Phylogenetic tree of lepidopteran VMPs.

<p>The un-rooted maximum likelihood tree was calculated on the basis of a ClustalW alignment of VMPs from different lepidopteran species. Bootstrap values are given in percentages at the internodes; Accession numbers refer to the entries of the dbEST database of Butterflybase. MS, <i>Manduca sexta</i>; BM, <i>Bombyx mori</i>; TN, <i>Trichopulsia </i><i>ni</i>; HA, <i>Helicoverpa armigera</i>; SF, <i>Spodoptera frugiperda</i>; PI, <i>Plodia interpunctella</i>.</p

Immunodetection of MsVmp1 in the posterior midgut of <i>M. sexta</i> larvae at different physiological conditions.

<p>Cryosections of posterior midguts were stained with CFW and immune-labeled with anti-VMP1 antibodies. Primary antibodies were detected with ALEXA 488-conjugated anti-guinea pig IgGs. Brightfield and fluorescence images (for anti-Vmp1 and CFW), and overlays of fluorescence images are shown. Cryosections were obtained from feeding 2^nd instar larvae (top), starving 2^nd instar larvae (middle) and larvae molting from the 2^nd to the 3^rd instar. C, cuticle; EC, ectoperitrophic space; EN, endoperitrophic space; PM, peritrophic matrix. Size bar, 100 µm.</p

Ultrastructure of mesocuticle and endocuticle from TcCHT7-deficient adults.

<p>Ultrastructure of rigid elytral dorsal cuticle from 1 d-old adults (A and B) and 3 d-old adults (C and D) that had been injected with dsRNA (200 ng per insect; n = 30) for <i>TcVer</i> (A and C) and <i>TcCHT7</i> (B and D) into the late instar larvae was analyzed by TEM. EN, envelope; EP, epicuticle; EXO, exocuticle; PCF, pore canal fiber; APMP, apical plasma membrane protrusion; MESO, mesocuticle; ENDO, endocuticle; PC, pore canal. Scale bar = 2 μm.</p

Wheat germ agglutinin (WGA)-gold labeling TEM of dorsal elytral cuticle of <i>T</i>. <i>castaneum</i>.

<p>Ultra-thin sections (~90 nm) of pharate adults (5 d-old pupae) that had been injected ds<i>TcVer</i> (200 ng per insect) in late instar larvae were incubated with gold-labeled (10 nm) WGA (EY Laboratories) to detect chitin in the dorsal side of elytral cuticle. dsRNA for <i>chitin synthase-A</i> (ds<i>TcChs-A</i>) which is required for cuticular chitin synthesis [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004963#pgen.1004963.ref030" target="_blank">30</a>] was injected as a negative control. Chitin was detected in both horizontal laminae and vertical PCFs of elytral cuticle from ds<i>TcVer</i>-treated insects (A and C). Few or no gold particles were observed in TcChs-A-deficient insects (B and D). EN: envelope, EP: epicuticle, PRO: procuticle, PCF: pore canal fibers, APMP: apical plasma membrane protrusion. Scale bar in A and B = 1 μm and C and D = 500 nm.</p

<i>Tribolium castaneum</i> RR-1 Cuticular Protein TcCPR4 Is Required for Formation of Pore Canals in Rigid Cuticle

<div><p>Insect cuticle is composed mainly of structural proteins and the polysaccharide chitin. The CPR family is the largest family of cuticle proteins (CPs), which can be further divided into three subgroups based on the presence of one of the three presumptive chitin-binding sequence motifs denoted as Rebers-Riddiford (R&R) consensus sequence motifs RR-1, RR-2 and RR-3. The TcCPR27 protein containing the RR-2 motif is one of the most abundant CPs present both in the horizontal laminae and in vertical pore canals in the procuticle of rigid cuticle found in the elytron of the red flour beetle, <i>Tribolium castaneum</i>. Depletion of TcCPR27 by RNA interference (RNAi) causes both unorganized laminae and pore canals, resulting in malformation and weakening of the elytron. In this study, we investigated the function(s) of another CP, TcCPR4, which contains the RR-1 motif and is easily extractable from elytra after RNAi to deplete the level of TcCPR27. Transcript levels of the <i>TcCPR4</i> gene are dramatically increased in 3 d-old pupae when adult cuticle synthesis begins. Immunohistochemical studies revealed that TcCPR4 protein is present in the rigid cuticles of the dorsal elytron, ventral abdomen and leg but not in the flexible cuticles of the hindwing and dorsal abdomen of adult <i>T. castaneum</i>. Immunogold labeling and transmission electron microscopic analyses revealed that TcCPR4 is predominantly localized in pore canals and regions around the apical plasma membrane protrusions into the procuticle of rigid adult cuticles. RNAi for <i>TcCPR4</i> resulted in an abnormal shape of the pore canals with amorphous pore canal fibers (PCFs) in their lumen. These results support the hypothesis that TcCPR4 is required for achieving proper morphology of the vertical pore canals and PCFs that contribute to the assembly of a cuticle that is both lightweight and rigid.</p></div

