Changes in the amount of free GrybiApoLp-III by AKH stimulation and starvation.

<p>(A) Representative data from native PAGE analyses of proteins in <i>G</i>. <i>bimaculatus</i> hemolymph before and after AKH injection. Free GrybiApoLp-III is indicated by a bar. Figures are representative data from experiments using three individual crickets [the lane number (#1, #2, and #3) indicates the sample from an individual cricket]. Reproducibility of this experiment was confirmed by different experimental trials using more than 30 individuals. (B) Representative data from native PAGE analyses of proteins in hemolymph of starved <i>G</i>. <i>bimaculatus</i>. Free GrybiApoLp-III is indicated by a bar. Numbers represent individual crickets (#1, #2 and #3). Data on the left (samples from fed crickets) is composed of three single gels. The reproducibility of this experiment was also confirmed by different trials using totally more than 30 individual crickets from different populations.</p

Identification of GrybiApoLp-III.

<p>(A) Analysis of hemolymph proteins from <i>G</i>. <i>bimaculatus</i> (lane 1) and <i>B</i>. <i>mori</i> (lane 2) separated by SDS-PAGE. <i>B</i>. <i>mori</i> ApoLp-III is indicated by a bar. The candidate for GrybiApoLp-III is indicated by an arrow. (B) The RP-HPLC profile of gel-digested proteins from the band corresponding to the GrybiApoLp-III candidate. Peaks A–D were subsequently subjected to amino acid sequence analyses. (C) Alignment of amino acid sequences of the resulting GrybiApoLp-III and <i>A</i>. <i>domesticus</i> ApoLp-III sequences. Bars above sequences indicate the results of amino acid sequence analyses. N-terminal sequence, from 1st residue (D) to 38th residue (L), and fragment sequences from peaks A–D in RP-HPLC indicate N and A–D, respectively. (D) Tissue distribution of <i>G</i>. <i>bimaculatus ApoLp-III</i> by RT-PCR. Elongation factor (<i>EF</i>) was used as an experimental control. FB, Fat body; FG, foregut; MG, midgut; HG, hindgut; MT, Malpighian tubules; TR, trachea; MS, muscle; OV, ovary; NS, nervous system; HC, hemocytes.</p

Efficiency of knockdown by <i>G</i>. <i>bimaculatus ApoLp-III</i>-dsRNA treatment.

<p>(A) Quantitative RT-PCR analysis of <i>GrybiApoLp-III</i> in <i>GrybiApoLp-III</i>-dsRNA-treated (<i>GrybiApoLp-III</i>^RNAi) crickets. RNA was prepared from the fat body of crickets 2 days after dsRNA treatment. <i>EGFP</i>-dsRNA was used as an experimental control (<i>EGFP</i>^RNAi). EF (elongation factor) was used as a reference of transcription. Mean + SD (n = 5), *: <i>P</i> < 0.01, Student’s-<i>t</i> test. (B) Representative data of native-PAGE analyses of hemolymph collected from crickets after <i>GrybiApoLp-III</i>-dsRNA treatment (<i>GrybiApoLp-III</i>^RNAi). Hemolymph was collected 0, 2, 4, and 6 days after dsRNA treatment, and was subjected to native-PAGE. <i>EGFP</i>-dsRNA was used as an experimental control (<i>EGFP</i>^RNAi). GrybiApoLp-III is indicated by a bar. Numbers represent individual crickets. The reproducibility of this experiment was confirmed by different trials totally using more than 30 individuals from different populations. We also confirmed no off-target effects by dsRNA encoding EGFP using different control gene (DsRed) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154841#pone.0154841.s002" target="_blank">S2 Fig</a>).</p

Schematic models to maintain the hemolymph lipid level.

<p>(A) For crickets feeding normally, the hemolymph lipid level is maintained by shuttling between HDLp to LDLp; however, the hemolymph lipid level was maintained at similar levels even in GrybiApoLp-III knockdown crickets. (B) For long-term starved crickets, the lowered hemolymph lipid level was recovered from the fat body lipids via LDLp. (C) For AKH-stimulated crickets, possibly under the condition of acute lipid requirements, hemolymph lipid levels increase by lipids of the fat body via LDLp by AKH stimulation.</p

Effect of <i>G</i>. <i>bimacutatus</i> ApoLp-III knockdown on hemolymph lipid levels.

<p>(A) Analysis of basal hemolymph DAG levels in <i>GrybiApoLp-III</i>^RNAi and <i>EGFP</i>^RNAi crickets. Values are mean + SD (n = 5). (B) Analysis of basal hemolymph DAG levels in <i>GrybiApoLp-III</i>^RNAi crickets and hemolymph DAG levels in <i>GrybiApoLp-III</i>^RNAi crickets after GrybiAKH injection or Ringer’s solution alone. Values are mean + SD (n = 5). Significant differences are denoted by an asterisk (*, <i>P</i> < 0.05 by Tukey’s PSD test). Bars without asterisks indicate that differences among levels are not significant.</p

Effect of <i>G</i>. <i>bimaculatus ApoLp-III</i> knockdown on food intake.

<p>Food intake of <i>GrybiApoLp-III</i>^RNAi adult females (A) and males (B). The amount of food intake was evaluated by counting the number of fecal pellets as previously observed [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154841#pone.0154841.ref023" target="_blank">23</a>]. There were no significant differences between dsGrybiApoLp-III-treated crickets and dsEGFP-treated crickets (<i>P</i> > 0.1 by Tukey’s PSD test). Values are mean ± SD (n = 6).</p

Preparation of Lp and the effect of Lp injection on hemolymph lipid levels and the increased duration to initiate feeding.

<p>(A, B) Preparation of GrybiLp. <i>G</i>. <i>bimaculatus</i> hemolymph was subjected to KBr density gradient ultracentrifugation. (A) After ultracentrifugation, 12 fractions were separated, and the hemolymph before ultracentrifugation was analyzed by SDS-PAGE. (B) Gradients were fractionated from low to high according to density, the specific gravity of each fraction (blue squares). The lipid level of each fraction was also measured (red diamonds). (C) Analysis of hemolymph lipid levels after injection of Lp fractions containing 180, 270, and 450 μg of lipid. Values are mean ± SD (n = 6). Significant differences compared to 0 h are denoted by asterisks (*, <i>P</i> < 0.05 by Dunnett’s test). (D) Measurement of duration to the initiation of feeding after injection of Lp fractions containing 90, 180, and 270 μg of lipid. Values are mean + SD (n = 6). Significant differences are denoted by asterisks (*, <i>P</i> < 0.05; **, <i>P</i> < 0.005; ***, <i>P</i> < 0.0005 for Mann-Whitney-test).</p