OF NEVADA submitted to
THE NUTRITIONAL ECOLOGY OF POLLINATION IN A WARMING WORLD: LINKING POLLEN LIPID CHEMISTRY AND BEE HEALTH
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1029706
Grant No.
2023-67013-38991
Project No.
NEVW-2022-08488
Proposal No.
2022-08488
Multistate No.
(N/A)
Program Code
A1113
Project Start Date
Feb 15, 2023
Project End Date
Feb 14, 2027
Grant Year
2023
Project Director
Leonard, A.
Recipient Organization
UNIVERSITY OF NEVADA
(N/A)
RENO,NV 89557
Performing Department
(N/A)
Non Technical Summary
Climate change is an urgent threat to the security of agricultural systems: its impacts on crop yield, the price of food, and the infrastructure to bring it to market are already major challenges. Animal-pollinated plants face a dual vulnerability to climate change because in addition to the direct effects of altered temperature and precipitation on plant performance, the health, diversity, and abundance of the pollinators they rely upon is also at stake. Pollinators, especially bees, obtain their nutrition from plant pollen and nectar. Nutrition is widely recognized as a key determinant of how both wild and managed pollinators cope with a wide variety of stressors including pathogens and pesticides; there are thus major initiatives by growers, agencies, and non-profits to develop plantings for pollinators in working lands, in part to provide high quality nectar and pollen as nutritional resources to support healthy pollinator populations. However, the extent to which the nutritional value of the nectars and pollens change in response to climate stressors such as elevated temperature is a major open question. Reciprocally, we know very little about how pollinators' nutritional requirements may shift in a warming world. Nectar and pollen are chemically complex, and at least in the few plant species that have been considered, their chemistry may change dramatically with temperature, presenting a threat to pollinator health. These effects are particularly noted in relation to lipid chemistry, which plays a critical role in bee physiology and reproduction, and in plants' response to environmental stress.To understand how climate change affects 1) the quality of nutritional resources plants provide for pollinators as well as 2) bees' nutritional needs, our project combines a new analytical pipeline for the study of pollen chemistry with physiological and behavioral research on agriculturally important bee species (bumble bees and mason bees). We use these approaches to chart changes in pollen chemistry and bee nutritional requirements under the temperature increases modeled for our region by midcentury. Ultimately, this work will inform screening methods for stakeholders to identify seed mixes used to create or restore pollinator habitat in working lands (or crop varieties) most nutritionally valuable to bees in a warmer world. Although we address basic questions about the chemical and nutritional ecology of pollination under climate stress, we frame our questions in terms of pollinator-friendly plantings in agricultural or restoration settings. We perform this work in lab and field settings in Reno, Nevada, a site identified as one of the country's most rapidly-warming cities. Stakeholders involve beekeepers, agency staff, non-profits and growers motivated to understand how best to support bee nutrition and thus promote climate resilience of food systems.
Animal Health Component
66%
Research Effort Categories
Basic
60%
Applied
20%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2113085106034%
1322420100066%
Goals / Objectives
Our primary goal is to characterize the effects of climate warming on both floral reward chemistry and bee health. To address this goal, we will investigate economically important bee species and plant species with economic, ecological, and cultural importance. A secondary goal is to develop standardized methods to characterize pollen composition and chart its climate-mediated chemical shifts relevant to bee health. Both goals extend beyond the scope of the specific species we consider, as we introduce a new approach broadly relevant to understanding the biochemical linkages between plants and bees in working lands. To accomplish these goals we will pursue the following objectives:Objective 1: Evaluate the nutritional stability of floral resources under warming, developing new methods to characterize and predict changes to pollen chemistry with a particular focus on lipids and lipid-derived metabolites.Objective 2: Determine how warming alters bee nutritional needs using bumble bees (Bombus impatiens, the Common Eastern Bumble Bee) and mason bees (the Blue Orchard Bee, Osmia lignaria).Objective 3: Explore how the combined effects of warming alter interactions between bees and plants to impact the performance of both parties.
