DOI: 10.5061/dryad.bk3j9kdqz
title: Data from: Requirement of group I lytic polysaccharide monooxygenase for turnover of chitinous cuticle during molting in two forest pest beetles, <em>Monochamus alternatus</em> and <em>Psacothea hilaris</em>

description: Lytic polysaccharide monooxygenases (LPMOs) that are capable of oxidative
 cleavage of glycosidic bonds in crystalline polysaccharides
 including chitin and cellulose are widely distributed among
 organisms. Insect LPMOs belong to auxiliary activity family
 15 (LPMO15/AA15) and have been classified further into at least
 four subgroups. However, unlike LPMOs from microorganisms and
 viruses, their physiological functions in insects have not been
 well studied. In the present work, we investigated the functions of two
 group I LPMO15s, MaLPMO15-1 and PhLPMO15-1, in chitinous cuticle turnover
 during molting of two important forest pest longhorn beetles, the
 Japanese pine sawyer beetle, Monochamus alternatus, and the
 yellow spotted longicorn beetle, Psacothea hilaris. Real-time qPCR
 showed a similar pattern of expression of MaLPMO15-1 and
 PhLPMO15-1 during late stages of development with high levels
 present at young pharate pupal and young pupal stages
 and declining thereafter. Injection of double-stranded RNA
 (dsRNA) for MaLPMO15-1 (dsMaLPMO15-1) or PhLPMO15-1
 (dsPhLPMO15-1) into last instar larvae of
 M. alternates and P. hilaris, respectively, did not
 affect subsequent larval-pupal molting and the resulting pupae
 exhibited normal development. However, the pupae were unable to eclose to
 the adult stage and died entrapped inside their old pupal
 cuticle. TEM analysis revealed that, unlike the respective
 dsEGFP-treated control insects, both dsMaLPMO15-1- and
 dsPhLPMO15-1-treated pharate adults failed to degrade the endocuticular
 layer of their pupal cuticle in which the horizontal chitinous
 laminae remained largely intact. These results demonstrate that
 the group I LPMO15-1 enzymes play a role in pupal cuticle chitin
 turnover, which is critical for molting to the adult. Because
 LPMO15-1 is highly conserved among many insect species,
 this gene/enzyme is a potential target for the control of
 populations of both M. alternatus and P. hilaris as well as other
 pest insect species.
description: # Data from: Requirement of group I lytic polysaccharide monooxygenase for
 turnover of chitinous cuticle during molting in two forest pest beetles,
 *Monochamus alternatus* and *Psacothea hilaris* Dataset DOI:
 [10.5061/dryad.bk3j9kdqz](10.5061/dryad.bk3j9kdqz) ## Description of the
 data and file structure **Data files: Requirement of Group I lytic
 polysaccharide monooxygenase for turnover of chitinous cuticle during
 molting in two forest pest beetles,** ***Monochamus alternatus***
 **and *Psacothea hilaris*** 
 
 We have submitted our raw data for the real-time qPCR analysis
 (Data_file_1.xlsx and Data_file_2.xlsx) and cuticle thickness measurement
 in MaLPMO15-1- and PhLPMO15-1-deficient pharate adults
 (Data_file_3.xlsx).  
 
 ### Files and variables #### File: Data_file_1.xlsx **Description:** Low
 data of real-time qPCR for analysis of expression profiles
 of *MaLPMO15-1* and *PhLPMO15-1* during late stages of development (Figure
 3a and b). 
 
 * Column A: **Gene**; *MaLPMO15-1* and *PhLPMO15-1* genes analyzed. *
 Column B: **Stage**; late stages of development analyzed. YPP, young
 pharate pupae; OPP, old pharate pupae; P0-P14, 0-14 d-old pupae; A1, 1
 d-old adults; A5, 5 d-old adults. * Columns C-F: **Samples 1-4**; total
 RNA was isolated from pools of three whole insects collected ranging from
 young pharate pupae (YPP) to 5 d-old adults (A5). Total RNA was
 independently isolated from each of four replications (Samples 1-4). The
 values indicate transcript levels of *MaLPMO15-1* and *PhLPMO15-1*
 relative to those of *M. alternatus* ribosomal protein S6 (*MaRpS6*) and
 *P. hilaris *ribosomal protein S6 (*PhRpS6*), respectively. * Column G:
 **Average**; value of average of the relative transcript levels of
 *MaLPMO15-1* and *PhLPMO15-1*. * Column H: **SEM**; value of standard
 error of the mean. #### File: Data_file_2.xlsx **Description:** Low data
 of real-time qPCR for analysis of knockdown levels of *MaLPMO15-1* and
 PhLPMO15-1 transcripts after RNAi (Figure 4a and b).  
 
 * Column A: **Figure**; data are shown in figures indicated. * Column B:
 **dsRNA**; double-stranded RNA (dsRNA) used. dsE*GFP*, dsRNA for enhanced
 green flourescent protein (negative control); ds*MaLPMO15-1*, dsRNA for
 *MaLPMO15-1*;  ds*PhLPMO15-1*, dsRNA for *PhLPMO15-1*. * Columns C-E:
 **Samples 1-3**; total RNA was isolated from three whole young pharate
 pupae and 1 d-old pupae that had been injected with ds*MaLPMO15-1* and
 ds*PhLPMO15-1* into last instar larvae of *M. alternatus* and *P.
 hilaris*, respectively. Total RNA was independently isolated from each of
 three replications (Samples 1-3). The values indicate expression levels of
 *MaLPMO15-1* and *PhLPMO15-1* relative to the levels in respective
 ds*EGFP*-treated controls. * Column F: **Average**; value of average of
 the relative transcript levels of *MaLPMO15-1* and *PhLPMO15-1*. * Column
 G: **SEM**; value of standard error of the mean. * Column H: **t.test**;
 Statistical analyses were performed using the Student *t*-test.
 Significant differences (*p*-values) between ds*EGFP*-controls and
 ds*LPMO15-1*-treated insects are shown. 
 
 #### File: Data_file_3.xlsx **Description:** Thickness measurement of
 newly forming adult and old pupal cuticles from ds*MaLPMO15-1* and
 ds*PhLPMO15-1*-treated pharate adults (described in section 3.5). ##### *
 Column A: **Species**; *M. alternatus* and *P. hilaris* used. * Column B:
 **dsRNA**; double-stranded RNA (dsRNA) used. dsE*GFP*, dsRNA for enhanced
 green flourescent protein (negative control); ds*MaLPMO15-1*, dsRNA for
 *MaLPMO15-1*;  ds*PhLPMO15-1*, dsRNA for *PhLPMO15-1*. * Column C:
 **Cuticle**: old pual and newly forming adult cuticles from the pharate
 adults of *M. alternatus* and *P. hilaris* that had been treated with
 ds*MaLPMO15-1* and ds*PhLPMO15-1* at last instar larval stage,
 respectively. * Columns D-L: **Samples 1-9**; The values indicate
 thickness (µm) of the old pupal or newly forming adult cuticles. Nine
 cuticle specimens (Samples 1-9) for each group prepared from 3-5
 dsRNA-treated pharate adults were analyzed. * Columns M: **Average**;
 Value of average of cuticle thickness (µm). * Columns N: **SEM**; value of
 standard error of the mean. * Columns O: **t.test**; Statistical analyses
 were performed using the Student *t*-test. Significant differences
 (*p*-values) between ds*EGFP*-controls and ds*LPMO15-1*-treated insects
 are shown. 
 
 ## Code/software Microsoft Excel (.xlsx) software is required to view the
 data.