The Red Imported Fire Ant (<i>Solenopsis invicta</i> Buren) Kept Y not F: Predicted sNPY Endogenous Ligands Deorphanize the Short NPF (sNPF) Receptor

<div><p>Neuropeptides and their receptors play vital roles in controlling the physiology and behavior of animals. Short neuropeptide F (sNPF) signaling regulates several physiological processes in insects such as feeding, locomotion, circadian rhythm and reproduction, among others. Previously, the red imported fire ant (<i>Solenopsis invicta</i>) sNPF receptor (<i>S. invicta</i> sNPFR), a G protein-coupled receptor, was immunolocalized in queen and worker brain and queen ovaries. Differential distribution patterns of <i>S. invicta</i> sNPFR protein in fire ant worker brain were associated both with worker subcastes and with presence or absence of brood in the colony. However, the cognate ligand for this sNPFR has not been characterized and attempts to deorphanize the receptor with sNPF peptides from other insect species which ended in the canonical sequence LRLRFamide, failed. Receptor deorphanization is an important step to understand the neuropeptide receptor downstream signaling cascade. We cloned the full length cDNA of the putative <i>S. invicta</i> sNPF prepropeptide and identified the putative “sNPF” ligand within its sequence. The peptide ends with an amidated Tyr residue whereas in other insect species sNPFs have an amidated Phe or Trp residue at the C-terminus. We stably expressed the HA-tagged <i>S. invicta</i> sNPFR in CHO-K1 cells. Two <i>S. invicta</i> sNPFs differing at their N-terminus were synthesized that equally activated the sNPFR, SLRSALAAGHLRYa (EC₅₀ = 3.2 nM) and SALAAGHLRYa (EC₅₀ = 8.6 nM). Both peptides decreased the intracellular cAMP concentration, indicating signaling through the G_αi-subunit. The receptor was not activated by sNPF peptides from other insect species, honey bee long NPF (NPY) or mammalian PYY. Further, a synthesized peptide otherwise identical to the fire ant sequence but in which the C-terminal amidated amino acid residue ‘Y’ was switched to ‘F’, failed to activate the sNPFR. This discovery will now allow us to investigate the function of sNPY and its cognate receptor in fire ant biology.</p></div

Amino acid sequences of the ligands tested on the <i>Si</i>sNPFR-C6E8 cell line and <i>S. invicta</i> peptides EC₅₀ values.

<p><i>S. invicta (Solenopsis invicta)</i>, Analogous sNP(F) (Analog of <i>S. invicta</i> sNPF1), <i>Drome (Drosophila melanogaster)</i>, <i>Apime (Apis mellifera)</i>.</p><p>*Sequence identical to <i>Apime</i> sNPF.</p><p>All other peptides tested were not active at 1 µM.</p><p>Amino acid sequences of the ligands tested on the <i>Si</i>sNPFR-C6E8 cell line and <i>S. invicta</i> peptides EC₅₀ values.</p

Detection of the fire ant sNPF (Partial sequence: ALAAGH) by MALDI-TOF MS.

<p>The Y-axis represents % intensity (as % of the highest charge detected) and the X-axis represents mass. The agarose electrophoresis analysis shows the amplified expected size band (377 bp) of the fire ant sNPF from the fire ant larvae cDNA. The peptide sequence analysis performed with the PeptideCutter tool at ExPASy Bioinformatics Resource Portal revealed several potential protease cleavage sites, such as those for proteinase K (thin arrows), thermolysin (thick arrows), and Arg-C proteinase, Clostripain, and trypsin (black arrow head). The detected fragment is predicted after thermolysis digestion.</p

Nucleotide and deduced amino acid sequence of <i>Solenopsis invicta</i> short neuropeptide F (GenBank accession number KJ812404).

<p>Arrowhead indicates the predicted post-translational cleavage site for processing the prepropeptide to active peptide(s). The predicted signal peptide is shaded in gray and boxed, the predicted active peptide sequence is in bold and the polyadenylation signal sequences in the 3′UTR are boxed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109590#pone.0109590-Liu1" target="_blank">[49]</a>. Poly (A) tail of the shorter cDNA begins after nucleotide ‘C’ shaded black.</p

Detection of <i>Solenopsis invicta</i> short neuropeptide F receptor (<i>Solin</i> sNPFR) in stably transformed CHO-K1 cell line <i>Si</i>sNPFR-C6E8.

<p>The HA-tagged <i>Solin</i> sNPFR was detected by fluorescence immunocytochemistry using an anti-HA-tag antibody. The cell line names are shown on the left panel, and the antibody used labels the top of the first column. For each row, the images on the center show the nuclear staining with DAPI (blue) of the same cells on the left, and those on the right are merged images of the two previous. The HA-tag (red signal) is detected in <i>Solin</i> sNPFR cells (A–C) but not in the vector-only transformed cells (D–F). Rhimi-CAP_2b-R cells (G–I) showing red signal were used as positive controls for the HA-tag labeling. No red signal was detected in <i>Solin</i> sNPFR cells incubated with pre-immune rabbit serum (J–L). An anti-α-tubulin antibody was used as positive control for the labeling of a cytoplasmic structural protein (M–O); the red pattern is different than for the HA-tag (contrast A and G to M). Scale bar, 50 µm.</p

sNPF peptides inhibition of forskolin-stimulated cAMP accumulation in CHO-K1 cells stably transformed with the <i>S. invicta</i> sNPF receptor (<i>Si</i>sNPFR-C6E8).

<p><b>A.</b> Independent dosage-response curves of <i>S. invicta</i> sNPF1 [SLRSALAAGHLRYa (13 a.a. residues); dashed line] and sNPF2 [SALAAGHLRYa (ten a.a. residues); solid line]. Cells were treated with a phosphodiesterase inhibitor previous to the simultaneous application of peptide and forskolin (10 µM), which was applied to elicit maximal cAMP production. The Y-axis represents the cAMP production as a percentage of the forskolin-stimulated maximum cAMP level, considered 100%. The X-axis shows the log molar (Log (M)) concentrations of peptides applied. Values indicate means ± S.E. of three independent biological replicates Half maximal effective concentration values (EC₅₀ values) are given on the graph. <b>B.</b> In the fire ant sNPF1 sequence, the change of the C-terminal amidated residue ‘Y’ to ‘F’ [sNP(F)] eliminates the ligand activity on the receptor. <i>S. invicta</i> sNPF1 (1 µM) decreased cAMP as expected (*, significantly different at <i>p</i>≤0.05 level). Data were analyzed by ANOVA followed by Tukey’s multiple comparison test. Each value is the average of three biological replicates with the error bars showing standard deviation of the average for each treatment. The standard error of the analysis is 19.8.</p

Detection of <i>S. invicta</i> sNPF receptor in cell membranes prepared from the <i>Si</i>sNPFR-C6E8 cell line by western blot analysis.

<p>Numbers in the left indicate the marker’s protein mass (kDa). Lane 1, the membrane fraction of the fire ant mated queen ovary (Ov) as a positive control; lane 2, the membrane fraction of <i>Si</i>sNPFR-C6E8 (C6E8) and lane 3, the membrane fraction of the vector-only (VO) (pcDNA3.1(-)) transfected CHO-K1 cells probed with the specific anti-sNPF receptor anti-peptide antibody <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109590#pone.0109590-Lu1" target="_blank">[44]</a>. Membrane protein (50 µg) was loaded in each lane. The antibody detects the 55 kDa receptor protein in lanes 1 and 2, as expected.</p

Alignment of insect sNPF sequences.

<p><b>A.</b> The unique amino acid sequence of the <i>S. invicta</i> short neuropeptide F active peptide(s) is compared to those of other insect species. Aligned sNPF sequences are from (top to bottom): three ant species <i>Solin</i> (<i>Solenopsis invicta</i> KJ812404), <i>Acrec</i> (<i>Acromyrmex echinatior</i> EGI59536.1), and <i>Camfl</i> (<i>Camponotus floridanus</i> EFN66516.1); the honey bee, <i>Apime</i> (<i>Apis mellifera</i> XP_003250155.1); a basal ant, <i>Harsa</i> (<i>Harpegnathos saltator</i> EFN85447.1); the locust, <i>Schgr</i> (<i>Schistocerca gregaria</i> AHH85823.1); the flour beetle, <i>Trica</i> (<i>Tribolium castaneum</i> EEZ97763.1); dipterans, <i>Anoga</i> (<i>Anopheles gambiae</i> ABD96048.1), <i>Aedae</i> (<i>Aedes aegypti</i> ABE72968.1), and <i>Drome</i> (<i>Drosophila melanogaster</i> AAN11060.1); the brown planthopper, <i>Nillu</i> (<i>Nilaparvata lugens</i> BAO00976.1), and three lepidopterans, <i>Helar</i> (<i>Helicoverpa armigera</i> AGH25568.1), <i>Danpl</i> (<i>Danaus plexippus</i> EHJ63336.1), and <i>Bommo</i> (<i>Bombyx mori</i> NP_001127729.1). Identical amino acid residues are shaded black. In the alignment, active peptides corresponding to the location of <i>S. invicta</i> sNPFs are underlined with a solid line, and additional orthologous peptides encoded in cDNAs from other species are underlined with dashed lines. Arrowheads indicate the predicted post-translational cleavage sites for processing the prepropeptide to active peptide(s). <b>B.</b> Alignment of partial sNPF nucleotide sequences encoding the active peptide in four ant species. Identical nucleotides are shaded black. Amino acid residues for the active sNPF peptide (shaded gray) are in the middle of the respective codon; for <i>S. invicta</i> are at the top and for the other three ant species, at the bottom. The arrowhead indicates a transversion from C to G in the codon first position in <i>S. invicta</i> resulting in Ala (A), and not Pro (P) as in other ants, and the insertion of six additional nucleotides also extends the peptide length. There is a conservative replacement of R for H in <i>S. invicta</i> and F (TTT or TTC) is replaced with Y (TAC). The sequence for C-terminal α-amidation of Y or F is X-Gly-basic residue (R), and Gly provides the nitrogen for the same <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109590#pone.0109590-Eipper1" target="_blank">[70]</a>.</p

Phylogenic tree of insect short neuropeptide F prepropeptide sequences from different insect species.

<p>Amino acid sequences were analyzed by MEGA (version 5.05); the phylogenetic tree was constructed using the neighbor-joining method. The number 86 indicates the bootstrap confidence level. Abbreviations on branches correspond to the genus and species name, and these are identical to those described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109590#pone-0109590-g002" target="_blank">Figure 2</a>, with the addition of the Southern house mosquito, <i>Culqu</i> (<i>Culex quinquefasciatus</i> EDS32332.1) sNPF and the predicted sNPF from the ant species <i>Attce (Atta cephalotes), Linhu (Linepithema humile),</i> and <i>Pogba (Pogonomyrmex barbatus)</i>. The honey bee, <i>Apime</i> NPY (NP_001161192.1) was also included in the analysis.</p