Project Methods
Efforts:Objective 1: Evaluate the nutritional stability of floral resources under warming, developing new methods to characterize and predict changes to pollen chemistry.To understand how predicted climate warming affects pollen and nectar of plants that support bees in working lands, we will compare how taxonomically diverse plants respond to the same warming treatment with respect to multiple dimensions of reward chemistry (Q1). We will also assess whether plasticity in leaf metabolites is a reasonable proxy for concomitant changes to pollen or nectar across plant species (Q2).Experiment 1: We will grow focal plant species at elevated vs. baseline temperatures, then collect and analyze their floral and leaf chemistry using a targeted metabolomics pipeline to characterize multiple dimensions of lipid chemistry. This involves Gas Chromatography-Mass Spectrometry and a flame ionization detector (GC-MS-FID). To assess whether focal plant species show consistent shifts in chemistry under warming we will use multivariate principal component analysis (PCA) based on spectral features and GC peak areas across samples, to determine if overall pollen chemical profiles differ between treatments within and across species. We will further identify and quantify individual lipids and lipid-derived metabolites from GC-MS-FID. This will allow us to assess total compositional features (chemical diversity and abundance) contributing to observed differences in the metabolite dataset and quantitatively assess of how specific metabolites important to bee health (such as fatty acids and sterols further explored in Objective 2) are affected across treatments and species (Q1). To determine whether warming-associated changes to leaf chemistry predict the degree of plasticity we observe in pollen or nectar chemistry (Q2) we will regress changes in key leaf lipids and associated metabolites against key reward lipids identified as changing under warming. This effort should give rise to at least one peer-reviewed journal article. Objective 2: Determine how warming alters bee nutritional needs using Bombus impatiens and Osmia lignaria. Here we assess how warming alters bees' nutritional needs. We focus particularly on lipids (fatty acids and sterols) given their dietary relevance to bee metabolism, lifespan, ovary development, and cognition. We will explore the roles of two focal lipid classes in bees' responses to experimental warming: 1) fatty acids, particularly omega-6:3 ratios (known for their effects on bee cognition and performance), and 2) pollen phytosterols abundant in our focal plant genera, 24-methylenecholesterol and ß-Sitosterol, known for their effects on bee longevity and the production of glycolipid proteins involved in responses to heat stress.Our design is based on a classic framework in nutritional ecology, the Geometric Framework (GF) long used to study the cause and consequences of dietary choice. A GF approach to understanding the nutritional drivers of foraging involves two complementary manipulations. In the restricted diet manipulation, subjects are restricted to a single (no-choice) feeding assay; across subjects, diet treatments vary in nutrient ratios, and performance is compared across treatments. From this, the intake target is identified as the nutrient ratio that maximizes a focal aspect of performance. In the paired choice manipulation, subjects are offered two simultaneous diets that vary in nutrient ratios. Subjects' consumption of these two diets is tracked to determine if and how effectively they regulate their intake of each to reach their intake target.We will house individual bees in experimental chambers, contained within incubators set at baseline vs. elevated temperatures. We will provide bees with artificial nectar diets manipulated to offer different ratios of the focal lipids described above. In Experiment 2a we focus on Bombus impatiens and in Experiment 2b we focus on Osmia lignaria. Each experiment combines a Restricted Diet Manipulation and a Paired Diet Manipulation alongside a host of organismal fitness and performance metrics. These experiments should generate at least one peer-reviewed journal article. Objective 3: Explore how the combined effects of warming alter interactions between bees and plants to impact the performance of both parties. Here, we combine what we have learned about how warming alters the pollen and nectar of focal plants (Exp. 1) and bees' nutritional needs (Exp. 2a,b). Though our approaches are tailored to each species, both involve a factorial design to explore how warming of both plants and bees gives rise to additive or synergistic effects on the performance of bees (Exp. 3a,b) and plants (Exp. 3b). Experiment 3a: We ask how pollen collected from warmed plants affects Bombus behavior, physiology, and survival, on its own and in combination with warming experienced by workers. We will house bees in individual chambers with identical "nectar" diets. According to treatment we will provision bees with pollen from the focal plant species, subjected to either the warmed or baseline treatment. Half of our subjects will be kept in an incubator at baseline and half at warmed temperatures. We will measure multiple aspects of bee performance including survival, cognition, and physiology (e.g. ovarian development, fat body size and content, and overall nutritional status). This experiment will yield a peer-reviewed journal article. Experiment 3b: We will