Effect of different serpin knockdown on larval mortality of <i>Plutella xylostella</i>.

<p>Each treatment was injected with 2 µl of a solution containing a total of 5 µg dsRNA. Percentage mortality 24 h after RNAi was calculated. Each value was shown as values ± S.E.M of three independent experiments. Significant differences were indicated with different letters at <i>P</i><0.05.</p

Hemolymph phenoloxidase (PO) activity in serpin silenced larvae of <i>Plutella xylostella</i>.

<p>Hemolymph was collected at 24(dsSerpin-2, dsSerpin-4 and dsSerpin-5). Larvae injected with dsGFP and saline buffer were used as control groups. The PO activity was measured using L-dopa and defined as ΔA₄₉₀ per mg total protein. Each value was shown as values ± S.E.M of three independent experiments. Significant differences were indicated with different letters at <i>P</i><0.05.</p

Melanization of <i>Plutella xylostella</i> treated with destruxin and dsSerpins.

<p>The treatment group was injected with 2 µl of a solution containing 200 µg/ml destruxin A and a total of 5 µg dsRNA. The control treatment was injected with 2 µl of PBS buffer.</p

Detection of the efficiency of RNAi and impact on serpin-2, serpin-4 and serpin-5 mRNA levels by Western Blot (A) and qRT-PCR (B).

<p>The mRNA and protein levels were normalized relative to the β-Actin. The different letters above the columns indicate significant differences in serpins genes expression during <i>P. xylostella</i> development (<i>P</i><0.05). Each point represents mean value ± S.E.M of three independent experiments with three individuals in each replicate. Western Blot analysis visualized by DAB.</p

Multiple alignment of serpin-4 and serpin-5 with other known serpins.

<p>Here serpin-2 (<i>P. xylostella</i>, BAF36820), serpin-4 (<i>Bombyx mori</i>, ACZ81437), serpin-4B (<i>Manduca sexta</i>, AAS68504), serpin-4A (<i>Manduca sexta</i>, AAS68503), serpin-4 (<i>Danaus plexippus</i>, EHJ70588), serpin-5A (<i>Manduca sexta</i>, AAS68507), serpin-4 (<i>Glossina morsitans</i>, AFG28186), serpin-77Ba (<i>Papilio xuthus</i>, BAM10360), serpin-006 (<i>Chilo suppressalis</i>, AFQ01142), serpin-5 (<i>Danaus plexippus</i>, EHJ70286) and serpin-7 (<i>Chilo suppressalis</i>, AFQ01143) are included. The reactive centre loop regions are boxed.</p

Nucleotide sequence (above) and deduced amino acid sequence (below) of the serpin-5 (GenBank accession No. KC505247).

<p>The serpin domain is shadowed. The asterisk (*) indicates the stop codon. Polyadenylation signal is bolded and italicized.</p

Expression of serpin-4 and serpin-5 after injecting dsSerpin-2 (A), expression of serpin-2 and serpin-5 after injecting dsSerpin-4 (B).

<p>Each value was shown as values ± S.E.M of three independent experiments. Significant differences were indicated with different letters at P<0.05.</p

Consensus Neighbour-Joining tree based on the sequences of serpins.

<p>Maximum likelihood tree reconstruction based on sequences of serpins (length approximately 449) of representative sequences from Genbank under the WAG+G substitution model was carried out. The estimated value of the gamma shape parameter (+G) was 1.70. The bootstrap values are indicated. Serpins number (abbreviated as S in tree), accession number and species name are shown along the sequence in the tree. Sequences used in this study are highlighted in the tree via grey colour.</p

Insecticidal efficacy of destruxin A and serpin knockdown on larvae of <i>Plutella xylostella</i>.

<p>Each treatment was injected with 2 µl of a solution containing 200 µg/ml (LC₅₀) destruxin A and a total of 5 µg dsRNA. The control treatment was injected with 2 µl of PBS buffer. Percentage mortality 24 h after treatment was calculated. Each value was shown as values ± S.E.M of three independent experiments. Significant differences were indicated with different letters at <i>P</i><0.05.</p

Expression of serpin-2, serpin-4 and serpin-5 in different lifestages of <i>Plutella xylostella</i> detected by Western Blot (A) and qRT-PCR (B).

<p>The mRNA and protein levels were normalized relative to the β-Actin. The different letters above the columns indicate significant differences in serpin gene expression during <i>P. xylostella</i> development (<i>P</i><0.05). Each point represents mean value ± S.E.M of three independent experiments with three individuals in each replicate. Western Blot analysis visualized by DAB.</p