Location: Horticultural Crops Disease and Pest Management Research Unit
Project Number: 2072-30400-001-016-A
Project Type: Cooperative Agreement
Start Date: Sep 1, 2026
End Date: Dec 31, 2028
Objective:
Obj 1. Quantify the effects of protein supplementation on honey bee colony strength, nutritional status, and foraging activity during blueberry bloom.
Obj 2. Evaluate the impact of improved colony nutrition on blueberry pollination, fruit set, fruit quality, and yield.
Approach:
Experimental Design: Eight commercial blueberry fields planted with similar varieties will be selected. In four fields, honey bee colonies will receive one pound of protein supplement every two weeks during bloom. The remaining four fields will serve as untreated controls. Colony strength, flower visitation, fruit set, and yield will be compared between treatment and control fields. This experiment will be repeated in two consecutive years.
Honey bee visitation: Bee visitation will be quantified using standardized visual observations conducted between 9:00 AM and 3:00 PM during peak bloom under favorable weather conditions. Observers will perform 5-minute walk between rows, with eight replicate observations per field.
Blueberry Yield and Quality Assessment: Within each field, twelve bushes will be randomly selected across three transects. Two branches per bush (approximately 50 flower buds per branch) will be marked. Fruit sets will be calculated by counting the number of fruits per branch. At harvest, fruits from marked branches will be collected and analyzed for fruit weight, fruit size, and seed number per fruit.
Colony Health Assessment: Eight colonies per field will be tagged and evaluated twice: placement in the field and prior to removal. Colony strength will be assessed by estimating adult bee population, brood area, and stored honey and pollen.
Nurse Bee Head Protein Content: Ten nurse bees per colony will be collected from brood combs at the second colony evaluation for protein analysis. Bee heads will be homogenized in 100 µL phosphate-buffered saline (PBS) using a 3-mm tungsten carbide bead, followed by centrifugation. The supernatant will be diluted 1:20 and analyzed using a bicinchoninic acid (BCA) protein assay. Total head protein content will be expressed as milligrams per bee.
RNA Extraction and Vitellogenin Gene Expression: To evaluate the effects of protein supplementation on nutritional health, the expression level of the vitellogenin gene in nurse bees will be measured. Fifty nurse bees will be collected from brood combs of each experimental honey bee colony and stored at -80 until RNA extraction. Total RNA will be extracted from pooled bee samples. Quantitative PCR will be conducted using SYBR Green Master Mix, with gene-specific primers for vitellogenin and RpS18 (reference gene). The relative expression of vitellogenin will be calculated using the ¿Ct method, comparing treatment groups against the control.
Statistical analysis: All analyses will be conducted using R, with field as the experimental unit. Treatment effects of protein supplementation on foraging activity, pollinator visitation, fruit set, yield, fruit quality, colony strength, nurse bee head protein content, and vitellogenin expression will be analyzed using mixed-effects models, with treatment as a fixed effect and year as a random effect. Changes in colony performance will be quantified as percent change relative to baseline values and analyzed as continuous response variables using linear mixed-effects models, with treatment as a fixed effect and year as a random effect. Statistical significance will be assessed at a = 0.05.