use mesocosms to consider how warming-related changes to Osmia foraging behavior and aspects of floral phenotypes beyond pollen chemistry scale up to impact plant reproduction. To accomplish this, we will set up mesocosms at the UNR Agricultural Experimental Station, combining the features of a field cage and an open top chamber (OTC) to warm plants and/or bees depending on treatment.Each mesocosm will consist of two field cages connected by a passageway. In a nest cage we will provision a standard number of male and female Osmia and nesting straws. The connected plant cage will house focal plant species including Brassica napus. This allows Osmia to freely forage while we use 1 or 2 OTCs to selectively warm the nest cage, plant cage, both, or neither (depending on treatment). We will collect data on bee foraging behaviors, and analyze nest pollen provisions for species composition (via microscopy) and chemical composition. We will also measure bee adult and larval performance (e.g. larval development, ovaries, cuticular hydrocarbons, nutritional status). To measure floral phenotypes and plant performance we will measure floral traits and seed set as well as B. napus seed lipid composition as a proxy for overall oilseed crop quality. This experiment will yield a peer-reviewed journal article. Evaluation:Scientific publications and presentations are the primary products of this project. To gain feedback on our work pre-publication, we would aim to have at least one presentation from the project team at an internal or invited seminar by a PD each year, as well as at least one conference talk. Measurable outcomes in the context of these efforts primarily relate to meeting (or surpassing) the number of annual presentations (at least one each year) and publications we envision producing (4 as described above) once the research is up and running.To receive guidance about how well we are meeting the interest of regional stakeholders, we will form an advisory group composed of local professionals offering extension, agency, and/or non-profit perspectives. After sharing with each of these potential advisors a brief summary of our keyprogress over the past year, we will ask them for specific feedback on the extent to which our efforts are regionally relevant, and seek their perspective on how we might better target stakeholders with either our science or our science communication

Progress 02/15/23 to 02/14/24

Outputs
Target Audience:The scientific community was a primary target for our efforts this year. We shared preliminary data and our overall conceptual framework through invited talks by PD Leonard at the 2023 Gordon Research Conference on Plant-Herbivore Interactions and co-PD Vaudo at the 2024 International Conference on Pollinator Biology, Health and Policy and "Pollinator State of the Science" USDA/EPA workshop. This introduced our projects to national and international audiences including experts in pollination ecology, plant biology, and nutritional/chemical ecology. More locally, we also reached members of the public interested in bees and beekeeping through several outreach efforts, including organizing an outreach event at a local Meadery to celebrate National Pollinator Week in July 2023, delivering a presentation about bee behavior at the Nevada State Beekeepers meeting, and presenting an online extension talk about bee biology for continuing education credit through UNR Extension. Finally, undergraduate students were one of our audiences this year, whom we reached by running a 3-hour lab activity on bee learning and biology for an upper-level undergraduate neuroscience course. The lab was co-run by a PhD student involved in the grant research, and we demostrated the learning assay we are using in our experiments on diet and bee performance. Changes/Problems:We hit a major delay in the arrival of both our postdoctoral reseacher (who started in July 2023) and a critical piece of equipment (an incubator designed to match the one currently in PD Leonard's lab, needed for the proposed experiments that involved keeping bees in otherwise identical set ups but at different temperatures). The incubator was orderd in Jan 2023, had an initial ship date of 6/30/23 but did not arrive until late October 2023 and was unable to be installed in PD Leonard's lab until December 2023. Needless to say this was a frustrating delay, which set back the initiation of the bee-focused experiments by ~ 6 months. While normally we might just have pivoted to focus on pollen chemistry and greenhouse work, the unexpected delay unfortunately coincided with co-PD Kosma's sabbatical in Europe. We now have the people and equipment in place, but the incubator delay was enough of a setback that we anticipate requesting a no-cost extension if possible. A second change to note is that co-PD Anthony Vaudo has maintained Adjunct Professor position at UNR, and his involvement with advising on the grant, but has accepted a federal Research Biologist position at the Rocky Mountain Research Station (USFS). We are happy for Anthony's career success, and Dr. Charles Dean has been hired as a postdoctoral researcher to fill this role on the grant. Dr. Dean brings expertise in chemical ecology and insect physiology that we believe will enhance the depth and breadth of grant research. What opportunities for training and professional development has the project provided?Training Activities: Grant personnel including Postdoc Dr. Charles Dean, PD Leonard, and