Progression of pathology in the midgut of <i>dsDvSnf7</i>-treated WCR larvae.

<p>Observed symptoms were summarized in four phases (1 = lightest to 4 = most severe). Highlighted are macroautophagic complexes (blue ellipses) (diagram, A′), gut lumen (L with orange glowing background) filled with cellular debris and loss of microvilli (white arrowheads) (diagrams, B, B″), large vacuoles (diagrams, B′, B″, C′) (green arrows), luminal membrane disintegration (black arrowheads) (diagram, C″) and infolding of the basal membrane (transparent black arrowheads) (diagrams, C″, D′, D″), progressive cell sloughing (asterisks) (diagrams, A″, B, C). Panels E-E″ show midgut of dsGFP-treated control larvae. Highlighted are endosomes (white arrows), lysosomes (magenta arrows) and autolysosomes (black arrow). refers to grey area denoting sectional artifact (cracks in the tissue section). Scale bars  = 4 µm (A-E) and 2 µm (A′-E′ and A″-E″).</p

Midgut ultrastructural profiles of untreated, dsGFP-treated and dsDvSnf7-treated western corn rootworm (WCR) second instar larvae fed for 5 days.

<p>The letter L with orange glowing background indicates the region of gut lumen, white arrows indicate endosomes, magenta arrows indicate lysosomes, black arrows indicate autolysosomes and MLB is multi-lamellar bodies. C′ is for the magnified view of macroautophagic complex in the midgut of dsDvSnf7-treated individuals. Scale bars  = 4 µm (a, b, c), 1 µm (a′, b′) and 500 nm (c′). D refers to macroautophagic complexes counted in enterocytes of untreated, dsGFP-treated and dsDvSnf7-treated larval midgut. Error bars represent mean ± S.D. of three individuals.</p

Percent identities of amino acid residues among the AChEs of <i>Tribolium castaneum</i>, <i>Torpedo californica</i> and other seven insect species.

<p>NOTE: TcaAce, <i>Torpedo californica</i>; TcAce, <i>Tribolium castaneu</i>m; SaAce, <i>Sitobion avenae</i>; DmAce, <i>Drosophila melanogaster</i>; AgAce, <i>Anopheles gambiae</i>; BgAce, <i>Blattella germanica</i>; HaAce, <i>Helicoverpa assulta</i>; SgAce, <i>Schizaphis graminum</i>; LdAce, <i>Leptinotarsa decemlineata</i>.</p

Separation of Arg from the catalytic triad in TcAce2 (A), TcAce1 (B), <i>Anopheles gambiae</i> AP-AChE (C), and human AChE (D).

<p>Separation of Arg from the catalytic triad in TcAce2 (A), TcAce1 (B), <i>Anopheles gambiae</i> AP-AChE (C), and human AChE (D).</p

The cDNA and deduced amino acid sequences of two <i>Ace</i> genes from <i>Tribolium castaneum</i>.

<p>The amino acid sequences were numbered from the start of the mature proteins. The start codon ATG were bold and underlined, and the stop codon TAA at the end of the coding region were bold and marked with asterisks. The putative signal peptides of the deduced amino acid sequences were underlined with red dots. Potential N-linked glycosylation sites were bold and shaded. The sequences were deposited in the GenBank (accession numbers: HQ260968 for <i>TcAce1</i> and HQ260969 for <i>TcAce2</i>).</p

Comparison of loop conformations of residues 146–154 and 493–509 in TcAce1 with the corresponding ones of residues 120–128 and 488–504 in TcAce2.

<p>Comparison of loop conformations of residues 146–154 and 493–509 in TcAce1 with the corresponding ones of residues 120–128 and 488–504 in TcAce2.</p

Schematic diagram of the organization of two <i>Ace</i> genes from <i>Tribolium castaneum</i>.

