Different Functions of the Insect Soluble and Membrane-Bound Trehalase Genes in Chitin Biosynthesis Revealed by RNA Interference

Abstract

BackgroundTrehalase, an enzyme that hydrolyzes trehalose to yield two glucose molecules, plays a pivotal role in various physiological processes. In recent years, trehalase proteins have been purified from several insect species and are divided into soluble (Tre-1) and membrane-bound (Tre-2) trehalases. However, no functions of the two trehalases in chitin biosynthesis in insects have yet been reported.Principal FindingsThe membrane-bound trehalase of Spodoptera exigua (SeTre-2) was characterized in our laboratory previously. In this study, we cloned the soluble trehalase gene (SeTre-1) and investigated the tissue distribution and developmental expression pattern of the two trehalase genes. SeTre-1 was expressed highly in cuticle and Malpighian tubules, while SeTre-2 was expressed in tracheae and fat body. In the midgut, the two trehalase genes were expressed in different locations. Additionally, the expression profiles of both trehalase mRNAs and their enzyme activities suggest that they may play different roles in chitin biosynthesis. The RNA interference (RNAi) of either SeTre-1 or SeTre-2 was gene-specific and effective, with efficiency rates up to 83% at 72 h post injection. After RNAi of SeTre-1 and SeTre-2, significant higher mortality rates were observed during the larva-pupa stage and pupa-adult stage, and the lethal phenotypes were classified and analyzed. Additionally, the change trends of concentration of trehalose and glucose appeared reciprocally in RNAi-mutants. Moreover, knockdown of SeTre-1 gene largely inhibited the expression of chitin synthase gene A (CHSA) and reduced the chitin content in the cuticle to two-thirds relative to the control insects. The chitin synthase gene B (CHSB) expression, however, was inhibited more by the injection of dsRNA for SeTre-2, and the chitin content in the midgut decreased by about 25%.Conclusions SeTre-1 plays a major role in CHSA expression and chitin synthesis in the cuticle, and SeTre-2 has an important role in CHSB expression and chitin synthesis in the midgut.

cDNA cloning and characterization of two trehalases from Spodoptera litura (Lepidoptera; Noctuidade)

“…This dramatic increase in expression is needed to prepare sufficient energy for pupation and hydrolyze trehalose for chitosan biosynthesis. These findings agreed with those of Chen et al (2010) that Treh1 regulates the chitosan biosynthesis in the epidermis. The reduction of Treh1 was related to the fact that storage was sufficient and the insect required less energy in pupal stage ( Figure 5A).…”

“…The reduction of Treh1 was related to the fact that storage was sufficient and the insect required less energy in pupal stage ( Figure 5A). Treh2 in the fat body was expressed more highly in the pupal stage, which may promote chitosan in gut consistent with the findings of Chen et al (2010) that Treh2 regulates chitosan biosynthesis in the gut. Treh1 and Treh2 were both highly expressed in 3rd-instar larvae.…”

“…Treh1 is located in intracellular hydrolyzing endogenous trehalose, and Treh2 is a transmembrane enzyme involved in the assimilation of exogenous trehalose to supply energy (de Almeida et al, 2009). Chen et al (2010) found that the expression of trehalose is increased and that of glucose is reduced with Treh1 reduction, whereas Treh2 occurs in S. exigua through the RNA interference.…”

Trehalases: A neglected carbon metabolism regulator?

“…[12][13][14][15][16][17][18][19] In addition, trehalase contrastingly different sub-cellular localizations have been reported, including the cytoplasm, the vacuole, the apoplast, or being bound to the plasma membrane and oriented to the apoplast. 16,[20][21][22][23] In fungi, trehalase activity and localization have been directly involved in regulating the trehalose content and its mobilization into the cytoplasm and during the symbiotic interaction of the ectomycorrhizal fungi several genes for trehalose metabolism are strongly upregulated. 23,24 In insects the regulation of trehalose degradation by trehalase is essential to supply the energetic requirements for the flight and the direct regulation for chitin biosynthesis during the insect development.…”

“…23,24 In insects the regulation of trehalose degradation by trehalase is essential to supply the energetic requirements for the flight and the direct regulation for chitin biosynthesis during the insect development. 6,16,18,25,26 In higher plants, trehalose content is regulated by trehalase activity, which impacts directly on sucrose levels by negatively correlating sucrose synthase 1 expression levels and starch content. 14,15 Apparently, since trehalose accumulation is regulated by trehalase, this enzyme could be directly involved in regulating carbon partitioning, as well.…”