PhD student Jessica Buelow participated in a series of plant identification workshops held in the herbarium of the UNR Natural History Museum. Dr. Charles Dean was trained in behavioral techniques, including general experimental design as well as more specific learning and memory assays used in grant experiments. Professional Development: PD Leonard led a reading group for Postdoc Dr. Charles Dean and PhD student Jessica Buelow focusing on nutritional ecology. Grant personnel (Postdoc Charles Dean and PhD student Jessica Buelow) gained experience in mentoring by co-mentoring an undergraduate involved in the research. The undergraduate (Peyton Benner) gained research experience, and prepared a proposal to fund an independent research project. In terms of more formal instruction experience, Dr. Dean provided a guest lecture for PD Leonard's Sensory Ecology undergraduate course, and PhD student Jessica Buelow helped develop and present a hands-on lab activity relating to bee behavior for an upper-division neuroscience course. Jessica Buelow and undergraduate Peyton Benner participated in the Meadery outreach event for National Pollinator Week, with Buelow giving a 20 minute talk about bee behavior and biology, and Benner helping exhibit a bumble bee colony. Buelow further developed her public speaking skills by presenting a general talk about bee behavior for the 2023 Nevada State Beekeepers Association meeting. How have the results been disseminated to communities of interest?We reached colleagues in the scientific community through the presentations described above (e.g. Entomological Society of America, Gordon Research Conference, USDA/EPA State of the Science Workshop). We reached Nevada Beekeepers, Master Gardeners, and members of the general public via public talks at the Nevada State Beekeepers meeting, a local meadery, and an online talk on bee biology delivered by PD Leonard (220 people in "attendance") through UNR Extension. What do you plan to do during the next reporting period to accomplish the goals?(numbers here refer to those in the "Accomplishments" section). Objective 1: Evaluate the nutritional stability of floral resources under warming, developing new methods to characterize and predict changes to pollen chemistry with a particular focus on lipids and lipid-derived metabolites. The return of PD Kosma from a year sabbatical will position us to address Obj. 1 in the next reporting period. 1) Methods development manuscript: We anticipate that this manuscript will be submitted or close to submission during the next reporting period. 2) Field-based project led by PhD student Jessica Buelow (warming Lupinus plants in a field setting). We anticipate that the majority of data/sample collection will be completed during the next reporting period. We are eager to begin the greenhouse element of our project, and this project will be led by Dr. Charles Dean over the next reporting period. While the timing will depend on the availability of specific plant species, we anticipate he will have begun data collection on this project. Objective 2: Determine how warming alters bee nutritional needs using bumble bees (Bombus impatiens, the Common Eastern Bumble Bee) and mason bees (the Blue Orchard Bee, Osmia lignaria). 3) We began a major lab-based experiment on Bombus impatiens to explore the performance consequences of variation in omega 3:6 fatty acid dietary ratios (in line with the "Restricted Diet Manipulation" experiment described in the project narrative). This effort is led by the postdoctoral researcher we hired on the grant, Dr. Charles Dean, with assistance from PhD student Jessica Buelow and an undergraduate helper Peyton Benner. We anticipate that analysis of the behavioral data will be completed, and the chemical analysis of physiological markers should be well underway or even complete during the next performance period. Dr. Dean is eager to expand into Osmia, so we will decide whether to expand out into this new-to-us system, or go into further depth with Bombus during this period, to be determined by the specific results of the initial experiment. PhD Jessica Buelow has developed a dissertation proposal that dovetails with this objective, but focuses more specifically on the potential that secondary metabolites found in nectar and pollen might have temperature-dependent toxicity to bees. She is also interested in the extent to which specific lipids found in pollen might mediate this toxicity. Jessica plans to initiate these experiments (while assisting with some of the main grant experiments) during the next reporting period. Objective 3: Explore how the combined effects of warming alter interactions between bees and plants to impact the performance of both parties. 4) PD Leonard plans to spearhead an effort to write a review paper focusing on how climate change might alter the nutritional basis of species interactions. A target journal and author list will be finalized during the reporting period, along with an organizational structure. 5) Co-PD Vaudo and Dean will run their accepted symposium on anthropogenic influences on insects at the 2024 Pacific Branch Meeting of the Ecological Society of America Progress on this third Objective as a whole depends on having some specific outcomes relating to how warming affects plant chemistry. We may or may not have that in an interpretable form by the end of the next reporting period, but we should have plants started growing in a greenhouse by then.

