Confirming in vivo PPIX function via synthetic PPIX administration to bees

While work was being done to express PPIX in our bacteria, we needed to ensure that the PPIX would have the desired effect of diminishing spore load in the bees without having a significant toxic effect on the bees.

To address PPIX-spore-bee interactions, we set up a PPIX feed experiment. We collected six cages of about 30-45 bees from our team hive. Given the difficulties associated with collecting bees, it was not possible to know exactly how many bees we had in each cage while they were alive. We administered different conditions whereby two cages were control cages, two were solvent system feed, and two were PPIX substituted feed. One cage in each treatment contained healthy bees whilst the other contained N. ceranae infected bees. It is important to note that after the N. ceranae infection was initiated in the bees, they were given one day to recover while being fed sugar water and to let the infection settle into their system before commencing the treatment. The experiment was started the next day. The bees were appropriately segregated to avoid cross contamination and were subsequently monitored for 10 days. Every 24 hours, any dead bees were collected and catalogued while the conditions of each cage were documented. Bee activity and behaviour was also documented during these check-ins. Observations included: bee activity level, cleanliness, appetite and general health appearance.

For the control group, the sugar feed consisted of 2M sucrose solution administered through 15 ml plastic centrifuge tubes with holes in the bottom. The solvent system feed was going to inform whether the bees could tolerate the PPIX solvent (1:1 0.1 M Tris and 10 mg/L EtOH) in their feed. It was administered in a 50:50 ratio with the sugar feed in the same way as the control bees. Essentially, the administration route needed to be confirmed as viable and non-toxic to the bees. As for the PPIX substituted feed, we were observing whether PPIX did indeed have a role to play in diminishing N. ceranae spore counts in the bees. To observe PPIX spore interactions, we mixed equal parts sugar feed and 50 mg/L PPIX (80 μM) in the solvent system. This was fed in the same manner as the other conditions.

After the experiment was concluded, 25% population triplicates of bees were collected from every experimental group and counted. To count spores in bees, we homogenized the bees in PBS (in a 1ml PBS: 1 bee ratio), filtered the resulting liquid and counted the spores via standard hemocytometer techniques.

What we found was that the PPIX solvent system was well tolerated by the bees and did not manifest in any observable signs of toxicity. It also had no effect on spore inactivation on its own. Additionally, we observed a statistically significant decrease in nosema spore counts in PPIX treated N. ceranae infected bees when compared to the sugar fed control bees. The treatment decreased spore loads to a healthy threshold. There was a 2 order of magnitude decrease in spore loads between PPIX treated infected bees and control infected bees (from millions of spores to tens of thousands of spores). This experiment confirmed the hypothesis that PPIX is effective in reducing N. ceranae spore load.

References

[1] S. J. Kwon, A. L. de Boer, R. Petri and C. Schmidt-Dannert, "High-Level Production of Porphyrins in Metabolically Engineered Escherichia coli: System Extension of a Pathway Assembled from Overexpressed Genes Involved in Heme Biosynthesis," Applied and Environmental Microbiology , vol. 69, no. 8, pp. 4875-4883, 2003.

[2] J. Zhang, K. Zhen, J. Chen and G. Du, "Optimization of the heme biosynthesis pathway for the production of 5-aminolevulinic acid in Escherichia coli," Scientific Reports , vol. 5, no. 8584, pp. 1-7, 2015.