Defining Mechanisms Underlying Mite Tolerance and Honey Bee Survival

Honey bee samples - Honey bees were in vitro reared (Schmehl et al 2016). This allowed for controlled development across samples, and also limited other unknown environmental variables that may affect DWV infection (including exposure to DWV from the colony). Bees were collected from a research colony with a single drone inseminated (SDI) queen - this allowed for approximately 75% relatedness between sister bees due to the honey bee's haplodiploid sex-determination system, and therefore, minimized the effect of differing honey bee host genetics influencing DWV infection. This SDI colony was inspected weekly to ensure health status (ie low virus infection and low parasite load) and confirm the presence of the original queen. 

Virus Isolation - Since it is not possible to general purified bee viruses, crude isolations were conducted to create two starting inoculums, as well as the subsequent inoculums from the viral passaging paradigm (see below, Virus Passaging). Virus was isolated from individual bees using the following protocol. 2ml screw-cap microcentrifuge tubes with 2mm zirconia beads within were sterilized with a UV-cross linker for 2 minutes. Bees (flash-frozen and kept at -80°C until processing) were placed into 2ml screw-cap microcentrifuge tubes, and 500ul of 1x PBS was added. Bees were homogenized using a Bead Ruptor Elite (Omni International, Kennesaw, GA) at 6.5 ms for 45 seconds. Tubes were then placed on ice then centrifuged for 3 minutes at max speed. Supernatant was removed and passed through a sterile 0.2um syringe filter to separate viral particles from honey bee cells (pellets were stored at -80°C). This crude isolate was kept at 4°C for no longer than a week (for injections and/or RNA extractions) and stored long-term at -80°C.

Virus Passaging - The starting inoculum was injected into in vitro reared honey bee pupae at the white-eyed stage (14 days post-egg-laying). Virus concentration was approximately 6 x 10^9 genome equivalents. 1.5ul inoculum was injected using a mouth aspirator with an attached 10ul capillary tube pulled into a needle. Needles were changed between treatments to avoid contamination between treatments. Two separate starting inoculums were utilized. To measure colony DWV levels and the effect of the injection itself on DWV levels, control bees (in vitro reared but further unmanipulated) and PBS-injected bees (injected with the stock viral isolation saline) were included as controls (these groups are labeled as "no inject" and "PBS"). 

Two additional treatments were incorporated into the virus passaging paradigm to immunocompromise bees infected with DWV. Bees were either injected with a nylon thread to induce melanization (an immune response supplementary to the main antiviral response, the RNA interference pathway) or had 1μl hemolymph removed (to mimic Varroa feeding). These groups were identified as "DWV+mel" and "DWV+hemo", respectively. Bees without any immunocompromisation were simply identified DWV+. Passaging virus through Varroa (the biological vector) was not included as proposed as mites could be successfully kept in the lab, but did not reproduce. To control for virus amplification triggered by the immunocompromisation protocol, bees were immunocompromised and injected with PBS (groups "DWV-mel" and "DWV-hemo").

Virus inoculums from samples were isolated (see above, Virus Isolation) four days after infection, and all groups were passaged five times. See Figure 1 for all 10 included groups. 

Figure 1. Groups included in the immunocompromised bees viral passaging paradigm. 

To passage virus, viral inoculums with confirmed DWV (see below, Virus Quantification) was injected into a new in vitro reared honey bee pupae at the white-eyed stage using the described method - 1.5ul injection using a mouth aspirator and capillary needles - and isolated again 4 days post-injection. Inoculums isolated from bees with additional treatments (ie hemolymph removal or triggered melanization) were again injected into bees with the same treatment as the previous passage (ex DWV+hemo inoculum into another bee with 1μl hemolymph removed, etc.). This method was repeated until virus populations were passaged 5 times. 

