Biochar Ingestion Is Associated with Hindgut Symbiotic Disruption and Mortality in Coptotermes formosanus: A Proposed Protist-to-Bacterial Cascade

Background: Biochar is being explored for pest management, but its potential as a subterranean termite bait and its mechanism of action on the gut symbiont community are poorly understood. Methods: We fed Coptotermes formosanus workers a diet of pine wood powder containing 0%, 5%, or 20% (w/w) rice-straw biochar. We then measured feeding preference, body weight, mortality, and analyzed changes in the hindgut flagellate community, bacterial 16S rRNA gene profiles, and host transcriptomics. Results: The 5% biochar diet did not deter feeding, whereas 20% reduced intake by ~18.75%. Both doses were associated with significant, time- and dose-dependent reductions in body weight and survival. The three major cellulolytic flagellate species (Holomastigotoides mirabile, Pseudotrichonympha grassii, Spirotrichonympha leidyi) declined sharply, coinciding with a restructuring of the bacterial community, notably a decrease in nitrogen-fixing Dysgonomonadaceae and an increase in opportunistic taxa. Host transcriptomics revealed exploratory, dose-dependent trends from anabolic growth toward catabolic stress and detoxification pathways, including upregulation of the proteasome and downregulation of ribosomal and energy metabolism pathways; several of these enrichment signals did not remain significant after multiple-testing correction and are therefore treated as hypothesis-generating. Conclusions: Our data are consistent with a proposed cascade hypothesis in which biochar ingestion is associated with disruption of essential gut flagellates, subsequent restructuring of bacterial symbiosis, and host metabolic stress. However, these links are inferred from parallel measurements and require continuous causal and rescue experiments for confirmation. Reduced intake at 20% and unmeasured biochar physicochemical properties remain alternative or non-exclusive explanations. Low-dose, non-deterrent biochar formulations represent candidate multi-mode active ingredients for termite bait development.