Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
(N/A)
Non Technical Summary
Managed honey bee colonies are experiencing unsustainable annual losses that are partly due toreduced immunocompetence that leads to acute viral outbreaks and mortality. To help restorehoney bee health despite the myriad of environmental stressors, we have focused onidentifying novel physiological pathways that can mitigate virus-mediated mortality throughincreased immune function. We previously demonstrated that a family of potassium ionchannels, termed KATP channels, mediate the survival of honey bees during infection fromIsraeli Acute Paralysis Virus (IAPV), suggesting KATP channels may drive antiviralimmunity. Interestingly, these channels have been linked to regulation of reactiveoxygen species (ROS), which are known to function as an immune system stimulator duringvirus infection. Therefore, the overarching goals of this proposal is to build a functionalmodel characterizing the relationship between KATP channels, ROS, and antiviralimmunity in bees and to determine the toxicological relevance of this pathway. Toachieve this goal, we propose the following objectives: 1) determine the ability of KATPchannel modulators to reduce mortality stemming from Deformed Wing virus; 2) determine ifKATP channels regulate ROS to control antiviral immunity; and 3) test the hypothesis thatKATP modulators will reduce virus prevalence at the level of the colony. Findingswill provide a fundamental understanding of bee immunity and will reveal approachesfor the development of novel antiviral therapeutics to address stakeholder needs. With theultimate aim being to increase the sustainability of managed bee colonies, this projectsupports the Pollinator Health (A1113) program priorities
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
0%
Goals / Objectives
Theoverarching goalsof this investigation are to 1) test the hypothesis that modulation of honey bee KATPchannels will stimulate production of ROS, which will serve as a secondary messenger to enhance immune function and reduce virus mediated mortality and 2) test the hypothesis that in-hive acaricides alter antiviral immunity and virus susceptibility of the individual bee.Collectively, these data will fill significant knowledge gaps pertaining to honey bee physiology and will provide tractable biochemical targets that can be exploited through product development campaigns (subsequent proposals). To achieve our goal, we will:Determine the relevance of KATPchannels to honey bee antiviral immunity and the impact of miticides to bee survivorship after inoculation with deformed wing virus;Determine the functional relevance of ROS to antiviral immunity and the functional linkage between KATPchannels and ROS levels to individual and colony immunity;Test the impact of KATPmodulators to virus dynamics at the level of the colony through controlled field studies?
Project Methods
Insects:We will use the Italian strain ofApis mellifera, which is currently maintained at LSU and the USDA-ARS Honey Bee Lab in Baton Rouge, LA.The bees and colonies used in this study will be standardized between the LSU and UNL by creating colonies from sister queens resulting from a single drone insemination event, which will eliminate the potential for differential genetics to prevent accurate data interpretation.The USDA-ARS Bee Laboratory routinely performs these techniques and is commonly used by M. Simone-Finstrom (co-PD).Pharmacological Tools:A combination of activators and inhibitors for KATPchannels will be used to ensure the phenotype that is observed is due to action at the expected target site and not through off-target effects. Obj 1:We will infect adult and larval bees with DWV and test the ability of KATPmodulators or miticides to alter the replication of DWV mRNA as well as alter the deformed wing phenotype.DWV is ideal for use in this study because the infection symptomology is easily identified by visual observation, whereas other viruses require multiple days to weeks to observe a phenotype from infected individuals.Virusreplication kinetics will be established by infecting age-matched newly emerged bees or age-matched larval bees with DWV via microinjection and then collecting groups for qRT-PCR analysis of viral replication in a time course experiment, as previously described. Groups of bees will be fed a 50% sucrose solution containing KATPactivators, KATPinhibitors, inactive analogs,or a vehicle control prior to infection with DWV. For all survival experiments, six replicates of 25 bees will be used for each treatment group and survival will be observed 12 h following injection and then every 24 h for 7 daysObj 2: Newly emerged bees (< 12-hours old) will be collected from a colony with known genetics at the USDA-ARS Honey Bee Lab.To study the impact of KATPchannel modulators or miticides to ROS regulation, we will isolate 50 newly emerged bees into a holding chamberand expose them to various treatment groups.Treatment groups will be as follows: 