Virus Quantification - RNA was extracted from 30ul of each virus inoculum using a Direct-zol RNA Miniprep kit (Zymo Research, Irvine, CA) following the manufacturer's protocol. cDNA was prepared from each sample using a High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (ThermoFisher Scientific, Maltham, MA) the following way : 1ul of 10x Buffer, 0.4ul of 25x dNTP mix, 1ul of 10x random primers, 0.5ul of Reverse Transcriptase, 0.5ul of RNAse Inhibitor, and 3.4ul of RNA. cDNA was diluted 1:20x to allow a sufficient amount of cDNA for all qPCR reactions. qPCR was conducted using PowerUp™ SYBR™ Green Master Mix (ThermoFisher) in the following way : 5ul SYBR, 1ul of 10x Forward Primer, 1ul of 10x Reverse primer, 1ul of water, and 2ul cDNA. qPCR was conducted using a 7900HT Fast Real-Time PCR system (Applied Biosystems) under the following parameters: 50°C for 2 minutes, 95°C for 10 minutes, then cycle 40x 95°C for 15 seconds and 60°C for one minute, and then a dissociation stage step for melting curve analysis. qPCR primers can be found in Table 1. 

Table 1. Primers used in this study to quantify DWV-A and DWV-B within viral populations. S8 was used to confirm successful RNA extraction and cDNA conversion. 

Arnot Forest Collections - Bees were collected from 13 sites across NY and PA (Figure 2). Collections were conducted between September 19 to October 14, 2019, between 10AM-5PM on sunny, warm (mid 60's-mid 70's°F) days, by Allyson Ray and Dr. Thomas Seeley. Bees were captured using insect nets and knocked out on dry ice and then placed into 15ml conical tubes by site and kept on dry ice. Wild Arnot Forest bees were collected while foraging on flowers (n = 5-12 bees/site), and bees from managed colonies were collected from colony entrances, preferentially selecting obvious foragers (indicated by pollen-filled corbiculae) when possible (n = 2-5 colonies/apiary, 10-15 bees/colony). Upon returning from the field, bees were placed at -80°C for long term storage. See Table 2 for details. 

Figure 2. Collection sites (From top to bottom, left to right : Site 3, 8, 9, 10, 11, 12, 13)

Table 2. 

Virus Isolation and Quantification - Virus from Arnot forest collections was isolated and quantified as described above, with the exception that, for Presence/Absence consideration, cycle threshold (ct) from qPCR experiments had to be less than 30 to be considered 'infected'. 

Sequencing of subset and analysis – As BQCV was in low abundance across our samples, we focused on DWV for deep sequencing analysis. RNA extracted from a subset of virus isolations with higher DWV levels were submitted to the Pennsylvania State Genomics Core Facility (University Park, PA, USA) for library preparation and sequencing. Libraries were prepared from viral populations of individual bee samples across the Arnot forest samples as well as the managed honey bees from New York and Pennsylvania. The 28 samples were sequenced on the Illumina Miseq platform, resulting in 150 nucleotide paired-end stranded mRNA reads. Total reads per sample ranged between 274,942 – 753,035. Reads were assessed for quality with FastQC and quality trimmed with Trimmomatic (option SLIDINGWINDOW:4:25).

DWV consensus DWV-A and DWV-B genomes were built using methods described in Ray et al 2021. Briefly, genomes were created by aligning reads from each sample to DWV-A and -B reference genomes from NCBI (Ref. NC_004830.2 and NC_006494.1, respectively) using Hisat2 (Pertea, Kim, Pertea, Leek, & Salzberg, 2016). Using bcftools (Li, 2011), variants were called and the consensus fastq sequence file generated, and from the resulting fasta files, bases with qualities less than 20 were converted to Ns using seqtk (Li, 2013). DWV levels were still mostly low in these samples, but full-length genomes were constructed for 10 samples. This resulted in 11 consensus genomes (one sample, 13-1-E was naturally co-infected with DWV-A and DWV-B). Reads were also aligned to a third variant of DWV, variant C, as well as other common bee viruses – less than 0.06% of reads within each sample aligned to DWV-C (CEND01000001.1), and less than 0.35% of reads within each sample aligned to other common bee viruses in the USA (acute bee paralysis virus (NC_002548.1), black queen cell virus (NC_003784.1), chronic bee paralysis virus (NC_010711.1), Israeli acute paralysis virus (NC_009025.1), Lake Sinai virus 2 (NC_035467.1), sacbrood virus (NC_002066.1), and thus these viruses were not further examined within the sequence data.

