Rearing methodology
Eristalis flower flies were reared in laboratory conditions from egg clutches laid by wild-caught females in the summer of 2019 (see Supplementary Materials for detailed rearing methodology). Only flies that emerged on the same day were used in the experiments. An artificial diapause protocol (see Supplementary Materials for detailed protocol) was used to prolong the lifespan of lab-reared flower flies, as adult Eristalis flower flies in lab colonies have shorter lifespans than adult Eristalis flies in the wild48. Once the adult flies emerged, all siblings were placed in artificial diapause in a refrigerator and fed 10% sucrose ad libitum until the experiment began. These Eristalis flower fly rearing and artificial diapause protocols are a modification of previously published protocols48,49.
Osmia lignaria (n = 50; Crown Bees, Woodinville, WA, USA) and Megachile rotundata (n = 50; Watts Solitary Bees, Bothell, WA, USA) were purchased and allowed to emerge in an incubator kept at 23 °C and 65% humidity. Bumble bees (Bombus impatiens) used as C. bombi source colonies or as uninfected sources of bees for the dose–response trials were purchased from Biobest (Biobest, Leamington, Ontario, Canada) and maintained in the lab by feeding sucrose and pollen from a mixture of honey bee-collected poly-floral pollen (Bee Pollen Granules, CC Pollen High Desert, Phoenix AZ, USA). To ensure the commercial colonies were free of parasites, we pulled 20 workers and screened them for parasites via microscopy. No parasites were found in any of the colonies used for the dose–response trials.
Evaluating whether the European drone fly, Eristalis tenax, is a host of Crithidia bombi
After breaking artificial diapause, the E. tenax flower flies were allowed to groom, but not feed, for one hour. Each fly was then placed abdomen-first into a 1.5 mL microcentrifuge tube harness to collect defecation events (Supplementary Figure S5). The size of these tubes allowed the flies to feed comfortably, but the tubes were also tapered at the bottom, which prevented the flies from stepping in their feces. Holes were placed along the side of the tubes so the fly could respire. One large hole was placed on the lid of the tube so the fly could be inoculated directly with a pipette.
Flies were randomly divided into treatment and control groups. E. tenax flies in a roughly 1:2 F:M sex ratio were used in both the treatment (n = 30) and control groups (n = 30), for a total of 60 replicates. The flies that emerged from the same egg clutch with this 1:2 F:M sex ratio were the only siblings that could accommodate the replicates needed for this experiment, which is why this sex ratio was used.
The C. bombi inoculum was made fresh from infected B. impatens individuals the morning of the experiment using established protocols. Briefly, we dissected the gut of infected B. impatiens workers from a laboratory source colony that sustained a strain collected from wild B. impatiens workers from Massachusetts, USA (GPS coordinates: 42.363911 N, – 72.567747 W). We homogenized the bee guts in distilled water and diluted the mixture to 1280 C. bombi cells μL−1, which we then combined 1:1 with 30% sucrose solution for an inoculum of 640 cells μL−1, a standard inoculum concentration for infecting bumble bees with C. bombi35,50. Control groups were fed 5 μL of a 30% sucrose and blue dye (Butler Extract Co., Lancaster, PA, USA) that in pilot experiments was not found to influence host or parasite survival. Treatment groups were inoculated with 5 μL (3200 cells total) of C. bombi, 30% sucrose and blue dye solution. The number of cells used in the inoculum is similar to levels of C. bombi found in the feces of bumblebees with recently established infections37. Blue dye was used to better visualize when fecal events occurred and flies that did not drink the entire 5 μL inoculum were not used in the experiment.
After feeding, the flies were monitored continuously until defecation occurred. As these flies recently emerged from artificial diapause and were starved pre-experiment, every hour post-inoculation the flies were fed a 30% sucrose and blue dye solution ad libitum to encourage defecation. Once a fly defecated, the feces were collected via pipette and diluted to a 10 μL solution with deionized water to observe and count parasites using Kova Glasstic slides. The fly was then placed in an individual 60 mL plastic portion cup with filter paper (Sigma–Aldrich, St Louis, MO, USA) and a 1.5 mL microcentrifuge tube feeder containing 500 μL of a 30% sucrose and blue dye solution for 10 days. Feeders and filter papers were replaced every 3 days to prevent mold growth. As C. bombi typically replicates in high numbers after 10 days in the guts of bumble bees51, both control and treatment flies were dissected and C. bombi gut counts were performed 10 days post-inoculation. Since actively swimming, and thus live, C. bombi is infective to susceptible bumble bee hosts35, only actively swimming C. bombi were counted. The fecal volume, dilution factor and counts of C. bombi were quantified for each individual fly to calculate the exact amount of C. bombi in the individual’s first defecation event.
