Comparative metagenomic study unveils bacterial communities and their putative involvement in ecological success of two pine-feeding Ips beetle holobionts

Climate change has recently boosted the severity and frequency of the pine bark beetle attacks. The bacterial community associated with these beetles acts as “hidden players”, enhancing their ability to infest and thrive on defence-rich pine trees. There is limited understanding of the environmental acquisition of these hidden players and their life stage-specific association with different pine-feeding bark beetles. There is inadequate knowledge on novel bacterial introduction to pine trees after the beetle infestation. Hence, we conducted the first comparative bacterial metabarcoding study comprehensively revealing the bacterial communities in the pine trees before and after beetle feeding and in different life stages of two dominant pine-feeding bark beetles, namely Ips sexdentatus and Ips acuminatus. We also evaluated the bacterial association between wild and lab-bred beetles to measure the deviation due to inhabiting a controlled environment. Significant differences in bacterial amplicon sequence variance (ASVs) abundance existed among different life stages within and between the pine beetles. Such observations endorsed that the bark beetle life stage shaped bacterial assemblage. Furthermore, lab-bred and wild-collected adult beetles had distinct bacterial assemblages, implying that the breeding environment induced crucial changes. Alteration of pine wood bacteriome after beetle feeding is an intriguing observation in the present study, which demands further investigation. We validated the relative abundances of selected bacterial taxa estimated by metagenomic sequencing with quantitative PCR.  Functional predictions revealed that these bacterial genera might execute conserved functions, aiding the ecological success of these beetles. Nevertheless, these findings shed new insights into bacterial associations and their putative metabolic roles in pine beetles under the influence of various drivers such as environment, host, and life stages and provide the foundation for future downstream functional investigations.