Recipient Organization
ARIZONA STATE UNIVERSITY
660 S MILL AVE STE 312
TEMPE,AZ 85281-3670
Performing Department
School of Life Sciences
Non Technical Summary
Despite their importance to agriculture and food supply, honey bees and pollinators are facing ongoing exposure to environmental hazards contributing to declining populations. Several common hazards are not well understood including exposure to fungicides applied to a variety of bee-pollinated crops during bloom. Understanding how commonly encountered environmental hazards, such as fungicides, affect pollinator health can help better inform growers, beekeepers and pesticide manufacturers on how to protect crops and their pollinators. Such measures may mitigate ongoing pollinator population declines and strength food security measures.To assess fungicide exposure on honey bee health I will expose honey bees to a commonly used fungicide at levels mimicking those encountered in field conditions. Such exposures will allow me to then examine resulting cell function, organ function and overall health inexposed honey bees. These health parameters will then inform a mathematical model which will be used to predict long term effects of exposure to Pristine fungicide, other agrochemicals and common environmental hazards. This intended work will allow for the precise characterization of how a fungicide affects honey bee health. This information will inform growers and beekeepers about potential hazards of current agrochemicals and stimulate dialogue on alternative options for the protection of pollinators, crops and food security.?
Animal Health Component
40%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Goals / Objectives
We have recently shown that a common fungicide, Pristine®, strongly reduces honey bee populations when consumed in the field. This is a very important finding that has the potential to change both our recommended methods for evaluating pesticide safety and our agricultural practices for use of pesticides while preserving pollinator populations. Convincing farmers and agrochemical companies of the need to change fungicide use will require clear evidence of the mechanisms of fungicide toxicity for bees, which is the focus of this research. By completing the proposed research objectives, we will arrive at a clearer understanding of the underlying effects of exposure, providing a compelling case for re-evaluation of current pesticide safety evaluation and use.Research Objectives:1.Assessment of the effect of consumption of Pristine® fungicide on vitellogenin levels and protein concentration2.Assessment of the concentrations of boscalid and pyraclostrobin in the tissues and hive components of Pristine®-treated hives3.Assessment of the effects of Pristine® on protein digestion rate4.Assessment of the effects of Pristine® on midgut mitochondrial oxidation rate5.Predictive modeling of fungicide impact on colony growth and survival
Project Methods
Methodology:Objective 1: To test if Pristine® ingestion alters protein concentration levels, newly emerged bees from all Pristine® dose groups and the control will be marked and collected at 3, 8, and 14 days of age. Five workers will be sampled at each age from each of three hives per treatment (4 treatments * 3 hives * 3 ages * 5 bees = 180 protein samples for hemolymph, whole body and rectal contents).Hemolymph samples, gutted bee carcasses, and rectal contents will be collected, diluted, homogenized and then a Bradford Assay will be used to assess protein content. Bee carcasses and rectal contents will be homogenized in deionized water (diH2O) through sonication and centrifugation. Homogenates of each sample will be loaded into a 96-well plate along with dye reagent, and the absorbance of each sample will be measured at 595nm using a microplate spectrophotometer. I predict that hemolymph and whole body protein levels will be reduced by Pristine® consumption, while rectal protein levels will increase due to poor protein digestionObjective 2:To assess the relative exposure of beemidgut epithelia andother tissues to the active ingredients of Pristine® (boscalid, pyraclostrobin), 16 week-old bees will be collected, four per treatment groups (two from each of two hives). The gut will be removed, the midgut lumen sampled, and the remaining carcass homogenized. As boscalid and pyraclostrobin must be analyzed separately, this will require analysis of 32 midgut lumen and 32 bee carcass samples. To test the dose of boscalid and pyraclostrobin received by larvae, worker jelly and pollen samples will be taken from larval cells, with four samples analyzed per compound per treatment group (two each from two hives in a treatment group). This will require analysis of 32 worker jelly and 32 pollen samples. Bee, jelly and pollen samples will be prepared using a QuEChERS sample prep, starting with homogenization in acetonitrile solution using metal beads. The resulting homogenate will then be shaken vigorously and mixed with citrate buffer before centrifugation. The resulting supernatant will be incubated for 15 hours at -18oC. An aliquot of the supernatant will then be centrifuged and dried at 40oC. The dried supernatant will then be resuspended in methanol and phenacetin solution from which a 40μL aliquot will be added to 160 μL of deionized water. The resulting solution will be analyzed using the Agilent GC-MS system at the Mass Spectrometry core lab at ASUObjective 3:To assess the capacity for protein digestion, midguts from ten three-day-old bees, reared on one of the four different concentrations of Pristine®, will be dissected, and midgut contents homogenized and sonicated, followed by measurement of maximal trypsin-like and chymotrypsin-like activity (Moritz and Crailsheim 1987) using a microplate spectrophotometer.Objective 4:To test whether Pristine® directly inhibits the oxidative activity of mitochondria from the honey bee midgut, ten three-day-old honey bees from each treatment group will be dissected and the midguts isolated. The oxygen consumption rate of midgut tissues will be measured with an Agilent Seahorse XFp Analyzer. Midguts will be exposed to various concentrations of boscalid and pyraclostrobin, based on the concentrations measured in midgut lumens as determined in Objective 2.Objective 5:To assess outcomes of fungicide exposure in the broader context of the hive environment and interaction with other stressors, data generated from all completed objectives will be input into the VARROAPOP + Pesticides model. VARROAPOP + Pesticides model is a mathematical model allowing for the prediction of honey bee colony growth or decline factoring in multiple potential stressors such as queen strength, Varroa population, weather and pesticide exposure. The use of data collected for the objectives described here will allow for the prediction of the sublethal effects of Pristine® exposure on colony survival under varying biotic and abiotic conditions. The development of such models will enable assessment of the effects of Pristine® exposure on colonies in a variety of environmental conditions. Further, predictive modeling may allow for examination of sublethal effects contributing to colony decline beyond the scope of field experiments.