Recipient Organization
OREGON STATE UNIVERSITY
(N/A)
CORVALLIS,OR 97331
Performing Department
Horticulture
Non Technical Summary
Poor nutrition has been cited as one of the major factors involved in both honey bee and native bee declines reported over the last decade. Nutrition plays a vital role in mitigating effects of biotic and abiotic stressors on bees. Since reports of significant bee declines began, much effort has been dedicated to researching the causes of such declines, but only a few studies have addressed the underlying, fundamental problems, particularly with regard to nutrition. As honey bee nutrition plays a vital role in mitigating the effects of biotic and abiotic stressors on bees, efforts to improve bee nutrition are critical.The long-term goal of this proposal is to improve bee nutrition by building a database of the pollen nutritional compositions (especially phytosterols), understanding phytosterol requirements and their impact on colony health and performance, and investigating impacts of sterol biosynthesis inhibitor fungicides on plant pollen sterol availability and bee health.Objective 1. Analyze phytosterol profiles, amino acids and metabolites in pollens from 100 major bee pollinated crops, predominant natural forage and commonly used ornamental plants available to bees in North America.Objective 2. Investigate the effects of 24-methylenecholesterol on (a) honey bee colony growth and survival and (b) honey bee physiology - across all stages of the life cycle (egg, larva, pupa and adult).Objective 3. Examine potential effects of SBI fungicides on pollens and bees by (a) examining the impacts of SBI fungicides on pollen phytosterol composition (b) investigating indirect effects of two major SBI fungicides (propiconazole and metconazole) on honey bee physiology and (c) determining if sterol supplementation can mitigate effects of SBI fungicides, if any.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
The long-term goal of this proposal is to improve bee nutrition by building a database of the pollen nutritional compositions (especially phytosterols), understanding phytosterol requirements and their impact on colony health and performance, and investigating impacts of sterol biosynthesis inhibitor fungicides on plant pollen sterol availability and bee health.Objective 1. Analyze phytosterol profiles, amino acids and metabolites in pollens from 100 major bee pollinated crops, predominant natural forage and commonly used ornamental plants available to bees in North America.Objective 2. Investigate the effects of 24-methylenecholesterol on (a) honey bee colony growth and survival and (b) honey bee physiology - across all stages of the life cycle (egg, larva, pupa and adult).Objective 3. Examine potential effects of SBI fungicides on pollens and bees by (a) examining the impacts of SBI fungicides on pollen phytosterol composition (b) investigating indirect effects of two major SBI fungicides (propiconazole and metconazole) on honey bee physiology and (c) determining if sterol supplementation can mitigate effects of SBI fungicides, if any.
Project Methods
Objective 1: Analyze phytosterol profiles, amino acids and metabolites in pollens from 100 major bee pollinated crops, predominant natural forage and commonly used ornamental plants available to bees in North America.We will collect pollen samples from 100 bee visiting flowering plants including major bee pollinated crops (almond, apple, blueberry, cherry, cranberry, pear, canola, sunflower, alfalfa, cucurbits, and vegetable seed crops such as radish, onion, carrot) predominant natural forage and commonly used ornamental plants. We will collaborate with stakeholders (beekeepers and growers) in finding appropriate fields for collecting pollen from the target crops. Flowers will be collected from each target crop / species to extract desired amount of pollen manually for sterol analysis as per established methods (Roulston et al. 2000; Costa and Yang 2009; Villette et al. 2015; Vaudo et al. 2020).(i) Mass spectrometry for assessing sterol profiles of plant pollensWe will use our previously established methods (Chakrabarti et al. 2019a).(ii) Mass spectrometry for quantifying amino acids in plant pollensThe OSUMSC is well equipped to conduct such analysis and already has methods established to quantify amino acids. Amino acid profiles: Briefly, 25 mg of the tissue samples are to be homogenized in a 50:50 solution of methanol and separated on an Inertsil Phenyl-3 stationary phase (GL Sciences) coupled to a quadrupole-time-of-flight mass spectrometer (Triple TOF 5600, AB SCIEX) with MS/MS spectra recorded using Information Dependent Acquisition-Mass Spectrometry (IDA-MS). Amino acids are identified using the OSU-IROA in-house library covering 420+ measured standards based on accurate mass, fragmentation pattern, isotope distribution and retention time. For constructing the calibration curves, authentic amino acid standards will be used (Millipore Sigma, USA).