Location: Invasive Species and Pollinator Health
Project Number: 2030-21000-055-004-T
Project Type: Trust Fund Cooperative Agreement
Start Date: Feb 9, 2021
End Date: Jun 30, 2024
Objective:
Arguably the most critical member of a honey bee colony is a mated queen, who is solely responsible for the production of fertilized eggs that hatch and develop to replace aging worker bees. If not readily replaced, a failing or underperforming queen can quickly result in declining worker populations and colony loss. For this reason, reports of high rates of queen loss are of great concern to the beekeeping community, necessitating investigation into potential causes and threats to queen health and productivity. However, one limitation to studying queens has been the availability of quantitative assays to monitor and track queen performance under controlled experimental scenarios. Recently, a technique using highly specialized Queen Monitoring Cages (QMC) to subject honey bee queens to agrochemical stressors through diet administered to workers has been used to demonstrate that insect growth regulators (IGRs) can result in impaired hatching rates of eggs, demonstrating that indirect agrochemical exposure can negatively affect queen fecundity. Now, we propose to utilize this system to examine the effects of another class of agrochemicals on honey bee queens. Organosilicone surfactants (OSS) are a common class of adjuvants that have recently been shown to exert harmful effects on honey bees by impairing immune function, impairing learning, and recently, by affecting the expression of genes related to hormone signaling, which may have dramatic consequences on colony reproduction. This proposal will describe an experiment using QMCs to subject honey bee queens and their worker attendants to field realistic levels of specific OSS compounds known to be present in commonly used adjuvant formulations and monitor reproductive output and worker-queen interactions.
Approach:
Assays will be conducted in plexiglass QMCs, which are essentially micro-hives containing removable honeycombed plates for egg laying that can be easily manipulated for research and maintained in an incubator for easier monitoring. Cages will be assembled with 60 newly eclosed bees and a single mated queen. QMCs will receive two 2 mL feeders containing 50% sucrose solution, one feeder of water, and a feeder of pollen supplement. Experimental diet (agrochemical mixture) will be administered in sucrose solution and pollen supplement for 14 days (i.e., chronic exposure) at doses based on field and colony levels of OSS reported in pollen and nectar. For each dose, fifteen cage replicates will be used. Queens will be added to QMCs after the workers; QMCs will then be transferred to an incubator set at 34.5 C and 75 RH and acclimatized for 24 hours prior to introducing agrochemical treatments in diet. Throughout the length of the QMC trials (15 days), mortality of worker bees will be monitored, and dead bees will be removed daily.
Assessing worker retinue behavior towards queens: We will monitor queen retinue responses using cameras placed inside of incubators under red light. On day 5 and day 9 of the exposure scenario, cameras will record images of workers and queens in each cage at 30-minute intervals for 24 hours. Methods to quantify retinue behaviors will be adapted from Rangel et al. (2016). Briefly, retinue responses will be evaluated based on the number of worker bees grooming, feeding, antennating, and surrounding the queen in each photo. By determining how agrochemical exposure affects queen tending behaviors and relevant worker physiology we will elucidate their effects on intra-colony dynamics.
Assessing physiological changes and hormone shifts in queen and worker honey bees: Workers - 10 worker bees per cage will be collected on days 7 (midway) and 15 (end) of the experiment. Hemolymph will be extracted from 5 bees per cage per sampling event and levels of juvenile hormone will be assessed by ARS in Maricopa, Arizona. Hypopharyngeal gland development of the five bees will be measured using the remaining tissue, and the five additional workers will be flash frozen for gene expression analysis using RT-qPCR. Briefly, genes involved in behavioral development such as Kr-h1 (brain), Ilp-1 (brain), Vg (fat body), mrjp 1-9 (HPGs) will be assessed in specific tissues. Expression of CYP305D1 and CYP6AS14 will be assessed in the fat body. On day 15, the remaining bees will be flash frozen, and used to assess pathogen abundance using RT-qPCR.
Queens - Honey bee queens will be sampled at the conclusion of the experiment. Hemolymph will be extracted from living queens and JH levels will be analyzed as described above. Queens will then be sacrificed by freezing, and ovaries will removed and weighed as a proxy for development.
A portion of the diets will be used to verify the concentrations of OSSs and plot their degradation in the diet using LC-MS-MS.
