Bark beetles play an important role as agents of natural renovation and regeneration in coniferous forests. Several studies have documented the metabolic capacity of bacteria associated with the gut, body surface, and oral secretions of these insects; however, little is known about how the bacterial community structure changes during the life cycle of the beetles. This study represents the first comprehensive analysis of the bacterial community of the gut of the bark beetle D. rhizophagus during the insect’s life cycle using 454 pyrosequencing. A total of 4 bacterial phyla, 7 classes, 15 families and 23 genera were identified. The α-diversity was low, as demonstrated in previous studies. The dominant bacterial taxa belonged to the Enterobacteriaceae and Pseudomonadaceae families. This low α-diversity can be attributed to the presence of defensive chemical compounds in conifers or due to different morpho-physiological factors in the gut of these insects acting as strong selective factors. Members of the genera Rahnella, Serratia, Pseudomonas and Propionibacterium were found at all life stages, and the first three genera, particularly Rahnella, were predominant suggesting the presence of a core microbiome in the gut. Significant differences in β-diversity were observed, mainly due to bacterial taxa present at low frequencies and only in certain life stages. The predictive functional profiling indicated metabolic pathways related to metabolism of amino acids and carbohydrates, and membrane transport as the most significant in the community. These differences in the community structure might be due to several selective factors, such as gut compartmentalization, physicochemical conditions, and microbial interactions.

Bark beetles from Dendroctonus genus promote ecological succession and nutrient cycling in coniferous forests. However, they can trigger outbreaks leading to important economic losses in the forest industry. Conifers have evolved resistance mechanisms that can be toxic to insects but at the same time, bark beetles are capable of overcoming tree barriers and colonize these habitats. In this sense, symbiont yeasts present in the gut of bark beetles have been suggested to play a role in the detoxification process of tree defensive chemicals. In the present study, genes related to this process were identified and their response to a terpene highly toxic to bark beetles and their symbionts was analyzed in the Cyberlindnera americana yeast. The genome and transcriptome of C. americana (ChDrAdgY46) isolated from the gut of Dendroctonus rhizophagus were presented. Genome analysis identified 5752 protein-coding genes and diverse gene families associated with the detoxification process. The most abundant belonged to the Aldo-Keto Reductase Superfamily, ATP-binding cassette Superfamily, and the Major Facilitator Superfamily transporters. The transcriptome analysis of non-α-pinene stimulated and α-pinene stimulated yeasts showed a significant expression of genes belonging to these families. The activities demonstrated by the genes identified as Aryl-alcohol dehydrogenase and ABC transporter under (+)-α-pinene suggest that they are responsible, that C. americana is a dominant symbiont that resists high amounts of monoterpenes inside the gut of bark beetles.

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Dendroctonus-bark beetles are natural agents contributing to vital processes in coniferous forests, such as regeneration, succession, and material recycling, as they colonize and kill damaged, stressed, or old pine trees. These beetles spend most of their life cycle under stem and roots bark where they breed, develop, and feed on phloem. This tissue is rich in essential nutrients and complex molecules such as starch, cellulose, hemicellulose, and lignin, which apparently are not available for these beetles. We evaluated the digestive capacity of Dendroctonus rhizophagus to hydrolyze starch. Our aim was to identify α-amylases and characterize them both molecularly and biochemically. The findings showed that D. rhizophagus has an α-amylase gene (AmyDr) with a single isoform, and ORF of 1452 bp encoding a 483-amino acid protein (53.15 kDa) with a predicted signal peptide of 16 amino acids. AmyDr has a mutation in the chlorine-binding site, present in other phytophagous insects and in a marine bacterium. Docking analysis showed that AmyDr presents a higher binding affinity to amylopectin compared to amylose, and an affinity binding equally stable to calcium, chlorine, and nitrate ions. AmyDr native protein showed amylolytic activity in the head-pronotum and gut, and its recombinant protein, a polypeptide of ~53 kDa, showed conformational stability, and its activity is maintained both in the presence and absence of chlorine and nitrate ions. The AmyDr gene showed a differential expression significantly higher in the gut than the head-pronotum, indicating that starch hydrolysis occurs mainly in the midgut. An overview of the AmyDr gene expression suggests that the amylolytic activity is regulated through the developmental stages of this bark beetle and associated with starch availability in the host tree.