Dose–response data
Crithidia bombi inoculum was made from infected B. impatiens individuals the morning of each trial using the protocols described above, with two exceptions. First, the C. bombi strain was collected from wild B. impatiens workers from New York, USA (GPS coordinates: 42.457350, − 76.426907). Second, a range of serially diluted doses were used to inoculate uninfected B. impatiens workers. The doses were: 25,000 cells, 12,500 cells, 6250 cells, 3125 cells, 1563 cells, 781 cells, 391 cells, 195 cells, 98 cells, 49 cells, 24 cells, and 12 cells. To obtain these doses, we homogenized bee guts in distilled water and diluted the mixture to 5000 C. bombi cells μL−1 with 30% sucrose solution. Serial dilutions were then conducted with a 10% sucrose solution to ensure the same osmolarity of each inoculum.
We conducted four replicate dose–response trials over a period of four weeks. Each week, five uninfected workers per dose from each of two colonies were administered 5 μL of C. bombi inoculum. The ten highest doses were administered for the first 2 weeks, and two additional doses (24 cells, and 12 cells) were added for the final 2 weeks. Inoculated bees were kept individually in vials and fed 30% sucrose ad libitum for 7 days at 23 °C and 65% humidity. After 7 days, the bees were dissected and C. bombi loads were quantified using a hemocytometer as described above. In addition, the right forewing was removed from each bee and marginal cell length was measured as a proxy for size52. In total, 220 bees were inoculated (20 replicates for each of the ten highest doses, 10 replicates for the two lowest doses).
Defecation patterns on a shared floral resource
All pollinators (O. lignaria, M. rotundata, E. arbustorum and E. tenax) were placed in individual 60 mL plastic portion cups lined with filter paper. Each pollinator received a 1.5 mL microcentrifuge tube feeder containing 500 μL of fluorescent dye via 2.5 g of fluorescent powder (Stardust Micas) dissolved into 500 mL 30% sucrose feeders to visualize fecal deposition on flowers. After 24 hours, filter papers were collected (for analysis of fecal volume and defecation frequency, see below) and a total of five, randomly selected pollinators of the same species were placed in 12 × 12 × 12″ mesh cages (Bioquip Products, Rancho Dominguez, CA, USA) containing inflorescences of similar sized Solidago dansolitum ‘Little Lemon’ goldenrod each replicate trial. Goldenrod was used in this experiment because both bees and flower flies were observed foraging on this abundant floral resource. Only pollinators with filter papers containing fluorescing defecation events were released in the mesh cages.
All E. arbustorum cages (n = 10) contained 2:3 F:M sex ratios, except one cage contained a 3:2 F:M sex ratio. All E. tenax cages (n = 20) contained 3:2 F:M sex ratios, except four cages contained 2:3 F:M sex ratios. All O. lignaria cages (n = 10) contained 4:1 F:M sex ratios, except one cage contained a 3:2 F:M sex ratio. For the two fly species, sample sizes and F:M sex ratios were determined by the greatest, same-day sibling emergence. For O. lignaria, sample sizes and F:M sex ratios were determined by emergence availability. M. rotundata floral deposition data was not collected, as the F:M emergence was heavily skewed to males that did not interact with, and therefore defecate on, the flowers.
After 24-hours, the pollinators were removed and the defecation events on the goldenrod from all cages were counted under a blacklight. The location of the defecation events on the goldenrod was recorded. The plant parts were divided into six categories: ‘inside’ the flower (inside the corolla), ‘outside’ the flower (surface of the corolla), on the sepal, on the bract (the leaflike structure beneath the flower), on the stem or on a leaf.
