BACKGROUND: Climate change has recently boosted the severity and frequency of pine bark beetle attacks. The bacterial community associated with these beetles acts as "hidden players," enhancing their ability to infest and thrive on defense-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 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. RESULTS: Significant differences in bacterial amplicon sequence variance (ASVs) abundance existed among different life stages within and between the pine beetles. However, Pseudomonas, Serratia, Pseudoxanthomonas, Taibaiella, and Acinetobacter served as core bacteria. Interestingly, I. sexdentatus larvae correspond to significantly higher bacterial diversity and community richness and evenness compared to other developmental stages, while I. acuminatus adults displayed higher bacterial richness with no significant variation in the diversity and evenness between the life stages. Both wild and lab-bred I. sexdentatus beetles showed a prevalence of the bacterial family Pseudomonadaceae. In addition, wild I. sexdentatus showed dominance of Yersiniaceae, whereas Erwiniaceae was abundant in lab-bred beetles. Alternatively, Acidobacteriaceae, Corynebacteriaceae, and Microbacteriaceae were highly abundant bacterial families in lab-bred, whereas Chitinophagaceae and Microbacteriaceae were highly abundant in wild I. accuminatus. We validated the relative abundances of selected bacterial taxa estimated by metagenomic sequencing with quantitative PCR. CONCLUSION: Our study sheds new insights into bacterial associations in pine beetles under the influence of various drivers such as environment, host, and life stages. We documented that lab-breeding considerably influences beetle bacterial community assembly. Furthermore, beetle feeding alters bacteriome at the microhabitat level. Nevertheless, our study revisited pine-feeding bark beetle symbiosis under the influence of different drivers and revealed intriguing insight into bacterial community assembly, facilitating future functional studies.

• Klíčová slova
• Ips acuminatus, Ips sexdentatus, amplicon sequence variances (ASVs), core bacteriome, holobiont, microhabitat,
• Publikační typ
• časopisecké články MeSH

The bark beetle, Ips typographus (L.), is a major pest of Norway spruce, Picea abies (L.), causing enormous economic losses globally. The adult stage of the I. typographus has a complex life cycle (callow and sclerotized); the callow beetles feed ferociously, whereas sclerotized male beetles are more aggressive and pioneers in establishing new colonies. We conducted a comparative proteomics study to understand male and female digestion and detoxification processes in callow and sclerotized beetles. Proteome profiling was performed using high-throughput liquid chromatography-mass spectrometry. A total of >3000 proteins were identified from the bark beetle gut, and among them, 539 were differentially abundant (fold change ±2, FDR <0.05) between callow and sclerotized beetles. The differentially abundant proteins (DAPs) mainly engage with binding, catalytic activity, anatomical activity, hydrolase activity, metabolic process, and carbohydrate metabolism, and hence may be crucial for growth, digestion, detoxification, and signalling. We validated selected DAPs with RT-qPCR. Gut enzymes such as NADPH-cytochrome P450 reductase (CYC), glutathione S-transferase (GST), and esterase (EST) play a crucial role in the I. typographus for detoxification and digesting of host allelochemicals. We conducted enzyme activity assays with them and observed a positive correlation of CYC and GST activities with the proteomic results, whereas EST activity was not fully correlated. Furthermore, our investigation revealed that callow beetles had an upregulation of proteins associated with juvenile hormone (JH) biosynthesis and chitin metabolism, whereas sclerotized beetles exhibited an upregulation of proteins linked to fatty acid metabolism and the TCA cycle. These distinctive patterns of protein regulation in metabolic and functional processes are specific to each developmental stage, underscoring the adaptive responses of I. typographicus in overcoming conifer defences and facilitating their survival. Taken together, it is the first gut proteomic study comparing males and females of callow and sclerotized I. typographus, shedding light on the adaptive ecology at the molecular level. Furthermore, the information about bark beetle handling of nutritionally limiting and defence-rich spruce phloem diet can be utilized to formulate RNAi-mediated beetle management.

• Klíčová slova
• DAPs, Ips typographus (L.), Scolytinae, comparative proteomics, conifer pests, detoxification, digestion, enzyme assay,
• Publikační typ
• časopisecké články MeSH