Fig 1.
Structural comparison of the digestive tract in firebrat (Thermobia domestica) and silverfish (Ctenolepisma longicaudata), and relative CMCase activity in Zygentoma compared to other insect orders.
A) Activity (U/mg) of gut digestive fluids against CMC substrate in the most active samples from species of Zygentoma (C. longicaudata), and representative species from Orthoptera (Conocephalus strictus), Lepidoptera (Halysidota tessellaris), Blattodea (formerly Isoptera): Rhinotermitidae (Reticulitermes flavipes), Hymenoptera (Neodiprion lecontei), Diptera (Monarthropalpus flavus), Dermaptera (Forficula auricularia), Coleoptera (Scolytinae spp.) and Blattodea (formerly Blattaria): Cryptocercidae (Cryptocercus spp.). Shown are the average activity and corresponding standard error from at least three biological replicates performed in triplicate for each species. All experiments were concurrent, but all activities except for the Zygentoma sample were taken from Oppert et al. [7]. Dissected digestive tracts of firebrat (top) and silverfish (bottom). Note the relatively larger size of the tract in silverfish compared to firebrat. C) Morphological parts of the digestive tract of firebrat. FG, foregut and crop; MG, midgut; HG, hindgut; PV, proventriculus; GC, gastric caecae; MT, Malpighian tubules.
Fig 2.
Histology of the digestive system regions in Ctenolepisma longicaudata.
A) Longitudinal section of the crop showing the monolayer of epidermal cells and the underlying circular muscle cells. B) Longitudinal section of proventriculus (PV). C) Longitudinal section of midgut showing peritrophic membrane, columnar cells lined with brush border membrane and intermitted by a group of nidi cells at the bottom. D) Longitudinal section of hindgut wall showing the monolayer of epidermal cells. All sections were stained with hematoxylin and eosin stain. GC, gut cavity; CM, circular muscle cells; PTM, peritrophic matrix; CC, columnar cells; BBM, brush border membrane.
Fig 3.
Detection of cellulase activity in digestive fluids of Thermobia domestica and Ctenolepisma longicaudata.
Zymograms with 0.2% carboxymethyl cellulose were used to detect the cellulase (CMCase) activity in head, foregut, midgut and hindgut tissues of T. domestica (left) and C. longicaudata (right). MM, pre-stained protein molecular marker; +ve, commercial cellulase used as positive control; H, head; FG, foregut; MG, midgut; HG, hindgut.
Fig 4.
Quantification of plant cell wall degrading enzyme activities in the fluids derived from head and foregut tissues of Ctenolepisma longicaudata and Thermobia domestica.
Fluids from head and foregut (FG) tissues of C. longicaudata and T. domestica fed on protein (BSA) or paper diet (see Materials and methods) were used in assays to detect A) endoglucanase activity against 4-nitrophenyl-β-D-cellopentaoside (BPNPG5), B) β-glucosidase activity against 4-nitrophenyl β-D-cellobioside (pNPC), C) β-xylosidase activity against 4-nitrophenyl β-D-xylpyranoside (pNPX), and D) polygalacturonase activity against pectin from citrus peel. Shown are the means and corresponding standard errors calculated from three biological and three technical replicates. Different letters above the bars indicate significant differences in the mean activity (P < 0.05). Units of specific enzyme activity are per mg of protein in all the graphs except in graph A) where it is expressed per g of protein. One unit of enzyme activity was defined as the amount of enzyme required to release 1 μmol of 4-nitrophenol from the respective substrate in all the graphs except in D), where it is 1 μmol of glucose.
Fig 5.
Partial purification of cellulase enzymes from gut fluids of T. domestica for proteomic analysis.
A) Chromatogram from fractionation of gut fluids from the whole digestive system of T. domestica adults using size exclusion chromatography, and testing for CMCase activity in aliquots from eluted fractions using an agarose plate assay. Consecutively eluted fractions are numbered in the plate, as well as endoglucanase from A. niger as CMCase positive control (STD). Fractions displaying relatively high levels of CMCase activity (fractions 4–12) were resolved by electrophoresis in semi-denaturing SDS-10%PAGE (B). After electrophoresis, gels were stained for total protein with Coomassie stain or used for zymography with CMC as substrate, as indicated in the figure panels. Three protein bands in the Comassie stained gel replica (denoted by white 1–3 numbers in the figure) that approximately corresponded to the region including CMCase activity in the zymogram (delimited in the zymogram with a black square) were cut from the stained gel and submitted for mass spectrometry.
Table 1.
Proteins identified by nanoLC/MS/MS in fractionated gut fluids from Thermobia domestica with CMCase activity.
The contigs identified in the transcriptome and the matching proteins in the NCBInr database are listed, as well as the normalized quantitative values NSAF in each of the electrophoretic bands analyzed, and the respective glycoside hydrolase family assigned by the CAZy database.