Impact of Sublethal Concentrations of Imidacloprid on Honey Bee Hypopharyngeal Glands on Two Methodical Levels: Reliability of Measurements and Lab Versus Field Conditions

After decades of use of neonicotinoid insecticides, it is proven that sublethal doses have behavioural and physiological effects on both Apis mellifera honey bee individuals and colonies. This work aimed a) to ring test the methodology of digital measurement of hypopharyngeal glands (HPGs) of honey bees and to validate the method against the subjectivity of the assessors and b) to assess possible differences between laboratory and field studies when examining the impact of sub-lethal levels of the neonicotinoid imidacloprid on worker honey bee HPGs size. We did this by employing young honey bees reared in colonies that were fed for one month with sugar solution and pollen paste, both contaminated with imidacloprid at concentrations of 2 ng/kg and 3 ng/kg, respectively. Newly emerged bees were marked and then maintained as adults in either their own colonies or in laboratory cages. Digital measurements of HPG acini diameter were performed by 10 independent assessors using the same software at days 10 and 15 post-emergence. We found that HPGs glands have a significantly greater diameter under field conditions than in laboratory conditions, and that fifteen-day-old bees had smaller acini diameter compared to 10-day-old workers when reared in the laboratory but not in actual colonies. Imidacloprid had a significant negative effect on acini diameter of 10-day old workers, both under laboratory and field conditions, but not on 15-day old workers in both laboratory and field conditions. The reliability of measurements undertaken by all assessors was greater than 95% and there was no significant difference among the assessors. In conclusion, we found that digital measurement of HPG acini was a highly reliable method for detecting changes in the development of the glands and followed the same pattern under laboratory and field conditions, always independently by human error. Furthermore, the digital measurement of the HPG diameter at day 10 of the bees' life is a very reliable tool to be used for lower tier risk assessment and extrapolated for higher tier assessment.