<p>The full lengths of the two genomic sequences were 2,986 bp for <i>TcAce1</i> and 32,243 bp for <i>TcAce2</i>. Genome structure showed that two different <i>Ace</i> genes in <i>T. castaneum</i> located on different chromosomes. <i>TcAce1</i> located on chromosome 5 and <i>TcAce2</i> on chromosome 2. <i>TcAce1</i> has two exons and one intron, whereas <i>TcAce2</i> has six exons and five introns.</p

Alignment of deduced AChE protein sequences encoded by TcaAce (CAA27169, <i>Torpedo californica</i> Ace); TcAce1 (HQ260968, <i>Tribolium castaneum</i> Ace1, this paper); TcAce2 (HQ260969, <i>T. castaneum</i> Ace2, this paper); SaAce1 (AY819704, <i>Sitobion avenae</i> Ace1); SaAce2 (AY707319, <i>S. avenae</i> Ace2); DmAce (X05893, <i>Drosophila melanogaster</i> Ace); AgAce1 (XM_321792, <i>Anopheles gambiae</i> Ace1); AgAce2 (BN000067, <i>A. gambiae</i> Ace2); BgAce1 (DQ288249, <i>Blattella germanica</i> Ace1); BgAce2 (DQ288847, <i>B. germanica</i> Ace2); HaAce1 (DQ001323, <i>Helicoverpa assulta</i> Ace1); HaAce2 (AY817736, <i>H. assulta</i> Ace2); SgAce1 (AF321574, <i>Schizaphis graminum</i> Ace1) and LdAce2 (L41180, <i>Leptinotarsa decemlineata</i> Ace2).

<p>Numbering of the amino acid sequences was from the N-terminus of mature proteins. Identical amino acids were indicated by asterisks and conservative substitutions by dots. The catalytic triad residues were marked with arrowhead. The number 1, 2, 3 on the boxed amino acids indicated the residues forming intramolecular disulfide bonds. The positions of aromatic residues lining the active site gorge in <i>T. californica</i> AChE were marked with block arrows. The cholinesterase signature sequence was underlined.</p

Close-up view of the active site of TcAce2 with a perspective looking down to acetylcholine and the catalytic triad at the bottom of the gorge.

<p>Close-up view of the active site of TcAce2 with a perspective looking down to acetylcholine and the catalytic triad at the bottom of the gorge.</p

Rooted phylogenetic tree of deduced <i>Ace</i> amino acid sequences from the Pacific electric ray (<i>Torpedo californica</i>), two-spotted spider mite (<i>Tetranychus urticae</i>) and 43 insect species constructed by the neighbor-jointing method.

