Probiotic effects on ectoparasitic mite infestations in honey bees ( <i>Apis mellifera</i> ) are modulated by environmental conditions and route of administration

Beneficial bacteria can improve the health of managed honey bees (<i>Apis mellifera</i>) via immune system support as well as direct inhibition of pathogens. However, our understanding of how environmental factors and delivery methods impact treatment outcomes is limited. Here, we evaluated how supplementation of a three-strain lactobacilli consortium (<i>Lactiplantibacillus plantarum</i> Lp39, <i>Apilactobacillus kunkeei</i> BR-1, and <i>Lacticaseibacillus rhamnosus</i>; LX3) affects ectoparasitic mite (<i>Varroa destructor</i>) and bacterial (<i>Paenibacillus larvae</i>) load. We measured this effect following delivery of LX3 in either edible (protein patty) or topical (spray) forms and did so across three distinct habitats (forage-rich, agricultural, and urban). Results demonstrate significant control of mite infestation levels following patty delivery, relative to untreated patty controls. Spray methods, by contrast, were ineffective. Specifically, a control group that received only an uninoculated patty carried more mites than any other group, suggesting excess protein within hives is a catalyst for mite proliferation. This effect, whereby the excess-protein group had the highest parasite load, was pronounced in the most natural (forage-rich) environment type, indicating a significant site-by-treatment interaction. No influence of LX3 on <i>P. larvae</i> loads was observed in the asymptomatic colonies studied, which is likely attributable to the already low levels of pathogenic spores present and challenges associated with detection limits. In summary, this multi-site field study suggests that an LX3-infusion of standard protein patties is an effective method to control <i>Varroa destructor</i> infestations in commercial honey bee colonies.IMPORTANCECommercial beekeeping operations typically have a narrow profit range that depends on maintaining healthy hives throughout the season. Unfortunately, parasitic <i>Varroa</i> mites and bacterial pathogens can contribute to colony losses. The plight of honey bees can in turn affect the price and availability of produce on the agri-food market. There is therefore a need for innovation in the beekeeping industry to help secure bee livestock from season to season. One relatively new approach to sustainable beekeeping is the use of beneficial bacterial supplements that beekeepers can feed to or otherwise apply to hives in aid of the bee's natural health and abilities as mediated through their gut-brain axis. Our multi-site field study applies this approach. We find that a pollen protein patty is an effective vehicle for delivering probiotic bacteria to commercial honey bee colonies and for helping to keep <i>Varroa</i> destructor infestation levels in check.