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.

Dendroctonus bark beetles comprise 20 taxonomically recognized species, which are one of the most destructive pine forest pests in North and Central America, and Eurasia. The aims of this study were to characterize the gut bacterial diversity, to determine the core bacteriome and to explore the ecological association between these bacteria and bark beetles. A total of five bacterial phyla were identified in the gut of 13 Dendroctonus species; Proteobacteria was the most abundant, followed by Firmicutes, Fusobacteria, Actinobacteria and Deinococcus-Thermus. The α-diversity was low as demonstrated in previous studies and significant differences in β-diversity were observed. The core bacteriome was composed of Enterobacter, Pantoea, Pseudomonas, Rahnella, Raoultella, and Serratia. The tanglegram between bacteria and bark beetles suggests that members of bacterial community are acquired from the environment, possibly from the host tree. These findings improve the knowledge about the bacterial community composition, and provide the bases to study the metabolic functions of these bacteria, as well as their interaction with these bark beetles.

Bark beetles (Coleoptera: Curculionidae: Scolytinae) are economically and ecologically the most important group of forest insects. They use several monoterpenes produced by their host plants to locate and colonize trees. Some of these compounds also are metabolized to produce sex, aggregation, or antiaggregation pheromones. Experimental studies have shown that certain terpenes are highly toxic to the insects, negatively affecting their development, reproductive success, and colonization of host trees. Nevertheless, the effects of these compounds on specific organs or anatomical systems are unknown. Based on relationship between bark beetles and monoterpenes of their host trees and the midgut morphological characterization performed previously, we studied the effects of α-pinene and myrcene on midgut cells of the red turpentine beetle, Dendroctonus Valens LeConte. Our results show that both α-pinene and myrcene induce an increase in the numbers of lysosomes and mitochondria. Outer and inner mitochondrial membranes were very conspicuous but were not disrupted. Both smooth and rough endoplasmic reticula were abundant and were found throughout the cytoplasm. Two kinds of secretory vesicles were observed: one associated with digestive material and the other associated with “flocculent” material. The Golgi complex was prominent but had no defined arrangement. Observed ultrastructural changes are indicative of intense cell activity and are associated with processes of digestion, synthesis, and excretion as well as of monoterpenes transformation but are not indicative of irreversible cellular damage or death.

Serratia proteamaculans CDBB-1961, a gut symbiont from the roundheaded pine beetle Dendroctonus adjunctus, displayed strong cellulolytic activity on agar-plates with carboxymethyl cellulose (CMC) as carbon source. Automatic genome annotation of S. proteamaculans made possible the identification of a single endoglucanase encoding gene, designated spr cel8A. The predicted protein, named Spr Cel8A shows high similarity (59–94 %) to endo-1,4-β-d-glucanases (EC 3.2.1.4) from the glycoside hydrolase family 8 (GH8). The gene spr cel8A has an ORF of 1113 bp, encoding a 371 amino acid residue protein (41.2 kDa) with a signal peptide of 23 amino acid residues. Expression of the gene spr cel8A in Escherichia coli yields a mature recombinant endoglucanase 39 kDa. Cel8A displayed optimal activity at pH 7.0 and 40 °C, with a specific activity of 0.85 U/mg. The enzyme was stable at pH from 4 to 8.5, retaining nearly 40–80 % of its original activity, and exhibited a half-life of 8 days at 40 °C. The K m and V max values for Spr Cel8A were 6.87 mg/ml and 3.5 μmol/min/mg of protein, respectively, using CMC as substrate. The final principle products of Spr Cel8A-mediated hydrolysis of CMC were cellobiose, cello oligosaccharides and a small amount of glucose, suggesting that Spr Cel8A is an endo-β-1,4-glucanase manifesting exo-activity. This is the first report regarding the functional biochemical and molecular characterization of an endoglucanase from S. proteamaculans, found in the gut-associated bacteria community of Dendroctonus bark beetles. These results contribute to improved understanding of the functional role played by this bacterium as a symbiont of bark beetles.

The bark beetle, Dendroctonus rhizophagus Thomas & Bright, is endemic to the Sierra Madre Occidental (SMOC) in México. This bark beetle is a majo