<p>The name is made up of a species abbreviation (first letter of the genus followed by the first one or two letters of the specific name). Sequences used: TcaAce (CAA27169, <i>T. californica</i> Ace); TcAce1 (HQ260968, <i>Tribolium castaneum Ace1</i>, this paper); TcAce2 (HQ260969, <i>T. castaneum Ace2</i>, this paper); BmAce1(EU262633, <i>Bombyx mandarina</i> Ace1); BmAce2 (EU262632, <i>B. mandarina</i> Ace2); SaAce1 (AY819704, <i>Sitobion avenae</i> Ace1); SaAce2 (AY707319, <i>S. avenae</i> Ace2); RpAce1 (AY667435, <i>Rhopalosiphum padi</i> Ace1); RpAce2 (AY707318, <i>R. padi</i> Ace2); DmAce (X05893, <i>Drosophila melanogaster</i> Ace); AgAce1 (XM_321792, <i>Anopheles gambiae</i> Ace1); AgAce2 (BN000067, <i>A. gambiae</i> Ace2); LdeAce1 (FJ647186, <i>Liposcelis decolor</i> Ace1); LdeAce2 (FJ647187, <i>L. decolor</i> Ace2); OvAce1 (FJ228227, <i>Orchesella villosa</i> Ace1); OvAce2 (FJ228228, <i>O. villosa</i> Ace2); LeAce1 (EU854149, <i>Liposcelis entomophila</i> Ace1); LeAce2 (EU854150, <i>L. entomophila</i> Ace2); BgAce1 (DQ288249, <i>Blattella germanica</i> Ace1); BgAce2 (DQ288847, <i>B. germanica</i> Ace2); BtAce1 (EF675188, <i>Bemisia tabaci</i> Ace1); BtAce2 (EF675190, <i>B. tabaci</i> Ace2); CqAce1 (XM_001847396, <i>Culex quinquefasciatus</i> Ace1); CqAce2 (XM_001842175, <i>C. quinquefasciatus</i> Ace2); BmoAce1 (NP_001037380 <i>Bombyx mori</i> Ace1); BmoAce2 (NP_001108113 <i>B. mori</i> Ace2); ApAce1 (XM_001948618, <i>Acyrthosiphon pisum</i> Ace1); ApAce2 (XM_001948953, <i>A. pisum</i> Ace2); NvAce1 (XM_001600408, <i>Nasonia vitripennis</i> Ace1); NvAce2 (XM_001605518, <i>N. vitripennis</i> Ace2); PhAce1 (AB266605, <i>Pediculus humanus corporis</i> Ace1); PhAce2 (AB266606, <i>P. humanus corporis</i> Ace2); CpAce1 (DQ267977, <i>Cydia pomonella</i> Ace1); CpAce2 (DQ267976, <i>C. pomonella</i> Ace2); HaAce1 (DQ001323, <i>Helicoverpa assulta</i> Ace1); HaAce2 (AY817736, <i>H. assulta</i> Ace2); AaAce1 (AB218421, <i>Aedes albopictus</i> Ace1); AaAce2 (AB218420, <i>A. albopictus</i> Ace2); AgoAce1 (AF502081, <i>Aphis gossypii</i> Ace1); AgoAce2 (AF502082, <i>A. gossypii</i> Ace2); CtAce1 (AB122151, <i>Culex tritaeniorhynchus</i> Ace1); CtAce2 (AB122152, <i>C. tritaeniorhynchus</i> Ace2); SgAce1 (AF321574, <i>Schizaphis graminum</i> Ace1); MpAce1 (AF287291, <i>Myzus persicae</i> Ace1); MpAce2 (AY147797, <i>M. persicae</i> Ace2); CpiAce1 (AJ489456, <i>Culex pipiens</i> Ace1); CpiAce2 (AM159193, <i>C. pipiens</i> Ace2); MdAce (AY134873, <i>Musca domestica</i> Ace); PxAce1 (AY970293, <i>Plutella xylostella</i> Ace1); PxAce2 (AY061975, <i>P. xylostella</i> Ace2); CsAce1 (EF453724, <i>Chilo suppressalis</i> Ace1); CsAce2 (EF470245, <i>C. suppressalis</i> Ace2); CmAce2 (FN538987, <i>Cnaphalocrocis medinalis</i> Ace2); PhcAce1 (AB266614, <i>Pediculus humanus capitis</i> Ace1); PhcAce2 (AB266615, <i>P. humanus capitis</i> Ace2); TuAce (AY188448, <i>T. urticae</i> Ace); AaeAce1 (EF209048, <i>Aedes aegypti</i> Ace1); LmAce1 (EU231603, <i>Locusta migratoria manilensis</i> Ace1); LbAce1 (FJ647185, <i>Liposcelis bostrychophila</i> Ace1); LbAce2 (EF362950, <i>L. bostrychophila</i> Ace2); NlAce (FM866396, <i>Nilaparvata lugens</i> Ace); CcAce (EU130781, <i>Ceratitis captitata</i> Ace); AmAce (AB181702, <i>Apis mellifera</i> Ace); BdAce2 (AY155500, <i>Bactrocera dorsalis</i> Ace2); HiAce (AY466160, <i>Haematobia irritans</i> Ace); CppAce1 (AY762905, <i>Culex pipiens pallens</i> Ace1); HarAce2 (AF369793, <i>Helicoverpa armigera</i> Ace2); BoAce (AF452052; <i>Bactrocera oleae</i> Ace); LcAce (U88631, <i>Lucilia cuprina</i> Ace); NcAce (AF145235, <i>Nephotettix cincticeps</i> Ace); LdAce2 (L41180, <i>Leptinotarsa decemlineata</i> Ace2).</p