SUPPLEMENTARY MATERIAL
Evidence of the Role of Honey bees (Apis mellifera) as Vectors of the Bacterial Plant Pathogen Pseudomonas syringae.
D.E. Pattemore, R. M. Goodwin, H.M McBrydie, S. M. Hoyte and J. L. Vanneste
Summary:
Initial pilot trials established appropriate methodology for assessment of Pseudomonas syringae survival on honey bees (Apis mellifera) and in beehives. This supplementary material provides the details of the trials that established survival rates of P. syringae on plastic discs (to be used as a control surface), as well as an initial trial involving bee collection of P. syringae-contaminated pollen.
Aims:
P. syringae survival on plastic discs
As honey bees are efficient at cleaning themselves and some hive products have antibacterial effect, we sought a surface that we could use to assess the potential survival of P. syringae in a hive environment in isolation from these two factors. The aim of this trial was to assess the survival of Psa and the selected PssSmr strain (for strain details, see ‘Bacterial Strains and Populations’ in the main document) on plastic discs.
Survival of PssSmr on foraging honeybees
The aim was to assess the survival of Pss on bees that had foraged on PssSmr-contaminated pollens in field beehives and the spread of the contaminated pollen to other foraging bees.
Methods and Materials:
P. syringae survival on plastic discs
Thirty-six 10 mm diameter, 5 mm thick, PVC discs were inoculated with 10 µL of either PssSmr or Psa suspension, and then stored on a tray inside plastic bags at room temperature in a laboratory for nine days. Three discs each of PssSmr and Psa were sampled at 0, 1, 2, 5, 8 and 9 d. Sampled discs were individually washed in 1 mL of sterile distilled water (SDW) with 0.05% Tween 80 and serially diluted 1/10th. Three 10-µl droplets from each dilution were deposited onto King’s B agar (KB) for Psa or KB supplemented with 100ppm of streptomycin (KBSm) for PssSmr.
Survival of PssSmr on foraging honeybees
Bees from six hives were trained to collect kiwifruit (Actinidia deliciosa) pollen at a feeding station 20 m from their hives. Twelve foraging bees were captured as a background control before the contaminated pollen was presented (for description of the contaminated pollen, see ‘PssSmr spread between foragers’ in main document).
After the contaminated pollen was placed in the feeding station, foraging bees were captured, anoxiated with carbon dioxide (CO2), marked with white acetone-based paint, and then released to return to their hives. Twenty bees that were captured feeding on pollen were taken as time zero samples, and ten of these were processed after their pollen pellets had been removed.
Marked bees were then recaptured from inside hives on 1, 2, 5, 9 and 13 d after the foraging event. Individual bees were washed in 1 mL of SDW with 0.05% Tween 80 and the solution was then serially diluted on plates of KBSm. Colony forming units were counted after 48 h of incubation at 20°C.
On the day the contaminated pollen was presented to bees, other pollen-foraging bees returning to the hives with different pollen (identifiable by colour of pollen pellets) were sampled to assess the degree of contamination within hives between different groups of pollen foragers (five bees from each of two hives). To compare the mean number of cfu mL-1 between groups of bees from day 0, we conducted pair-wise t-tests using Bonferonni’s correction to derive a significance level of P = 0.0167 from an initial desired α = 0.05.
Results:
P. syringae survival on plastic discs
Both PssSmr and Psa survived for more than nine days when inoculated onto plastic discs (Figure S1), although Psa declined 10,000-fold (R2 = 0.97, P<0.0001, one-tailed t-test of significance of regression coefficient of log-transformed data) over the nine days, while PssSmr declined just 10-fold (R2 = 0.23, P=0.0231, one-tailed t-test of significance of regression coefficient of log-transformed data).
Figure S1. Mean number of colony forming units (cfu) of streptomycin-resistant Pseudomonas syringae pv. syringae (‘PssSmr’, red squares and red line, R2 = 0.23) and Pseudomonas syringae pv. actinidiae (‘Psa’, blue diamonds and blue line, R2 = 0.97) on plastic discs in ambient laboratory conditions.
Survival of PssSmr on foraging honeybees
One hundred and seventy-six bees foraging on PssSmr contaminated pollen were marked over approximately 6 h. Counts of marked bees in hives after 24 h found 54 bees in Hive No. 2, 15 in Hive No. 1, and just one marked bee in Hive No. 4. Because of the low number and the unequal distribution of marked bees, we only present data here from Hive No. 2, as well as the initial bacterial counts from day 0.
No PssSmr bacteria were found on bees that were caught before the contaminated pollen was presented. A mean of 1.1 x 107 cfu bee-1 was recovered from bees that had foraged on the contaminated pollen and collected enough to fill their corbiculae (Figure S2). When pollen sacks were removed from the bees before testing, the average count fell significantly to 1.6 x 105 cfu bee-1 (P = 0.014, one-tailed t-test, Figure S2). Other pollen foragers caught at the entrance of hives during the experiment with different pollen showed a mean of 1.0 x 103 cfu bee-1, which was significantly lower than the amount on bees without pollen sacks that had foraged directly on the contaminated pollen (P = 0.012, one-tailed t-test, Figure S3), but the range of recorded bacterial counts overlapped for these two groups, indicating that the PssSmr had been spread rapidly to bees that had been foraging elsewhere.
Figure S2. Mean number of colony forming units (cfu) of streptomycin-resistant Pseudomonas syringae pv. syringae recovered on day 0 from bees that had been foraging on contaminated pollen (with and without pollen sacks), and from bees that had been foraging on a different pollen source. Boxes show 25th and 75th percentiles and medians, error bars show 5th and 95th percentiles and dots show outliers.
The mean number of cfu of PssSmr on marked bees in Hive No. 2 after 24 h was 10,000 times lower than those recorded on bees whose pollen sacks had been removed after foraging on contaminated pollen on day 0 (Figure S3). PssSmr was still detected on one marked bee from this hive on Day 13.
Figure S3. Mean number of colony forming units (cfu) of streptomycin-resistant Pseudomonas syringae pv. syringae bacteria on marked bees from Hive No. 2 following presentation of contaminated pollen to pollen-foraging bees. Day 0 values are from bees that had their pollen sacks removed before testing for bacteria.
Conclusions:
P. syringae survival on plastic discs
The survival of Psa and PssSmr on plastic discs confirmed that this was a viable surface to inoculate for the purposes of assessing the survival of the bacteria in a beehive. The experiments proceeded with this strain of PssSmr despite its better survival than Psa, as we considered that the testing of a worst-case scenario for bacteria survival was preferable to selecting a weak Pss strain that might not survive as well as Psa.
Survival of PssSmr on foraging honeybees
Training bees to forage on a tray of pollen did not lead to an equal number of recruits per hive. Only one hive had enough marked foragers present to permit recapture on subsequent days, although all hives were checked thoroughly on each day of sampling. The remaining marked bees may have originated from a different hive, or might have been foraging out of the hive when samples were collected.
Because of the uneven distribution of marked bees through the six hives and low recapture rate, we proceeded with a different experiment to provide better replication of the transmission of contaminated material between marked and unmarked bees and the survival of PssSmr in field hives (See “PssSr spread between foragers” in main document). This second trial allowed us to control the number of marked and contaminated bees per hive to maximise the recovery rate and evenness of sampling between hives.