Spore Count

Nosema ceranae spores were originally obtained from infected A. florea and A. cerana workers in Chon Buri, Thailand and fed to A. mellifera workers in La Jolla, California to ensure a fresh stock, renewed weekly, for our experiments. Spore-producing bees were not fed pollen, only pure 2.0 M sucrose solution (55% sucrose w/w) to ensure that gut contents consisted mainly of spores. To obtain spores, we dissected out adult honey bee midguts, homogenized them in sterile double distilled water (ddH₂0), and vacuum-filtered them through Fisherbrand P8 filter paper with 20–25 μm pores. We collected the filtrate in microcentrifuge tubes that we centrifuged (Eppendorf 5415D centrifuge) at 9279 g (Relative Centrifugal Force) for 15 min, a modified method of Webster et al. [70 (link)]. We then removed the supernatant and resuspended the pellet in sterile ddH₂0 water. This procedure resulted in fairly pure spore preparations as determined with a microscope. We measured spore concentrations with a hemocytometer in a compound microscope (Zeiss Axioskop), making two independent measures of each sample and recording the average spore count [47 ]. We made appropriate dilutions with sterile ddH₂O to obtain the necessary treatment doses.
DNA was extracted to confirm we were using N. ceranae. We centrifuged dissected honey bee gut tissues with an estimated 40,000 spores per microliter at 9279 g to form a pellet. We then crushed the pellet using liquid nitrogen and extracted DNA with the Bioneer Accuprep Genomic DNA extraction kit. Using standard PCR methods, we sequenced the resulting spore DNA, using Genbank sequences to confirm their identity. DNA extracts for N. ceranae were screened using primer pairs NoscRNAPol-F2 and NoscRNAPol-R2 [71 (link)]. All PCR reactions were carried out in 20 μl reactions containing at least 5 ng total DNA, 10X PCR buffer (750 mM Tris-HCL [pH 8.5], (NH₄)₂SO₄ (Apex Life Science company), 0.25 mM of each dNTP (Promega), 0.5 μM of each primer (Allele Biotech), 1% Tween, 50 mM MgCl₂, and 1 unit of Taq DNA polymerase (Apex Life Science Co.). Cycling conditions were an initial 5 min at 95°C, then 35 cycles of 1 min at 95°C, 1 min annealing at 58°C, 1 minute at 72°C, and a final 10 min extension at 72°C. The 662 base pair amplicon was separated through gel electrophoresis and visualized using SYBR-safe (New England Biolabs). DNA was sequenced by Retrogen, Inc. with an Applied Biosystems 3730xl DNA Analyzer.

Wheat NIL-R and NIL-S were grown in greenhouse at 25±3°C with 70±10% relative humidity and 16 h of light & 8 h of darkness. F. graminearum (Schwabe) isolate 15–35 (obtained from Dr. S. Rioux, CEROM, Quebec) was maintained on PDA media. For spore production, cultures were grown on rye B agar media, under UV light and darkness, for 16 h and 8 h, respectively, at 25°C. Macroconidia were harvested and the spore count was adjusted to 1×10⁵ macroconidia ml⁻¹. Wheat spikelets were point inoculated with 10 µl of spore suspension (Approx. 1000 macroconidia per spikelet) at 50% anthesis, using a syringe with an auto dispenser (GASTIGHT 1750DAD W/S, Hamilton, Reno, NV, USA). For disease severity assessment, a pair of alternative spikelets, approximately at the middle of spike was inoculated. For metabolic/protein profiling, three alternate pairs of spikelets (six spikelets per spike), around the middle of spike, were inoculated. Ten spikes from 6 plants were inoculated for each treatment (pathogen or mock) per replication. The inoculated plants were covered with moistened plastic bags to maintain a saturated atmosphere to facilitate infection, and the bags were removed 48 h post inoculation (hpi).

For the 10 individual and 10 social lines, we determined the induced host mortality as a measure of virulence and the outgrowing spore number as transmission stage production under their matched and non-matched current host conditions. We exposed the workers as in the selection treatment, kept them either alone or with two untreated nestmates, and monitored their mortality daily for 8 d. Again, ants dying in the first 24 h after treatment and dying nestmates were excluded from the analysis. In total, we obtained survival data of 797 ants (19–20 ants exposed for each of the 10 replicates from each of 4 combinations of selection history and current host condition). Dead ants were treated as above and their outgrowing spores collected by a needle dipped in sterile 0.05% Triton X-100 directly from the carcass, and resuspended in 100 µl of sterile 0.05% Triton X-100. The number of spores per carcass was counted individually using the automated cell counter, as described above (n = 215; median of 5 per replicate). We excluded one outlier carcass(from replicate I5) where we expected a counting error as this single carcass showed approx. 100-fold higher spore count than the other carcasses of this replicate. Exclusion of this outlier did not affect the statistical outcome. The proportion of ants dying per replicate line for each combination of selection history and current host condition and the number of spores produced by all carcasses per replicate were respectively used as measures of virulence and transmission (mean carcass spore load per replicate plotted in Fig. 2).

