Fig 1.
Effect of colony hygienic status and N. ceranae dose on relative N. ceranae infection levels in bees over time.
There was a significant effect of sampling day (χ²₁ = 42.87, p < 0.001) and N. ceranae dose (χ²₂ = 79.11, p < 0.001) on relative N. ceranae infection levels (ΔΔCt, log-transformed), with a significant interaction effect of sampling day and N. ceranae dose (χ²₂ = 15.56, p < 0.001). There was no effect of colony hygienic status (χ²₁ = 1.22, p = 0.27). Purple points/lines represent hygienic bees; green points/lines represent non-hygienic bees. Panels correspond to N. ceranae doses (High = 5 × 10⁴ spores/bee; Low = 1 × 10⁴ spores/bee; Control = 0 spores/bee). Sample sizes are denoted at each time point as n = hygienic bees/ non-hygienic bees.
Fig 2.
Effect of N. ceranae dose on relative N. ceranae infection levels in hygienic and non-hygienic bees.
There was a significant main effect of N. ceranae dose on relative infection levels (χ²₂ = 79.11, p < 0.001), but no significant effect of colony hygienic status (χ²₁ = 1.22, p = 0.27) or an interaction effect between colony hygienic status and N. ceranae dose (χ²₂ = 0.062, p = 0.938). Purple boxes represent hygienic bees; green boxes represent non-hygienic bees. Panels correspond to N. ceranae doses (High = 5 × 10⁴ spores/bee; Low = 10⁴ spores/bee; Control = 0 spores/bee). Infection levels are shown as relative N. ceranae score (ΔΔCt, log-transformed). Sample sizes are denoted above each box and varied based on bee mortality. Significance between pairs is denoted as ‘.’ < 0.1, ‘*’ < 0.05, ‘**’ < 0.01, ‘***’ < 0.001.
Fig 3.
Effect of colony hygienic status and N. ceranae dose on the volume of sugar syrup inoculant consumed by bees in hoarding cages.
There was a significant main effect of colony hygienic status on the volume of inoculant consumed by bees in hoarding cages (Kruskal-Wallis test, χ² = 4.3104, df = 1, p = 0.038), but no effect of N. ceranae dose (χ² = 4.0909, df = 2, p = 0.129) nor interaction between colony hygienic status and N. ceranae dose (χ² = 9.228, df = 5, p = 0.101). There was a marginal difference in the volume of inoculant consumed between hygienic and non-hygienic bees in the high N. ceranae dose group (p = 0.055). Purple boxes represent hygienic bees; green boxes represent non-hygienic bees. Panels correspond to N. ceranae doses (High = 5 × 10⁴ spores/bee; Low = 1 × 10⁴ spores/bee; Control = 0 spores/bee). Inoculant consumption is shown in milliliters (mL). Sample sizes are denoted above each box and varied based on bee mortality. Significance between pairs is denoted as ‘.’ < 0.1, ‘*’ < 0.05, ‘**’ < 0.01, ‘***’ < 0.001.
Fig 4.
Effect of colony hygienic status on Vitellogenin (Vg) gene expression across time and N. ceranae doses.
There were significant main effects of colony hygienic status (χ²₁ = 7.62, p = 0.006) and sampling day (χ²₄ = 22.78, p < 0.001), as well as a significant interaction between colony hygienic status and day (χ²₄ = 19.79, p < 0.001) on Vitellogenin expression levels. Vitellogenin expression levels are shown as the relative Vg score (ΔΔCt, log₁₀-transformed). Purple bars indicate hygienic bees; green lines indicate non-hygienic bees. Panels correspond to sampling days post-inoculation. Error bars represent standard errors of the mean. Sample sizes are denoted above each bar pair as n = hygienic bees/non-hygienic bees. Significance between pairs is denoted as ‘.’ < 0.1, ‘*’ < 0.05, ‘**’ < 0.01, ‘***’ < 0.001.
Fig 5.
Effect of N. ceranae infection levels on Vitellogenin (Vg) gene expression by colony hygienic status.
Vg gene expression levels (ΔΔCt, log-transformed) were impacted significantly by main effects of colony hygienic status (χ²₁ = 6.27, p = 0.012), N. ceranae infection levels (χ²₁ = 4.81, p = 0.028), and sampling day (χ²₄ = 14.27, p = 0.006). There was no significant interaction effect between colony hygienic status and N. ceranae infection levels (χ²₁ = 1.37, p = 0.242). Purple points/lines represent hygienic bees; green points/lines represent non-hygienic bees. Sample sizes are denoted as n = hygienic bees/ non-hygienic bees.
Fig 6.
Effect of N. ceranae infection levels on Hymenoptaecin (Hym) gene expression by colony hygienic status.
Hym gene expression levels (ΔΔCt, log₁₀-transformed) were impacted by an interaction effect of colony hygienic status and N. ceranae infection levels (χ²₁ = 5.805, p = 0.016). No main effects of N. ceranae infection levels (χ²₁ = 0.05, p = 0.831) or colony hygienic status (χ²₁ = 0.00, p = 0.997) on Hym expression levels were detected. Purple points/lines represent hygienic bees; green points/lines represent non-hygienic bees. Sample sizes are denoted as n = hygienic bees/ non-hygienic bees.
Fig 7.
Survival probability of bees by colony hygienic status and treatment group over time.
Among N. ceranae-inoculated groups, hygienic bees had significantly better survival odds than non-hygienic bees (χ² = 8.3, df = 1, p = 0.004; HR = 1.53, 95% CI: 1.15–2.04). Among control bees, there was no significant difference by colony hygienic status (χ² = 0.4, df = 1, p = 0.50; HR = 0.83, 95% CI: 0.46–1.50). Purple lines indicate hygienic bees; green lines indicate non-hygienic bees. Dashed lines represent control bees; solid lines represent N. ceranae-inoculated bees. Sample sizes are denoted beside each line.
Fig 8.
Effect of N. ceranae dose on survival probability over time.
There was a significant effect of N. ceranae dose on survival probability (χ²₂ = 28.3, df = 2, p = 7 × 10 ⁻ ⁷), where N. ceranae exposure significantly increased the hazard of death compared to control bees. Bees that received the low and high dose N. ceranae inoculant did not differ significantly in survival probability (p = 0.93). Red line indicates high N. ceranae dose (5 × 10⁴ spores/bee), orange line indicates low N. ceranae dose (10⁴ spores/bee), and blue line indicates control group (0 spores/bee). Sample sizes are n = 240 for each group.