For phylogenetic analyses, Multi-sequence alignments of generated consensus genomes and additional reference genomes (DWV-A reference (NC_004830.2), DWV-B reference (NC_006494.1), and DWV-C (CEND01000001.1)) were generated with Clustal Omega using default settings. Multisequence alignment was then imported into MEGAX for Maximum likelihood tree construction with default settings and bootstrapped using 1000 replicates, (Kumar, Stecher, Li, Knyaz, & Tamura, 2018). Consensus genomes, as well as raw sequence reads, will be uploaded to the NCBI Genome and SRA database (accession numbers to be assigned).

Honey bee samples for experimental infections – Bees were collected in August through September 2021 from a Penn State University research apiary. Two different colony (genotypes) were utilized in this study. One colony was led by a single drone inseminated (SDI) queen – this allowed for approximately 75% relatedness between sister bees due to the honey bee’s haplodiploid sex-determination system (Winston, 1991), and therefore, minimized the effect of differing honey bee host genetics influencing DWV infection. The other colony was led by a naturally (i.e. multiply) mated queen. Prior to infection studies, colonies were assessed for viral infection via PCR and found no-low indication of common bee viruses. Both colonies were inspected weekly to ensure health status (i.e. no obvious signs of viral disease and low parasite load) and confirm the presence of the original queen.

Experimental infections – DWV populations from the sequencing analysis were additionally assessed for infection phenotype in the Summer of 2021. In order to reach a high titer of virus, inoculums were propagated in pupae collected from a DWV-free (assessed via qPCR) colony. Pupae at the white-eyed stage (14 days post egg-laying) were injected with the viral isolates, then collected 4 days post-injection (4DPI) as described above, and aliquoted to minimize the number of free-thaws. Virus was quantified (as described above) and assessed for co-infected bee viruses including BQCV, SBV, and the paralysis suite of bee viruses. Prior to injections, propagated virus populations were then normalized to two doses : approximately 10^6 genome equivalents per microliter and approximately 10^2 genome equivalents per microliter. Virus being actively used was kept at 4C for no longer than 3 days.

Pupal collections and infections were conducted in a UV sterilized hood to minimize contamination by mold and other microbes. Virus populations were injected into honey bee pupae at the white-eyed stage. White eye pupae were collected from the colony by uncapping worker cells and carefully removing pupae from their cell. Pupae that showed eye pigmentation (indicating older than 14 days old), melanization (indicating injury during collection), or Varroa within their cell were not utilized for infections. 1ul inoculum was injected using a mouth aspirator with an attached 10ul capillary tube pulled into a needle. Needles were changed between inocula to avoid contamination. To measure colony DWV levels and the effect of the injection itself on DWV levels, control bees (collected from the colony but further unmanipulated) and PBS-injected bees (injected with the saline solution used for the stock viral isolation) were included as controls (these groups are labeled as ‘Control’ and ‘PBS-inject’, respectively). 

Injected pupae were kept in 48-well plates and placed at 34.5C and approximately 75% R.H. in a desiccator within an incubator. Subsets of samples were collected at 3 days post-injection (DPI) to assess viral titers via qPCR. Samples were monitored daily for mortality and nearing eclosion timing (approximately 7DPI), monitored every 8-12 hours for successfully eclosed bees. ‘Successfully’ eclosed bees were assessed via pigmentation and high mobility, measured by noticeable movement around their respective well. Once eclosed, bees were removed from their well with sterilized forceps, accessed for deformed wings, and placed into plexiglass cages, split by group, noting the time of transfer. Cages were provided 30% sugar water and honey, ad libitum, and placed within an incubator at 34.5C and approximately 40-60% R.H. Cages with adult bees were monitored for survival daily, removing bees which had perished.

Virus quantifications from experimental infections – RNA was isolated from abdomens from 3DPI pupae collected during the experimental infection experiments using the Qiagen RNeasy Mini kit following manufacturers’ protocol including a DNAse incubation step and quantified using a Nanodrop. cDNA was prepared from each sample using a High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (ThermoFisher Scientific, Maltham, MA) from 200ng RNA following manufacturers’ protocol. cDNA was diluted 1:20x to allow a sufficient amount of cDNA for all qPCR reactions. qPCR was conducted using PowerUp™ SYBR™ Green Master Mix as described above.