Chronic oral toxicity of lithium citrate in honeybees (Apis mellifera L.): impacts on survival, midgut histopathology and gut microbiota.

The search for effective alternatives to conventional acaricides has positioned lithium as a promising experimental candidate for controlling Varroa destructor; however, its physiological impact on the honeybee (Apis mellifera) remains insufficiently characterized. This study evaluated the chronic oral toxicity of lithium citrate over a 10-day exposure period, focusing on survival, midgut histopathological changes, and gut microbiota dynamics. Newly emerged bees were exposed to sugar syrup containing 12.5, 25, and 50 mM lithium citrate. Mortality increased monotonically with dose: 10-day survival was 0.94 in the control and 0.84, 0.22, and 0 at 12.5, 25, and 50 mM, respectively (all pairwise contrasts significant, Benjamini-Hochberg correction). Histological analysis of the midgut revealed pronounced epithelial damage, characterized by severe desquamation and cellular shedding into the lumen, even at the lower dose of 12.5 mM, with the highest severity at 12.5 mM rather than 25 mM; the peritrophic membrane remained intact in all groups. Furthermore, 16S rRNA gene sequencing revealed variation in the relative abundance of several core gut bacteria across treatment groups, although neither alpha- nor beta-diversity differed significantly among groups. At the highest lithium concentration (25 mM), Gilliamella showed a markedly lower median relative abundance, whereas Frischella was relatively more abundant; however, these patterns were not statistically supported by differential abundance analysis and did not indicate a consistent concentration-dependent response. Given the limited number of biological replicates (n = 3 per group), these compositional patterns should be regarded as preliminary. These findings suggest that lithium citrate, despite its varroacidal potential, acts as a significant physiological stressor, compromising the structural integrity of the digestive tract and disrupting microbial homeostasis. Consequently, the development of lithium-based treatments must carefully consider both lethal and sublethal effects at the individual level, and colony-level consequences remain to be evaluated.