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ARS Home Northeast Area Beltsville, Maryland (BARC) Beltsville Agricultural Research Center Bee Research Laboratory Research





- Cook, Steven
- Crop Production (305)

Research Project: **Location:
                            Bee Research Laboratory** Project Number: 8042-21000-291-068-I
                        
                        Project Type: Interagency Reimbursable Agreement Start Date: Mar 1, 2023
                        
                        End Date: Sep 30, 2023 **Objective:**
                        
                        The overarching goal of these projects is to obtain data that will inform American beekeepers of the effects of different treatment regimens against mites on the transmissibility/infectivity of DWV, and to provide a potential explanation for why some treatments against Varroa do show expected efficacy in the field. The objective of project (1) above is to determine changes in DWV infectivity when vectored by Varroa mites that have or have not been restricted to a dispersal stage for an extended period.  For this, we will evaluate the genomic copies (GE) of DWV in naïve honey bee pupae exposed to Varroa mites that have or have not been restricted to a dispersal stage for an extended period. The objective of project (2) above is to determine the effects of Varroa management strategies on the transmissibility/infectivity of DWV.  For this, we will treat honey bee colonies with chemical and/or cultural means of Varroa control then measure the GE of DWV in naïve honey bee pupae exposed to Varroa mites collected from these differentially-treated honey bee colonies. The objective of project (3) above is to identify factors related to host use that confer tolerance, and thus survivability, of mites exposed to toxicants. **Approach:**
                        
                        For Objective 1, ten groups (50-100 individuals) of newly emerged adult honey bees will be housed in cages in the laboratory and either exposed or not to a ~30% infestation of Varroa mites (five cages each treatment).  After 12 days, 10 mites per cage will be collected and used in infectivity assays with naïve honey bee pupae.  Ten remaining live bees from each of the cages will be collected and stored at -80 C. Mites will be placed on naïve pupae for six days, after which mites and pupae will be collected and stored at -80° C.  RNA will be extracted from a subset of adult bees from cages and the pupae exposed to mites, and RT-qPCR runs conducted to quantify DWV loads in both samples.  

For Objective 2, twenty full-size honey bee colonies will be first assessed for their Varroa mite loads using alcohol washes of 200-300 adult bees collected from the colonies.  Colonies will also be assessed for their populations of both adult and developing bees, and also their stores of honey and pollen.  From these data, colonies will be separated into four treatment groups (5 colonies per group) that are distinguished by the type of Varroa treatment regimen applied.  These are: (1) Apivar treatment (chemical, Amitraz), (2) Queen caging, (3) Apivar + Queen caging, and (4) no treatment. Samples of both Varroa mites and adult bees will be collected from each colony before treatments are applied and samples stored at -80° C for later analyses.  Also before treatments are applied, ten mites will be collected per the twenty colonies and used in infectivity assays with naïve honey bee pupae.  After 21 days, the Queens will be released from their cages.  After 42 days, the Apivar treatments are removed.  After the first 21 days, samples of mites and bees will be collected and stored at -80° C.  Also, Varroa are collected from each of the colonies and used in infectivity assays with naïve honey bee pupae as hosts.  We will expose the pupae to the mites for six days, after which both live mites and pupae will be collected and stored at -80° C.  Varroa levels are checked again for each colony in order to gauge effect of treatments on mite levels. RNA will be extracted from the exposed honey bee pupae and field-collected adult bees and RT-qPCR runs will be conducted with these samples to quantify the GE of DWV.

For Objective 3, Varroa mites will be collected from honey bee field colonies and returned to the lab for use in toxicological assays using Amitraz as a toxicant.  Toxicological assays will use the standardized ‘vial assay’ wherein mites are exposed for 4 hours to a toxicant (different doses) that coats the inside surface of the vials, then mites are removed and placed on a honey bee pupa host for an additional 20 hours.  Mites will be assessed for their survival at each of the two time points.  After these time points, any dead mites will be promptly stored at -80 C, and the live mites after a total of 24 hours. Protein will be extracted from mite and host samples and quantified and checked for integrity (agarose gel). The same amount of protein per sample will be sent to the University of Virginia proteomics lab for proteomic analysis.


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