Linden (Tilia spp.), a profusely flowering temperate tree that provides bees with vital pollen and nectar, has been associated with bumble bee (Bombus spp.) mortality in Europe and North America. Bee deaths have been attributed, with inadequate evidence, to toxicity from mannose in nectar or starvation due to low nectar in late blooming linden. Here, we investigated both factors via untargeted metabolomic analyses of nectar from five T. cordata trees beneath which crawling/dead bumble bees (B. vosnesenskii) were observed, and of thoracic muscle of 28 healthy foraging and 29 crawling bees collected from linden trees on cool mornings (< 30C). Nectar contained the pyridine alkaloid trigonelline, a weak acetylcholin-esterase inhibitor, but no mannose. Principal component analysis of muscle metabolites produced distinct clustering of healthy and crawling bees, with significant differences (P<0.05) in 34 of 123 identified metabolites. Of these, TCA (Krebs) cycle intermediates were strongly represented (pathway analysis; P<0.01), suggesting that the central metabolism is affected in crawling bees. Hence, we propose the following explanation: when ambient temperature is low, bees with energy deficit are unable to maintain the thoracic temperature required for flight, and consequently fall, crawl, and ultimately, die. Energy deficit could occur when bees continue to forage on linden despite limited nectar availability either due to loyalty to a previously energy-rich source or trigonelline-triggered memory/ learning impairment, documented earlier with other alkaloids. Thus, the combination of low temperature and nectar volume, resource fidelity, and alkaloids in nectar could explain the unique phenomenon of bumble bee mortality associated with linden.
Bibliographical noteFunding Information:
Funded by S.R. Exploratory Research Grant 2016-67030-25338, United States Department of Agriculture, National Institute of Food and Agriculture, https://nifa.usda.gov/. J.F.S. Grant 1S10OD018518, S10RR027878 and S10RR022589. National Institute of Health. https:// www.nih.gov/. J.F.S. Grant 2014162, M. J. Murdock Charitable Trust. https://murdocktrust. org/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was supported by the Exploratory Research Grant 2016-67030-25338/project accession no. 1009859 from the USDA National Institute of Food and Agriculture. We acknowledge the support of the Oregon State University NMR Facility (NIH Grant 1S10OD018518), the Oregon State University Mass Spectrometry Center (NIH Grants S10RR027878 and S10RR022589), and the M. J. Murdock Charitable Trust grant #2014162. We thank Dr. Kevin Ahern and Dr. Robert Durst for the many thoughtful discussions throughout this project, and Dr. Michael Goblirsch and Dr. Tim Kurtti for their insights on early versions of the manuscript.
© 2019 Lande et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.