1) untreated bees (control), 2) feed KATPmodulators alone, 3) feed paraquat alone, 4) miticides alone, 5) pre-feed bees with KATPmodulators for 24 hours and then include paraquat, 6) expose bees to miticides at label rates and 24 hours later feed paraquat via sugar solution and 7) pre-feed with KATPmodulators for 24 hours and then expose to paraquat and miticide.A total of 20 bees will be collected per replicate at the following time points: 0, 12, 24, 48, 72, and 96 h.Chemicals dissolved in sugar water will be replaced every 12 hBiochemical Assays:Individual bees collected from each treatment group will be snap frozen in liquid nitrogen and then pulverized to maximize substrate and protein concentrations.The powdered bee will be reconstituted in assay buffer and used for the biochemical assays. ROS generation and subsequent cellular damage is highly variable among individual bees and thus, we will perform biochemical analyses on individual bees to provide greater resolution of the data. We will use commercial kits to measure the total antioxidant levels and the lipid peroxidization that are available from Cayman Chemical Company.To measure DNA oxidative damage, genomic DNA will be isolated from bees and examined for oxidative stress damage by measuring the presence of 8-oxoguanine, a primary target of reactive oxygen species.This is an ELISA based assay that utilizes a coated plate and a conjugated antibody for detection of 8-oxoguanine.Obj 3:Bee Stock:Nucleus colonies will be established early spring in hives with new frames having undrawn foundation.Queen failure has the potential to alter virus titers in the colony and, thus, we will take measures to reduce the probability of queen failure. For bee stock generation, we will inseminate sister queens with equal aliquots of pooled semen collected from 200 drones. This will reduce the probability of queen failure and maintain similar genetic structure of all test colonies. Colonies will be segregated into distinct apiaries by treatment group andseparated by>2 milesto avoidpossible drift from heavy virus-infected colonies to those with less virus infection.Mite management will not be performed in these hives to ensure mite treatments do not impact data.Brood disease and mite levels will be monitored in all experimental colonies.Field analysis:we willdivide the colonies equally into the following treatment groups:Untreated Control (UTC; vehicle only), Positive Control (POS, vehicle plus IAPV or DWV), Treatment 1 (T1; pinacidil only), Treatment 2 (T2;pinacidil+IAPV or DWV), Treatment 3 (T3; tolbutamide only) and Treatment 4 (T4;tolbutamide+IAPV or DWV). Prior to treatment, 200 newly emerged bees will be removed from each colony and the titers of IAPV and DWV in the pool of 200 bees will be quantified to establish a baseline infection level.Importantly, it has been shown that a pool of 90 bees gets 99% confidence of virus detection with 5% prevalence, thus a pool of 200 bees is more than ample to establish baseline infections104.After establishing baseline infection rates, colonies will be pre-treated with KATPmodulators only or solvent control for 24 hours prior to the introduction of IAPV or DWV into POS, T2, T4.KATPmodulators will be present throughout the duration of the study.A total of 6 treatment groups with 5 colonies per treatment will be used and they will be sampled bi-weekly for a two-month period and a total of 5 sampling periods. By conducting this trial in spring and early summer, this will ensure that transmission of viruses by varroa mites will be less influential in this more controlled field experiment.Obj 3.2:These data will extend data collected in Objective 3.1 by studyingnatural infestationsof virus. We will sample the following bee viruses: DWV, Chronic Bee Paralysis Virus, AKI (complex of Acute Bee Paralysis, Kashmir, and IAPV), and Black queen cell virus using primers that have been published1,95.These viruses represent the most commonly detected viruses in US honey bees. Colonies will be established in spring following established procedures where 5-frame colony "splits" will be made from overwintered colonies at the USDA Bee Lab using the previously described "tower" technique1. This method creates a set of colonies that are homogenous, equalized for adult and brood populations, and have similar pathogen and parasite loads. These colonies will be established with sister queens as described for Obj. 3.1. We will treat 15 colonies with pinacidil, 15 colonies with tolbutamide, and 15 colonies with vehicle (0.01% DMSO) only to serve as a control. Baseline levels of virus in colonies will be determined (see Obj. 3.1) and changes in viral infection over time will be analyzed. We will assess the colonies once per month from March - September for overall health (quantity of eggs and pupae, stored honey, and lipid levels in individuals) by removing 90 worker bees and 90 pupae from each colony to be assessed for virus titers following established protocols in our laboratory (see general methods).?