(iii) Total soluble protein and lipid content analyses of pollen samplesTotal soluble proteins in pollens will be estimated based on the protocols by Corby-Harris et al. 2018.Objective 2 (a) investigate the effects of 24-methylenecholesterol on honey bee colony growth and survivalNucleus honey bee colonies, each containing approximately 5,000 bees and a sister queen will be installed in flight cages at the Oregon State University apiary in Corvallis, OR, and supplemented with artificial diets. Diets for the experimental treatment group colonies will contain 0.1%, 0.5% and 1.0% (dry diet weight) of 13C labeled 24-methylenecholesterol (concentrations proposed here are derived from lab study using caged bees, published in Chakrabarti et al. 2019b). Colonies in a positive control treatment group will be fed diets made from mixed natural pollens (Mixed Floral Pollen, CA, USA). Each treatment will be replicated five times, hence there will be a total of 20 nucleus colonies (4 treatments and five replications). The study will be conducted for 8 weeks, inside flight cages.Formulation of artificial diets and measuring consumptionBased on the modified protocols of Herbert et al. 1980, the artificial diets will be formulated similar to the diets used in our lab study described earlier (Chakrabarti et al. 2019b, 2020).Colony evaluationBrood production in the nucleus hives will be measured, using a combination of imaging (Delaplane et al. 2013) and acetate sheet methods (Human et al. 2013).Survival in the treatment groupsSurvival probability of each hive, across the various treatment groups, will be calculated based on previous studies (Khoury et al. 2013; Russell et al. 2013). Using generalized linear models (McCullagh and Nelder 1989), we will analyze the production rates of eggs and the conversion rates of eggs to larvae and larvae to pupae. Both treatment effects and time effects (e.g., trend) will be examined.Objective 2 (b) assess the effects of 24-methylenecholesterol on honey bee physiology - across all stages of the life cycleThe samples for this objective will be collected periodically from all the experimental colonies described above in objective 2a, immediately frozen and stored in -80°C freezers for further molecular analyses.(i) ProteomicsProtocols for proteomic sample preparations and analyses have been established at the OSUMSC based on previously published studies (Troyer et al. 2017).(ii) Sterol analysesPhytosterol profiles of honey bee tissues will be analyzed using LC-Atmospheric Pressure Chemical Ionization-Multiple Reaction Monitoring (LC-APCI-MRM) methods at the OSUMSC as described before. A standard for 13C-24-methylenecholesterol (Avanti Polar Lipids, USA) will be used in addition to the other sterol standards to quantify the labeled sterol in the honey bee tissues, eggs and brood food.Objective 3. (a) Examine the impacts of SBI fungicides on plant pollen phytosterol compositions.We propose to study the impacts of two major, commonly used SBI fungicides - metconazole (MTZ) and propiconazole (PPZ). The Dept. of Horticulture, Oregon State University, will provide the access to the experimental farms and greenhouses for testing the effects of PPZ and MTZ on plant pollen sterol composition. Blueberry plants will be used for this experiment similar to our preliminary study, as we have designed a robust protocol for pollen collection from blueberry flowers. This study will be carried on for three years. Fifty potted blueberry plants will be sprayed each year with field spray doses (as per label) of each of the following fungicides - PPZ, MTZ and a mixture of PPZ and MTZ. A control set of potted plants will also be included in the experiment. Pollen samples will be collected from 2,000 flowers from each treatment and control groups during the blooming season for all three years, and tested for the sterol compositions. We will use the same methods described earlier for sterol analysis.Objective 3. (b) Investigate the indirect effects of two major SBI fungicides (propiconazole and metconazole) on honey bee physiology; and (c) Determine if sterol supplementation can mitigate effects of SBI fungicides, if any.We will test the same SBI fungicides (PPZ and MTZ) mentioned in objective 3a to investigate their indirect impacts on the physiology of honey bees. One hundred newly emerged adult honey bees from sister-queen colonies (to minimize genetic variation) will be released in to each of the three hardware cloth cages for each of the seven treatment groups. We will use the following fungicide concentrations based on LD50 values (propiconazole oral LD50 1,000,000 ppb and metconazole oral LD50 850,000 ppb), to assess the worst-case scenario effects. The study will be conducted for three weeks.(i) Formulation of dietsFor appropriate treatment groups, diets will contain propiconazole (Millipore Sigma, USA) and metconazole (Millipore Sigma, USA) - dissolved in acetone - to obtain the fungicide concentrations noted above. Fungicides will be added to diets as an acetone solution mixed into the dry sugar powder and evaporated. Honey bees will be provided with 2 g of dry sugar powder mixed with acetone only (for controls) and fungicides in acetone solution (for treatment groups) and water for one day to ensure complete consumption of the sugar powder. After 24 hours, only sugar syrup and water will be provided to all groups. After one week, based on the treatment groups, some will receive pollen diets (mixed floral pollen to provide additional sterol supplementation) with sugar syrup and water, while others will continue to receive only sugar syrup and water (no additional sterol supplementation) for the remaining two weeks of the experiments.(ii) Parameters to be measuredSurvival of adult honey bees, consumption of diets, sterol profiles in honey bee tissues (LC-MRM and MS) and vitellogenin gene expression (as a measure of fat body functioning) will be measured across all the experimental groups. Sterol profiles will be measured using methods described earlier.