Rapid but Unreliable: Cross-Generational Restructuring of the Tribolium castaneum Gut Microbiome under Contrasting Nutritional Regimes

Microbial communities are frequently exposed to environmental disturbances, yet recovery does not always lead to a single, predictable outcome. Distinguishing resilience, the capacity to recover following perturbation, from reliability, the predictability of that recovery, is essential for understanding microbiome dynamics. Here, we investigated cross-generational gut microbiome responses in the red flour beetle, Tribolium castaneum , a globally significant stored-product pest exposed to frequent environmental and dietary shifts. Using a four-generation experimental design (G0–G3), we examined how gut microbial communities respond to a major environmental transition from commercial to laboratory rearing, followed by dietary perturbations. The shift to laboratory conditions produced the largest change in gut microbiome composition, occurring within a single generation and exceeding the effects of dietary shifts. Following this disturbance, microbial communities stabilized across subsequent generations, with reduced temporal variability in alpha and beta diversity, consistent with strong ecological resilience in community composition. However, recovery trajectories and endpoints differed among treatments, with replicate lineages converging on multiple, history-dependent community states rather than a single shared configuration, indicating low reliability of the recovery process. Although diet influenced the abundance of specific bacterial taxa, these effects were smaller than the generational shifts shaping overall community composition. Together, these results show that the T. castaneum gut microbiome is resilient but not strictly reliable, stabilizing after rapid reorganization into apparently stable, history-dependent community states over the timescale of our experiment. These findings underscore the role of environmental history in shaping host-associated microbiomes and suggest that microbial flexibility may contribute to the ecological success of insect pests in variable environments.