Expression of 31 serine protease transcripts in eight different tissues including foregut (FG), hindgut (HG), midgut (MG), haemolymph (HL), Malpighian tubules (MT), carcass (CA), fat bodies (FB) and silk gland (SG) from <i>O. nubilalis</i> larvae.

<p>The expression levels of <i>OnTry13</i>, <i>OnCTP15</i> and <i>OnCTP11</i> were too low to be detected by RT-PCR. <i>O. nubilalis</i> ribosomal protein S3 (<i>OnRps3</i>) was used as a reference gene.</p

Primers used in RT-PCR and qPCR analyses of 34 putative trypsin and chymotrypsin and homolog genes in <i>O. nubilalis</i> larvae.

*<p>These deduced protein sequences have low bootstrap values (<75) and are not included in groups.</p

Characteristics of 12 putative trypsins and four homologs deduced from full-length cDNAs derived from the larval midgut of <i>O. nubilalis</i>.

<p>Characteristics of 12 putative trypsins and four homologs deduced from full-length cDNAs derived from the larval midgut of <i>O. nubilalis</i>.</p

Designations and key amino acid residues used to distinguish putative trypsins and chymotrypsins and their corresponding homologs from the larval gut of <i>Ostrinia nubilalis</i>.

*<p>OnTry14 and OnCTP17 are derived from partial cDNA sequences.</p

Multiple alignments of 17 deduced amino acid sequences of putative chymotrypsins and chymotrypsin homologs from <i>O. nubilalis</i> larvae using Clustal W2.

<p>The predicted signal peptide is underlined in brown; the catalytic triads and conserved regions are boxed in blue; the conserved catalytic triads are marked with blue arrows at the top; the autocatalytic site is marked with green arrow at the top; the conserved residue in the S1 pocket is marked with purple arrow at the top; and the six cysteine residues were marked with purple stars at the top.</p

GenBank accession numbers for other insect trypsins, chymotrypsins, and homologs that were used in phylogenetic analysis.

<p>GenBank accession numbers for other insect trypsins, chymotrypsins, and homologs that were used in phylogenetic analysis.</p

Characteristics of 12 putative chymotrypsins and four homologs deduced from full-length cDNAs derived from the larval midgut of <i>O. nubilalis</i>.

<p>Characteristics of 12 putative chymotrypsins and four homologs deduced from full-length cDNAs derived from the larval midgut of <i>O. nubilalis</i>.</p

Relative expression ratios of putative trypsin and chymotrypsin transcripts in the gut of <i>O. nubilalis</i> early third instar larvae fed Cry1Ab protoxin at the LC₅₀ concentration relative to larvae fed control diet at four different feeding periods.

<p>The asterisk “*” indicates that the transcript expression was significantly different at that feeding period (<i>P</i><0.05), whereas the positive or negative values of each column indicates up- or down-regulation, respectively.</p

Phylogenetic analysis of amino acid sequences of 34 putative serine proteases from <i>O. nubilalis</i> with serine proteases from <i>P. interpunctella</i>, <i>C. fumiferana</i>, <i>H. armigera</i>, <i>M. sexta</i>, <i>B. mori</i>, <i>A. ipsilon</i>, <i>S. nonagrioides</i>, <i>S. frugiperda, S. litura</i>, <i>H. zea, B. mandarina, H. punctigera</i>, <i>M. configurata</i>, <i>S. exigua</i>, <i>L. oleracea</i>, <i>G. mellonella</i>, <i>O. furnacalis</i>.

<p>Bootstrap values are obtained by neighbor-joining method using 5000 replications. The blue “▴” indicated the trypsin genes that have already been submitted to the NCBI database; and the red “▴” indicates new cDNA sequences that were revealed from this study. The GenBank accession numbers of the sequences used in this analysis are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044090#pone-0044090-t002" target="_blank">Table 2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044090#pone-0044090-t003" target="_blank">3</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044090#pone-0044090-t004" target="_blank">4</a>.</p

Molecular and Functional Analysis of UDP-<i>N</i>-Acetylglucosamine Pyrophosphorylases from the Migratory Locust, <i>Locusta migratoria</i>

<div><p>UDP-<i>N</i>-acetylglucosamine pyrophosphorylases (UAP) function in the formation of extracellular matrix by producing <i>N</i>-acetylglucosamine (GlcNAc) residues needed for chitin biosynthesis and protein glycosylation. Herein, we report two <i>UAP</i> cDNA’s derived from two different genes (<i>LmUAP1</i> and <i>LmUAP2</i>) in the migratory locust <i>Locusta migratoria</i>. Both the cDNA and their deduced amino acid sequences showed about 70% identities between the two genes. Phylogenetic analysis suggests that <i>LmUAP1</i> and <i>LmUAP2</i> derive from a relatively recent gene duplication event. Both <i>LmUAP1</i> and <i>LmUAP2</i> were widely expressed in all the major tissues besides chitin-containing tissues. However, the two genes exhibited different developmental expression patterns. High expression of <i>LmUAP1</i> was detected during early embryogenesis, then decreased greatly, and slowly increased before egg hatch. During nymphal development, the highest expression of <i>LmUAP1</i> appeared just after molting but declined in each inter-molting period and then increased before molting to the next stage, whereas <i>LmUAP2</i> was more consistently expressed throughout all these stages. When the early second- and fifth-instar nymphs (1-day-old) were injected with <i>LmUAP1</i> double-stranded RNA (dsRNA), 100% mortality was observed 2 days after the injection. When the middle second- and fifth-instar nymphs (3- to 4-day-old) were injected with <i>LmUAP1</i> dsRNA, 100% mortality was observed during their next molting process. In contrast, when the insects at the same stages were injected with <i>LmUAP2</i> dsRNA, these insects were able to develop normally and molt to the next stage successfully. It is presumed that the lethality caused by RNAi of <i>LmUAP1</i> is due to reduced chitin biosynthesis of the integument and midgut, whereas <i>LmUAP2</i> is not essential for locust development at least in nymph stage. This study is expected to help better understand different functions of <i>UAP1</i> and <i>UAP2</i> in the locust and other insect species.</p></div