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.

• Keywords
• Ips acuminatus, Ips sexdentatus, amplicon sequence variances (ASVs), core bacteriome, holobiont, microhabitat,
• Publication type
• Journal Article MeSH

We investigated gene expression patterns in Lutzomyia and Phlebotomus sand fly vectors of leishmaniases. Using quantitative PCR, we assessed the expression stability of potential endogenous control genes commonly used in dipterans. We analyzed Lutzomyia longipalpis and Phlebotomus papatasi samples from L3 and L4 larval stages, adult sand flies of different sexes, diets, dsRNA injection, and Leishmania infection. Six genes were evaluated: actin, α-tubulin, GAPDH, 60 S ribosomal proteins L8 and L32 (RiboL8 and RiboL32), and elongation factor 1-α (EF1-α). EF1-α was among the most stably expressed along with RiboL8 in L. longipalpis larvae and RiboL32 in adults. In P. papatasi, EF1-α and RiboL32 were the top in larvae, while EF1-α and actin were the most stable in adults. RiboL8 and actin were the most stable genes in dissected tissues and infected guts. Additionally, five primer pairs designed for L. longipalpis or P. papatasi were effective in PCR with Lutzomyia migonei, Phlebotomus duboscqi, Phlebotomus perniciosus, and Sergentomyia schwetzi cDNA. Furthermore, L. longipalpis RiboL32 and P. papatasi α-tubulin primers were suitable for qPCR with cDNA from the other four species. Our research provides tools to enhance relative gene expression studies in sand flies, facilitating the selection of endogenous control for qPCR.

• Keywords
• Lutzomyia, Phlebotomus, Endogenous control gene, Gene expression, Gene stability, Reference gene,
• MeSH
• Genes, Essential * MeSH
• Insect Vectors genetics   MeSH
• Genes, Insect MeSH
• Larva genetics   MeSH
• Leishmania genetics   MeSH
• Phlebotomus * genetics   MeSH
• Psychodidae genetics   MeSH
• Gene Expression Profiling methods   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Eurasian spruce bark beetle (Ips typographus [L.]) causes substantial damage to spruce forests worldwide. Undoubtedly, more aggressive measures are necessary to restrict the enduring loss. Finishing genome sequencing is a landmark achievement for deploying molecular techniques (i.e., RNA interference) to manage this pest. Gene expression studies assist in understanding insect physiology and deployment of molecular approaches for pest management. RT-qPCR is a valuable technique for such studies. However, accuracy and reliability depend on suitable reference genes. With the genome sequence available and the growing requirement of molecular tools for aggressive forest pest management, it is crucial to find suitable reference genes in Ips typographus under different experimental conditions. Hence, we evaluated the stability of twelve candidate reference genes under diverse experimental conditions such as biotic (developmental, sex and tissues) and abiotic factors (i.e., temperature and juvenile hormone treatment) to identify the reference genes. Our results revealed that ribosomal protein 3a (RPS3-a) was the best reference gene across all the experimental conditions, with minor exceptions. However, the stability of the reference gene can differ based on experiments. Nevertheless, present study provides a comprehensive list of reference genes under different experimental conditions for Ips typographus and contributes to "future genomic and functional genomic research".

• MeSH
• Coleoptera * genetics   MeSH
• Gene Expression MeSH
• Plant Bark MeSH
• Weevils * genetics   MeSH
• Reproducibility of Results MeSH
• Picea * genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH