<i>In vitro</i> competitive binding assay of rITPL and TK-4 to BNGR-A24.

<p>(A–D) Microscopic imaging. The binding of RR-labeled ligands (A and C, rITPL; B and D, TK-4) and CHO cells expressing EGFP-fused BNGR-A24 was examined in the presence of potential competitors (A, TK-4; B, rITPL; C and D, spantide I [SP]). EGFP and RR fluorescences were observed by confocal microscopy. Co-localization is presented as yellow in the merged images (Merge). Representative images of cells from at least two independent experiments are shown. (E–H) Relative fluorescent intensity obtained from experiment images (A)–(D), respectively. RR fluorescent intensity was normalized by EGFP intensity per image and is indicated as fold change over that without competitor (mean + S.D.; <i>n</i> = 6). Asterisks indicate significant differences compared to ‘no competitor’ data (<i>P</i> < 0.05, Dunnett’s test).</p

EC₅₀ of BNGR responses to <i>B</i>. <i>mori</i> TRPs.

<p>EC₅₀ of BNGR responses to <i>B</i>. <i>mori</i> TRPs.</p

Responses of BNGR-A24 and BNGR-A32 to <i>B</i>. <i>mori</i> TRPs.

<p>The response of HEK293T cells co-expressing BNGR-A24 (A) or BNGR-A32 (B) and promiscuous mouse Gα15 to the TRPs was monitored using the Ca²⁺ imaging technique. Dose-response curves are depicted as relative fluorescent intensity (mean ± S.D.; <i>n</i> = 3).</p

Effects of TK-4, rITPL, and spantide I on cyclic nucleotide levels in BmN cells.

<p>Intracellular cGMP (A) and cAMP (B) levels in BmN cells following 30-min incubation with the peptide and reagent of interest at the indicated concentrations were measured. (A) Effects of TK-4 and spantide I on the response of BmN cells to rITPL. The cGMP levels of the cells exposed to 100 nM rITPL with TK-4 or spantide I (SP) were determined. Responses to TK-4 alone, spantide I alone, rITPL alone, and vehicle treatment (-) were also examined. (B) The cAMP levels of the cells after incubation with 1 μM of five TRPs or rITPL. Responses to vehicle treatment (-) and 10 μM forskolin (FSK; positive control) were also examined. Data are presented as the mean + S.D. (<i>n</i> = 3 or 4). Different letters indicate significant differences (<i>P</i> < 0.05; Tukey’s HSD).</p

Effect of spantide I on the response of BNGR-A24 to rITPL and TK-4.

<p>Spantide I (<i>SP</i>), a potential competitor, was added together with rITPL or TK-4 and the response of BNGR-A24 was monitored using the Ca²⁺ imaging assay. Dose-response curves of the responses are presented as relative fluorescent intensity (mean ± S.D.; <i>n</i> = 3). The response curve of TK-4 with no competitor is the same as the one shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156501#pone.0156501.g002" target="_blank">Fig 2A</a>. Asterisks indicate significant differences for treatment with the same ligand without spantide I (<i>P</i> < 0.05; Dunnett’s test).</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

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

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

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