Assessment of Mitochondrial Function in the AmE-711 Honey Bee Cell Line: Boscalid and Pyraclostrobin Effects

Authors: MARTINOVIC-WEIGELT, DALMA - University Of St Thomas; DANG, MINH-ANH - University Of St Thomas; MORD, ALEX - University Of St Thomas; Goblirsch, Michael

Submitted to: Environmental Toxicology and Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/13/2024
Publication Date: 3/15/2024
Citation: Martinovic-Weigelt, D., Dang, M., Mord, A., Goblirsch, M.J. 2024. Assessment of Mitochondrial Function in the AmE-711 Honey Bee Cell Line: Boscalid and Pyraclostrobin Effects . Environmental Toxicology and Chemistry. http://doi.org/10.1002/etc.5847.
DOI: https://doi.org/10.1002/etc.5847

Interpretive Summary: Fungicides are used extensively to control fungal diseases that cause damage to crops that humans and livestock rely on for food. Importantly, some of these crops depend on pollination by honey bees and other organisms to sustain yields and production quality. While it was traditionally thought that fungicides posed little harm to non-target organisms, such as honey bees, a growing body of evidence has led to a reevaluation of this paradigm. Exposure, especially chronic, to fungicides, as well as their interaction with pathogens and other pesticides, has been shown to contribute to several negative effects for honey bee health. Studies conducted in the field and laboratory that have exposed individual bees or entire colonies to field-relevant doses of fungicides have help characterize these negative effects. However, characterizing the effects of fungicides at the level of the honey bee cell, isolated from the complexities of whole organism and whole hive life, would further resolve the link between the cellular mechanisms of toxicity and bee/hive morbidity due to fungicide exposure. Our objective was to develop and optimize a cell-based mitochondrial toxicity platform using the honey bee as a mode pollinator and the formulation, Pristine, which is applied to flowering crops visited by honey bees, as a model fungicide. Using cell-based techniques, we were able to expose honey bee cells in culture to a range of concentrations of the active ingredients found in Pristine, boscalid and pyraclostrobin, and isolate the effects of these active ingredients on honey bee cell viability and mitochondrial function. Results show that AmE-711 cell viability, mitochondrial membrane potential, and respiration were unaffected by short and extended exposures to boscalid. On the other hand, exposure to pyraclostrobin had a concentration-dependent response on viability and inhibited mitochondrial function.

Technical Abstract: There is a growing concern that chronic exposure to fungicides, as well as their interaction with pathogens and other pesticides, contributes to an array of negative effects on honey bee development, lifespan, and behavior. Field and caged-bee studies help characterize the outcome of field-relevant exposure, but linking these pathologies to proximate mechanisms of toxicity could benefit from the use of readily controllable, simplified host platforms like cell lines. Our objective was to develop and optimize an in vitro-based mitochondrial toxicity assay suite using the honey bee as a model pollinator and active ingredients of Pristine (boscalid and pyraclostrobin), which is applied to crops visited by honey bees, as a model fungicide. We measured effects of short (circa 30 mins) and extended exposure (16-24 hrs) to boscalid and pyraclostrobin on AmE-711 honey bee cell viability and mitochondrial function. AmE-711 cell viability, mitochondrial membrane potential, and cellular respiration were unaffected by short and extended exposures to boscalid. Short exposure to pyraclostrobin did not affect viability, while extended exposure reduced viability in a concentration-dependent manner (LC50 = 4,175 ppb). Mitochondrial function was inhibited by pyraclostrobin in both short (EC50 = 529 ppb) and extended exposure (EC50 = 989 ppb) scenarios. Short exposure to 10 and 1,000 ppb pyraclostrobin resulted in a rapid and significant decrease in the OCR; approximately 24% reduction by 10 ppb relative to the baseline OCR, and 64% by 1,000 ppb. Extended exposure to 1,000 ppb pyraclostrobin reduced all respiratory parameters whereas 1 and 10 ppb treatments had no significant effects. Present study demonstrates that the honey bee cell line AmE-711, when applied to cell viability, MMP, and extracellular flux-based bioenergetic assays, which include analyses of electron transport chain functionality, can provide high-throughput, cost-effective platform for mitochondrial toxicity screening relevant to bees.