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 Dendroctonusrhizophagus 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.

Keywords: Dendroctonus; RT-qPCR; bark beetle; heterologous expression; modelling; α-amylase.

Figure 1

Maximum-likelihood tree of α-amylases from coleopteran amino acid sequences of D. rhizophagus and GenBank and PDB sequences. The analysis was performed using the amino acid substitution model LG + G + I + F; G = 1.355, I = 0.141. The accession numbers of GenBank or PDB sequences are shown at the end of each branch, bootstrap values after 1000 pseudoreplicates are shown at nodes. AmyDr is boxed in red. The α-amylase sequence of Spodoptera frugiperda (AAO13754.1) was used as outgroup.

Figure 2

Structural model of the α-amylase of D. rhizophagus: AmyDr. (A) The cartoon structure: Letters a, b, and c indicate α-amylase domains, the arrows indicate β-sheet structures, helical structures indicate α-helices, the flexible loop is coloured in orange, the catalytic site is indicated in green, the calcium binding site in blue, and chlorine binding site in yellow. (B) Ramachandran plot: residues in the most favoured regions (A, B, L), residues in additional allowed regions (a, b, l, p), residues in generously allowed regions (~a, ~b, ~l, ~p), residues in disallowed regions (white region). (C) Error-values* plotted by Errat: the error function is based on the statistics of non-bonded atom-atom interactions in the structure.

Figure 3

Predicted docked interactions of AmyDr protein from D. rhizophagus. Cartoon diagram highlighting the interaction with ligands is shown to the centre. (A) Ligand: amylopectin. (B) Ligand: amylose. (C) Ligand: calcium ion. (D) Ligand: nitrate and chlorine ions.

Figure 4

Zymogram of amylolytic activity. (A) scheme of D. rhizophagus indicating head-pronotum and gut samples. (B) amylolytic activity visualized in the native-PAGE. The gut sample was loaded in lane 1, while the head-pronotum sample was loaded in lane 2. All sections (S1–S4) were analyzed in the MALDI-TOF/TOF. The arrows indicate the subsections within S3 from gut (a,b) and head-pronotum (a’,b’) lanes analyzed.

Figure 5

AmyDr cDNA and translation sequence. Identification of peptides. Peptides identified by MALDI-TOF/TOF in S2 and subsections a, b from the gut and a’, b’ from the head-pronotum are boxed in green. Peptides identified in S2 and only in a subsection a from S3 are boxed in yellow, peptides identified only in the section b from S3 are boxed in red. The predicted signal P is boxed in blue. Potential O-glycosylation sites are indicated with the symbol *. A potential N-glycosylation site is indicated with the symbol **.

Figure 6

Relative expression of the AmyDr gene. The expression pattern of the AmyDr gene was determined by RT-qPCR using cDNA from the fifth-instar larvae (L5), pupa (PU), preimage (PI), and pre-emergent adult (AD) of D. rhizophagus. GAPDH gene was used as a reference gene for RT-qPCR normalization and the expression profile was relative to the egg stage. The means of 2^−ΔΔCt were graphed, three technical replicates were performed for each biological replicate for each developmental stage. Biological replicates were integrated by a pool of ten guts or ten head-pronotum. Significant differences between states are indicated with letters and the standard error with bars.

Figure 7

Analysis of total protein extracts from Sf9/Bac_AmyDr in 10% SDS-PAGE. Line 1, MW protein standards; line 2, Sf900-II culture medium; line 3, AmyDr, and line 4, (NH₄)₂SO₄ precipitate from Sf9 cells extract after dialysis. Lines 2 and 4, negative controls.

Figure 8

Effect of metal (Ca²⁺ and Cl⁻¹) and non-metal ions (NO₃⁻¹) on AmyDr activity at two different concentrations (1 and 5 mM). (A,B) cells extract and (C,D) Sf9 AmyDr, both incubated with CaCl₂, NH₄Cl, Ca(NO₃)_2, and KNO₃ (1 and 5 mM each) on 1% (w/v) starch agar plates during 12 h at 37 °C.