We sourced the sodium hypochlorite solution used in the experiments from Merck (105614, Sigma Aldrich), stored at 4 ${}^{\circ }$ C and diluted as appropriate for the experiments.
Each of the 3 isolates was decontaminated with 0.1%, 0.5% and 1.0% sodium hypochlorite (1,000, 5,000 and 10,000 ppm active chlorine), for 9 experimental sets in total. The decontamination procedure for each sample was as follows. 100 $\mathrm{\mu }$ l of spore suspension in water at a concentration of ${10}^9$ spores/ml was mixed with 100 $\mathrm{\mu }$ l of double concentrated NaOCl (0.2%, 1.0% and 2.0%) and left for 10 minutes. The biocide was then neutralised with 0.5% sodium thiosulphate as described previously [21 (link)]. The spores were then washed with deionised water, by centrifuging and discarding the supernatant twice, to remove reacted chemicals.
The reduction in viable spore count was determined by spreading 100 $\mathrm{\mu }$ l culture onto BHIS-ST plates. Spores were grown in anaerobic conditions at 37 ${}^{\circ }$ C for 48 hours and colonies were counted from a plate with appropriate dilution. There were 3 biological replicates for each experimental set.

The murine macrophage cell line J774.2 was cultivated in Dulbecco's minimal essential medium (DMEM) (Lonza, Basel, Switzerland) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Biosera, Kansas City, MO, USA) and 1% penicillin–streptomycin solution (Sigma-Aldrich, St. Louis, MO, USA) at 37°C, 5% CO₂, and 100% relative humidity. The same medium was used in all the interaction experiments. Four hours before the experiment, J774.2 cells (2 × 10⁵ cells/mL) were freshly harvested and stained with CellMask deep red plasma membrane stain (Thermo Fisher Scientific, Waltham, MA, USA) following the instructions of the manufacturer, and then seated on a 24-well plate. Fungal spores were freshly collected from 1-week-old MEA cultures and stained with Alexa Fluor 488, carboxylic acid, and succinimidyl ester (Invitrogen, Waltham, MA, USA). Labeled macrophages and spores were coincubated at a multiplicity of infection (MOI) of 5:1 for 90 min at 37°C and 5% CO₂. For analysis, collected samples were centrifuged with 1,000 × g for 10 min and then resuspended in 200 μL PBS supplemented with 0.05% Tween 20. Interaction and phagocytosis were measured using a FlowSight imaging flow cytometer (Amnis ImageStream X Mk II imaging flow cytometer; Amnis, Austin, TX, USA) and evaluated with IDEAS software (Amnis ImageStream X Mk II imaging flow cytometer; Amnis, Austin, TX, USA). Data from 10,000 events per sample were collected and analyzed. The number of engulfed cells was determined by examining 200 images of individual macrophages, while the phagocytic index (PI) was determined using the following formula: PI = (mean spore count per phagocytosing cell) × (% of phagocytosing cells containing at least one fungal spore).
To analyze the phagosome acidification of J774.2 cells by flow cytometry, fungal spores were labeled with pHrodo Red succinimidyl ester (Invitrogen, Waltham, MA, USA), according to the manufacturer’s instructions. CellMask Deep Red Plasma Membrane stain (Thermo Ficher Scientific, Waltham, MA, USA) labeled macrophages and FITC labeled spores were coincubated at an MOI of 5:1, for 120 min at 37°C and 5% CO₂. The ratio of the phagocytosing cells was also determined at 120 min under the same conditions of interactions, except the fungal spores were stained with CFW. Phagosome acidification was calculated as follows: [(% of pHrodo Red⁺ cells)/(% of phagocytosing cells)] × 100.
To assay the survival of the fungal spores, the interaction of J774.2 cells and the spores was performed at an MOI of 5:1 for 180 min. After the interaction, cells and spores were collected and macrophages were lysed with sterile distilled water. Serial dilutions were prepared from the spore suspensions and plated on MEA to quantify the CFU. Survival of spores was calculated using the following formula: survival = (CFU_interaction × 100)/CFU_control, where CFU_interaction is the CFU of samples coincubated with macrophages, while CFU_control is the CFU of control samples, incubated under the same conditions but without macrophages.