MsVmp1 is extensively <i>O</i>-glycosylated.

<p>(A) <i>MsVMP1</i> was expressed in insect Sf21 cells and purified by Ni-NTA chromatography. After SDS-PAGE, the proteins were transferred to a nitrocellulose membrane and stained with Ponceau Red (left) and subsequently with different lectins (right lanes). GNA, <i>Galanthus nivalis</i> agglutinin; PNA, peanut agglutinin; SNA, <i>Sambucus nigra</i> agglutinin; DSA, <i>Datura stramonium</i> agglutinin; MAA, <i>Maackia amurensis</i> agglutinin. (B) For deglycosylation, MsVmp1 (expressed in insect cells) was treated with PNGase F or <i>O</i>-glycosidase in the presence of protease inhibitors. The reaction products were separated by SDS PAGE and stained with Coomassie Blue. <i>Left </i><i>lane</i>, MsVmp1 input without addition of glycosidase; Middle lane, PNGase F treatment; Right lane, <i>O</i>-glycosidase treatment. Std, standard proteins with indicated molecular masses in kDa. Arrows point to reaction intermediates and the terminal deglycosylation product with indicated molecular masses.</p

Ultrastructure of elytral dorsal cuticle from TcCHT7-deficient pharate adults.

<p>Ultrastructure of rigid elytral dorsal cuticle from pharate adults (5 d-old pupae) that had been injected with dsRNA (200 ng per insect; n = 30) for <i>TcVer</i> (A-D) and <i>TcCHT7</i> (E-H) into the late instar larvae was analyzed by TEM. The outer (B and F), middle (C and G) and inner (D and H) parts of elytral cuticle are enlarged from A and E. EN, envelope; EP, epicuticle; EXO, exocuticle; PCF, pore canal fiber; APMP, apical plasma membrane protrusion. Scale bar in A and E = 1 μm; B-D and F-H = 200 nm.</p

Immunodetection of MsVmp1 in PM preparations from anterior and posterior midguts of feeding larvae.

<p>(A) PM proteins were extracted by SDS treatment and separated by SDS-PAGE. Then the proteins were transferred to nitrocellulose and reacted with polyclonal anti-Vmp1 antibodies. Std, standard proteins with molecular masses indicated in kDa. (B) Immunodetection of Vmps using anti-Vmp1 antibodies. The PM preparations from the anterior and posterior parts of the midgut were washed several times with PBS buffer, blocked with bovine serum albumin and stained with the anti-Vmp1 antibodies. Cy3-conjugated anti-guinea pig IgGs were used as secondary antibodies. The PM was transferred to a microscope slide and mounted with Vectashield under a cover slip. The specimens were viewed under a fluorescence microscope using appropriate excitation an emission filters.</p

Immunoblotting detects MsVmp1 in the midgut of feeding, starving and molting larvae.

<p>Crude protein extracts from the anterior, median and posterior midgut were separated by SDS-PAGE, blotted onto nitrocellulose and stained with anti-VMP1 antibodies. The given molecular mass was estimated using standard proteins of known molecular masses. </p

ClustalW alignment of valine-rich midgut proteins from <i>M. sexta</i> (MsVmps).

<p>Highly conserved or identical amino acids are highlighted with light grey, grey or black shadings. The consensus sequence is given below. The accession numbers are as follows: MsVmp1 (Msex012254-PA), MsVmp2 (central part of Msex012257-PA), MsVmp3 (C-terminal part of Msex012257-PA), MsVmp4 (N-terminal part of Msex012261-PA), MsVmp5 (N-terminal part of Msex012257-PA), MsVmp6 (Msex011100-PA), MsVmp7 (C-terminal part of Msex012261-PA), MsVmp8 (Msex012260-PA), and MsVmp (Msex012262-PA).</p