Comparison of carboxylesterase numbers identified among <i>Locusta migratoria</i> (Orthoptera), <i>Drosophila melanogaster</i> (Diptera), <i>Anopheles gambiae</i> (Diptera), <i>Culex pipiens quinquefasciatus</i> (Diptera) and <i>Trialeurodes vaporariorum</i> (Hemiptera).

<p>Classifications of different carboxylesterases were based on Oakeshott et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094809#pone.0094809-Oakeshott1" target="_blank">[4]</a> and Karatolos et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094809#pone.0094809-Karatolos1" target="_blank">[25]</a>.</p

Tissue-dependent expression patterns of four CarE genes in <i>L. migratoria</i>.

<p>The analyses were conducted in nine different tissues, including the brain (BR), foregut (FG), gastric caeca (GC), midgut (MG), hindgut (HG), Malpighian tubules (MT), fat bodies (FB), muscle (MU) and hemolymph (HE). The mRNA level in each tissue was normalized by <i>β-actin</i> as a reference gene. Results are mean and standard errors (SE) of three biological replications (n = 3), each with three technical replications. Different letters above the vertical bars indicate significant differences among different tissues based on Fisher's LSD multiple comparison test (<i>P</i><0.05).</p

Accession numbers of other insect carboxylesterases used in phylogenetic analysis.

<p>Accession numbers of other insect carboxylesterases used in phylogenetic analysis.</p

Alignment of deduced amino acid sequences of selected <i>L. migratoria</i> CarE genes along with those known in other insect species.

<p>A) Alignment of deduced amino acid sequences of three <i>L. migratoria</i> CarE genes along with DmCG4382 and DmCG3841 in clade D. B) Alignment of deduced amino acid sequences of a <i>L. migratoria</i> CarE gene along with AgCOEjhe2E, AgCOEjhe3E and DmJHE in clade F. C) Alignment of deduced amino acid sequences of two <i>L. migratoria</i> CarE gene along with AgCOE7o and DmCG5397 in clade I. The multiple sequence alignment was performed using GeneDoc software. The predicted signal peptide is underlined in purple, the conserved motifs are boxed in red, N-terminal conserved Cys for disulfide bond is marked with purple star at the top, residues for oxyanion hole are linked with red line, the catalytic triads are marked with blue arrows at the top, and potential <i>N</i>-glycosylation sites are in orange.</p

Predicated biochemical characteristics of 39 carboxylesterases deduced from <i>Locusta migratoria</i> cDNAs.

<p>Predicated biochemical characteristics of 39 carboxylesterases deduced from <i>Locusta migratoria</i> cDNAs.</p

Primer sequences and predicted sizes of PCR products for full-length cDNA cloning, RT-qPCR analysis and dsRNA synthesis of four <i>LmCarE</i> genes along with those in dsRNA synthesis for the control gene (<i>GFP</i>) and the reference gene (<i>β-actin</i>) in RT-qPCR analysis.

<p><b>*</b>The lowercase letters represent the T7 promoter sequences for efficient in vitro transcription in dsRNA synthesis.</p

Key amino acid residues (RF, DQ motifs, residues for nucleophilic elbow, catalytic triads, N-terminal conserved Cys for disulfide bond and oxyanion hole) of 39 carboxylesterases deduced from <i>Locusta migratoria</i> cDNAs.

<p>Key amino acid residues (RF, DQ motifs, residues for nucleophilic elbow, catalytic triads, N-terminal conserved Cys for disulfide bond and oxyanion hole) of 39 carboxylesterases deduced from <i>Locusta migratoria</i> cDNAs.</p

Phylogenetic analyses of insect carboxylesterases (CarEs).

<p>MEGA 5 was used to construct the phylogenetic tree with neighbor-joining method. Nodes with distance bootstrap values greater than 50% (1000 replicates) are shown. The nomenclatures of the clades are according to Oakeshott et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094809#pone.0094809-Oakeshott1" target="_blank">[4]</a>. Sequences from the <i>D. melanogaster</i> and <i>A. gambiae</i> genomes are annotated as in Ranson et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094809#pone.0094809-Ranson2" target="_blank">[71]</a>.The 39 deduced CarEs from <i>L. migratoria</i> are marked with filled circles. The GenBank accession numbers for various CarEs of insects used in this tree were generally taken from NCBI database (<a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>). The tree included twelve clades named A-N (except for C and J clade). The abbreviations used for insect species are: <i>Drosophila melanogaster</i> (Dm), <i>Anopheles gambiae</i> (Ag), <i>Culex quinquefasciatus</i> (Cq), <i>Aedes aegypti</i> (Aa), <i>Lucilia cuprina</i> (Lc), <i>Haematobia irritans</i> (Hi), <i>Musca domestica</i> (Md), <i>Myzus persicae</i> (Mp), <i>Antheraea polyphemus</i> (Ap), <i>Manduca sexta</i> (Ms), <i>Choristoneura fumiferana</i> (Cf), <i>Bombyx mori</i> (Bm), and <i>Tribolium castaneum</i> (Tc).</p

Changes in susceptibility of the locusts to different insecticides after the injection of <i>LmCesA3</i>, <i>LmCesA20</i>, <i>LmCesD1</i> or <i>LmCesE1</i> dsRNA in second instar nymphs.

<p>Insecticides bioassays were conducted 24(SE) of three independent experiments (n = 3). The symbol “*” indicates significant difference between the control and treatment in Student <i>t</i>-test (<i>P</i><0.05).</p

Alignment of deduced amino acid sequences of 20 <i>L. migratoria</i> CarE genes along with Agae5G and DmCG9858 in clade A.

<p>The multiple sequence alignment was performed using GeneDoc software. The predicted signal peptide is underlined in purple, the conserved motifs are boxed in red, N-terminal conserved Cys for disulfide bond is marked with purple star at the top, residues for oxyanion hole are linked with red line, the catalytic triads are marked with blue arrows at the top, and potential <i>N</i>-glycosylation sites are in orange.</p