Defecation frequency and fecal volumes
The diameter of the smallest and largest defecation events per filter paper was measured by a digital caliper and an average diameter was calculated from these two values for all pollinators. The average diameter of the defecation events was converted to an average volume (in μL) using a standard curve (Supplementary Figure S6; R2 = 0.99 for the calibration data). The collected fecal volumes defecated by control flies from the E. tenax inoculation experiment (see above) were compared to the average fly fecal volumes calculated here. This was done to analyze whether flies in a confined environment, where they were inoculated with C. bombi, defecated similar volumes to flies allowed to move freely in an individual cup, which the average volumes were estimated from. In addition, the number of defecation events (frequency) over a 24-hour period on the collected E. arbustorum (n = 46) and E. tenax (n = 100) filter papers were counted for each fly.
Statistical analyses
For the E. tenax inoculation experiment, we evaluated the amount of C. bombi cells in the first defecation event using a negative binomial generalized linear model (GLM), with fly sex as predictor. We chose negative binomial over Poisson to account for overdispersion, which we evaluated using Pearson residuals. Significance of sex was evaluated using a likelihood ratio test (LRT).
Data from the B. impatiens inoculation experiment were used to fit two dose–response curves, the first for infection probability, and the second for infection intensity among infected bees. Infection intensity was defined using the loads estimated from the hemocytometer. A bee was considered infected if the counts were nonzero. We first tested whether the dose ingested, wing length (as a proxy for body size) and the colony the bee came from affected its response. For infection probability, this was done using a GLM with log10(dose), colony, wing length and their interactions as predictors, and infection status as the Bernoulli response. For infection intensity, this was done using a linear model (LM) with the same predictors, and log10(intensity) as response, using only infected bees. Doses were log-transformed in accordance to how the experimental doses were varied, while intensities were log-transformed to achieve normality of the residuals. Significance of predictors were tested in accordance with the principle of marginality.
While we found that wing length and colony were significant predictors, in practice the colony-specific response of a wild bee is unknown (since it would not have come from any of the experimental colonies), while the dependence on wing length is only useful in a size-based epidemiological model. Hence, we generated dose–response curves by marginalizing across colony and wing length. Finally, we tested whether linear relationships between the link function and log10(dose), assumed in LMs and GLMs, were sufficient to capture the shape of the dose–response curves, by fitting the data to shape-constrained additive models and then comparing AIC values53. SCAMs are generalized additive models (GAMs) on which additional constraints such as monotonicity have been imposed; being more flexible, they can better capture the shapes of the dose–response curves should the linear relationships be inadequate.
We evaluated whether fecal volume depended on pollinator species and sex with a linear model (LM), fitted using weighted least squares to account for unequal variances between group (detected using Levene’s test). Since the transformation from diameter (of feces on filter paper) to volume introduced a noticeable skew to the distribution, we transformed the volume back to diameter and further performed a Box-Cox transformation to achieve normality54, which we verified using the Shapiro–Wilk and D’Agostino’s K2 test. The transformed volume was used as the response in the abovementioned linear model.
For E. tenax, fecal volume was also manually collected from the 1.5 microcentrifuge tubes during the inoculations experiment. We compared the fecal volume from the two methods using a LM with method and fly sex as well as their interaction as predictors. Volumes were log-transformed to achieve normality, while the linear model was fitted using ordinary least squares since Levene’s test indicated no significant deviation from the assumption of equal variance.
We evaluated whether defecation frequency depended on pollinator species and sex with a LM, again fitted using weighted least squares to account for unequal variances between groups. While two of the groups showed deviation from normality using the Shapiro–Wilk test, the deviations were only marginally significant and hence not expected to qualitatively affect the results55.
Finally, we evaluated defecation patterns on goldenrod using a negative binomial GLM, with feces counts as the response, and pollinator species, plant location and their interaction as predictors. We did not use a mixed model with cage number as a random effect since there was only one count value per cage per location, so pseudo-replication was not an issue. Significance of predictors were evaluated using LRT in accordance to the principle of marginality56 (i.e., main effects were tested only when their interactions were insignificant and hence dropped). Post-hoc tests of pairwise contrasts with Tukey corrections were performed for predictors that were significant. We recognize that the principle sex ratio and its interactions with other predictors could also be included among the predictors; however, since each species had cages with predominantly one sex ratio (E. tenax 3F:2M; E. arbustorum 2F:3M; O. lignaria mix of 4F:1M and 5F:0M), this meant that species and sex ratios were highly correlated, making it impossible to separate their effects. Nonetheless, since female Eristalis flies do not provision their brood, the differences between sexes (e.g., time spent foraging on plants) may be less pronounced than in bees.
Source: Ecology - nature.com