Impacts
What was accomplished under these goals? Objective 1: Evaluate the nutritional stability of floral resources under warming, developing new methods to characterize and predict changes to pollen chemistry with a particular focus on lipids and lipid-derived metabolites. We made progress in several areas relating to this goal, (but prioritized Obj. 2 this year due to Co-PD Kosma's sabbatical as his lab and greenhouse access will play a major role in this area). We have numbered these efforts for cross-reference across this report. 1) Methods development manuscript: Co PD Vaudo continued to prepare this manuscript for publication, which describes a new pipeline for characterizing the external lipid chemistry of pollen samples of different starting masses. Given that there are often constraints on collecting sufficient material, we started with different masses of commercial pollens (1, 5, or 10 mg) and compared how starting material impacted our estimate of external lipid chemistry. Ultimately we show that starting masses as low as 5mg well-capture the same patterns that 10 mg returns. We believe this finding will be impactful because it will help make the study of pollen lipids more widely possible, particularly in situations where researchers want to include a large number of species or samples. Given the growing recognition of lipids' nutritional importance for bee health, we are eager to facilitate more researchers focusing on them. 2) Field-based project led by PhD student Jessica Buelow. Jessica is a PhD student in the UNR Ecology, Evolution and Conservation Biology program, who is supported by an NSF Graduate Research Fellowship. Jess has developed a dissertation proposal that connects to several grant themes, for example deploying open top chambers to artificially warm Sierra Nevada wildflowers known to be important nutritional sources for local bees, followed by pollen collection and lipid analysis. She piloted this approach on Lupinus argentus plants (one of our focal genera) at the UNR Whittell Forest & Wildlife Area during the reporting period. We believe Jessica's project will be impactful because Lupinus and other Fabaceous plants are well-known to be nutritionally important to a wide variety of bees, but we know almost nothing about what will happen to the nutritional value of these plants' pollen under warmer temperatures. Will their pollen become 'better' for bees (e.g. with more protein or lipids), 'worse' (e.g. with higher alkaloid content) in a warmer world? Jess' dissertation aims in part to answer these questions. Objective 2: Determine how warming alters bee nutritional needs using bumble bees (Bombus impatiens, the Common Eastern Bumble Bee) and mason bees (the Blue Orchard Bee, Osmia lignaria). This is the Objective we spent most of our efforts on during the reporting period: 3) We began a major lab-based experiment on Bombus impatiens to explore the performance consequences of variation in omega 3:6 fatty acid dietary ratios (in line with the "Restricted Diet Manipulation" experiment described in the project narrative). This effort is led by the postdoctoral researcher we hired on the grant, Dr. Charles Dean, with assistance from PhD student Jessica Buelow and an undergraduate helper Peyton Benner. Briefly, we are keeping individual workers in chambers housed within incubators where we can keep them at baseline or elevated temperature conditions for 5 days, while manipulating the ratio of these fatty acids across five dietary treatments experienced by bees at the two different temperatures. We then measure performance on an associative learning assay, and sacrifice bees while collecting a variety of metrics and samples for further analysis (e.g. ovarian development, fat body size and composition, and cuticular hydrocarbon profile). Due to shipping delays with the incubator required to properly run this experiment, we began data collection in earnest in December 2023, so during the reporting period collected about 1/3 of the required data. This project addresses the major question of how warming impacts bees' nutritional requirements, and as such, we believe that its results will be of major interest. Understanding whether at warmer temperatures bees need (or do not need) the specific fatty acids we focus on can help shape recommendations for what plants (whose pollens can vary widely in fatty acid content) best match bees' needs in a warmer environment. Objective 3: Explore how the combined effects of warming alter interactions between bees and plants to impact the performance of both parties. The empirical component of this Objective depends upon having some data in relation to the previous two Objectives, so most of our efforts during the reporting period related to more conceptual efforts. 4) We began planning for a conceptual framework or review paper focusing on how climate change might alter the nutritional basis of species interactions. Postdoctoral researcher Charles Dean began a bee-specific literature review on this topic during his first few months of the project. We are surprised that this question appears to be largely absent from the conservation or management literature, so envision providing a general framework that could inspire others to ask related questions in their focal systems (e.g. pollinators and beyond). 5) Co-PD Vaudo and Dean put together a successful proposal for a symposium on anthropogenic influences on insects at the 2024 Pacific Branch Meeting of the Ecological Society of America. Face to face discussion of the connection between nutritional ecology and global change can bring about new insights and form new connections for the early career scientists involved in this research. This symposium will provide Drs. Vaudo and Dean a chance to present and gain feedback on their work from potential reviewers and future collaborators.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Leonard, A.S. "Floral Reward Complexity and Pollinator Behavior" Invited Talk. Gordon Research Conference: Plant Herbivore Interactions in Action: Fundamentals to Applications. Ventura, CA Feb 26-March 3 2023.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Vaudo, A.D., Grames, E., Kosma, D.K. and Leonard, A.S. "The known unknowns of wild bee nutritional ecology and conservation" Talk. Entomological Society of America Meeting. National Harbor, MD Nov 5-8 2023.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Vaudo, A.D. "Pollen nutrition structures bee-wildflower community interactions". Invited Talk. 2023 International Conference on Pollinator Biology, Health, and Policy. State College, PA. June 2-6 2023.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Vaudo, A.S. "Pollen nutritional ecology of bee-flower interactions". USDA/EPA State of the Science Workshop. August 30-31 2023.