The Bark Beetle Dendroctonus rhizophagus (Curculionidae: Scolytinae) Has Digestive Capacity to Degrade Complex Substrates: Functional Characterization and Heterologous Expression of an α-Amylase

Abstract

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.

Scots pine defensins inhibit Ips acuminatus α-amylase activity

“…Recombinant Scots pine defensins. Recombinant proteins PsDef1, PsDef2, and PsDef5.1 were obtained as detailed in our previous reports (Kovaleva et al, 2011;Hrunyk et al, 2019;Shalovylo et al, 2021).…”

“…ac.uk/entry/E7DYB1). For docking, we utilized the solution NMR structures of PsDef1 (PDB code: 5NCE), PsDef2 (PDB code: 7LNS), and the structure of PsDef5.1, which was modeled in a previous study (Shalovylo et al, 2021). The 3D structures of Scots pine defensins were docked onto the α-amylase structure using the ClusPro 2.0 web server (Kozakov et al, 2017), which employs rigid body docking, root-mean-square deviation-based clustering, and energy minimization of the final structures.…”

Purification, properties, and characterization of amylase: a review

“…In addition, not all species' amylases can be expressed with high efficiency under ideal conditions, but amylase production can be achieved by heterologous expression techniques. For example: Soto-Robles et al [36] in order to explore the structure and properties of α-amylase in the intestinal tract of Bark Beetle (Curculionidae: Scolytinae), they expressed it heterologously based on an E. coli vector; El-Sayed et al [37] also used an E. coli vector to express α-amylase of Laceyella sp. DS3 by Heterologous expression in order to efficiently purify amylase and explore its enzymatic properties after immobilization.…”

Metabarcoding of mycetangia from the Dendroctonus frontalis species complex (Curculionidae: Scolytinae) reveals diverse and functionally redundant fungal assemblages

“…Beetles breed in the inner bark and feed on the phloem of living trees of genera Pinus, Larix, Pseudotsuga, and Piceae (Family: Pinaceae). This strategy requires them to have metabolic and nutritional capabilities to overcome chemical defenses of trees and exploit a substrate rich in complex polysaccharides (e.g., cellulose, hemicellulose, starch, pectin) and other polymers (e.g., lignin) that are not readily available to them (Krokene, 2015;Chiu et al, 2019;Pace, 2019;Soto-Robles et al, 2021). Dendroctonus bark beetles are associated with facultative microbes (e.g., filamentous fungi, yeasts, bacteria) that may aid them in the tasks of detoxifying terpenes (DiGuistini et al, 2011;Adams et al, 2013;Xu et al, 2015;Dai et al, 2022), degrading complex molecules (Valiev et al, 2009;Cano-Ramírez et al, 2016;Briones-Roblero et al, 2017a), uric acid recycling and fixing nitrogen (Ayres et al, 2000;Bleiker and Six, 2007;Goodsman et al, 2012;Morales-Jiménez et al, 2012, producing semiochemicals (Boone et al, 2013;Cale et al, 2019), and regulating ecological interactions between symbionts and pathogens (Cardoza et al, 2006;Adams et al, 2009;Cheng et al, 2015Cheng et al, , 2018Xu et al, 2016).…”