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

L Viridiana Soto-Robles,Gerardo Zúñiga,María Fernanda López,Verónica Torres-Banda,Gabriel Obregón-Molina,Claudia Cano-Ramírez

doi:10.3390/ijms22010036

L Viridiana Soto-Robles, Gerardo Zúñiga + Show 4 more

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https://doi.org/10.3390/ijms22010036

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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.

Highlights

Dendroctonus-bark beetles (Curculionidae: Scolytinae) play a fundamental role in the structure of coniferous forests, because they colonize and kill sick, damaged, and weakened trees, contributing to community succession, nutrient cycling, and canopy thinning [1].some of their species are disturbance agents because, under certain forest stress conditions, outbreaks occur that affect hundreds of thousands of conifer trees of the genera Larix, Picea, Pinus, and Pseudotsuga from North and Central America [2,3,4].Dendroctonus-bark beetles are phytophagous insects that spend most of their life cycle under the bark of host plants, where they reproduce, feed on phloem, and interact with microbial symbionts and invertebrates
Our findings showed that AmyDr is a functional enzyme encoded by an open reading frame (ORF) of 1452 nucleotides and is present both in the gut and head-pronotum of Dendroctonus rhizophagus
In silico analysis showed that this protein has a 51.51 kDa molecular mass, similar to the α-amylase from other scolytines, such as D. ponderosae (51.37 kDa, XP_019767850.1), Ips typographus (51.84 kDa, ADQ54210.1; 52.19 kDa, ADQ54211.1), and Hypothenemus hampei (51.24 kDa, AHY03307), curculionids, such as Antohonomus grandis (50.87 kDa, AAN77138.1; 52.28 kDa, AAN77139.1) and Sitophilus oryzae (51.32 kDa, ADM73187.1), other coleopterans, such as Tenebrio molitor (51.24 kDa, P56634) and Tribolium castaneum (51.60 kDa, AAA03708), and other insects, such as Spodoptera frugiperda

Summary

Introduction

Dendroctonus-bark beetles (Curculionidae: Scolytinae) play a fundamental role in the structure of coniferous forests, because they colonize and kill sick, damaged, and weakened trees, contributing to community succession, nutrient cycling, and canopy thinning [1].some of their species are disturbance agents because, under certain forest stress conditions, outbreaks occur that affect hundreds of thousands of conifer trees of the genera Larix, Picea, Pinus, and Pseudotsuga from North and Central America [2,3,4].Dendroctonus-bark beetles are phytophagous insects that spend most of their life cycle under the bark of host plants, where they reproduce, feed on phloem, and interact with microbial symbionts and invertebrates. Dendroctonus-bark beetles (Curculionidae: Scolytinae) play a fundamental role in the structure of coniferous forests, because they colonize and kill sick, damaged, and weakened trees, contributing to community succession, nutrient cycling, and canopy thinning [1]. Some of their species are disturbance agents because, under certain forest stress conditions, outbreaks occur that affect hundreds of thousands of conifer trees of the genera Larix, Picea, Pinus, and Pseudotsuga from North and Central America [2,3,4]. Some of microbial symbionts are capable of degrading different substrates such as starch, esters, and lipids [12], cellulose [13], recycling uric acid [7], and degrading or transforming different monoterpenes from the host trees of these insects [14,15,16]

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