Fig 1.
Aminoacid sequence alignment of Apime-ASTA-R and its Drosophila and human homologs.
Sequence alignments of the predicted allatostatin A receptor in the honey bee Apis mellifera (Apime-ASTA-R, GenBank: XP_006560262.1) and of its homologs in the fruit fly Drosophila melanogaster (DAR1, GenBank: NP_726877.2 and DAR2, GenBank: NP_524544.1) and human (GALR1, NP_001471.2). The amino acid position is indicated on the left. Conserved residues are in red, and conservative changes in blue. Vertical bars (|) indicate locations of putative introns; grey bars indicate putative trans-membrane regions (TM1–TM7). Amino acids that are characteristic of class A GPCRs are indicated by *, open diamonds (◊) indicate putative N-linked glycolysation sites, # indicate cysteine residues for disulfide bridge (between TM2—TM3 and TM4 –TM5) or palmitoylation (intracellular domain), and dots (●) indicate conserved putative phosphorylation sites for PKA/C. Sequences are based on transcripts (cDNA).
Fig 2.
Aminoacid sequence alignment of Apime-ASTC-R and its Drosophila and human homologs.
Sequence alignments of the predicted allatostatin C receptor in the honey bee Apis mellifera (Apime-ASTC-R, GenBank: XP_006560939.1) and of its homologs in the fruitfly Drosophila melanogaster (Drostar1, GenBank: NP_649040.2 and Drostar2, GenBank: NP_649039.4), and human (SSTR2, NP_001041.1). The amino acid position is indicated on the left. Conserved residues are in red, and conservative changes in blue. Grey bars indicate putative trans-membrane regions (TM1–TM7). Amino acids that are characteristic of class A GPCRs are indicated by *, open diamonds (◊) indicate putative N-linked glycolysation sites, # indicate cysteine residues for disulfide bridge (between TM2—TM3 and TM4 –TM5) or palmitoylation (intracellular domain), and dots (●) indicate conserved putative phosphorylation sites for PKA/C. Sequences are based on transcripts (cDNA).
Fig 3.
Phylogenetic tree of A. mellifera and D. melanogaster allatostatin receptors with respect to their closest mouse homologs.
Drosophila gene names are indicated. For M. musculus receptors, abbreviations are: GALR (Galanin receptor), KISS1R (kisspeptin receptor), OPR (opioid receptor), MCHR (melanin concentrating hormone receptor), NPBWR (Neuropeptide B and W receptor), SSTR (somatostatin receptor). The bottom bar indicates the phylogenetic distance scale. The bootstrap values are displayed in red.
Fig 4.
Functional characterization of Apime-ASTA and Apime-ASTC receptors in vitro.
HEK293 cells were transiently transfected with Apime-ASTA or Apime-ASTC receptors and a chimeric G protein allowing artificial coupling to PLC (see materials and methods). Receptor activation following stimulation by different concentrations of peptides was determined by monitoring intracellular calcium levels (indicated by relative fluorescence units, RFU). Concentration-response curves of Apime-ASTA on Apime-ASTA-R (A), Apime-ASTC on Apime-ASTC-R (B), and Apime-ASTCC on Apime-ASTC-R (C). Numbers below the X axis indicate the molar concentration (M). (D) Apime-ASTA is a specific ligand of Apime-ASTA-R; Apime-ASTC and Apime-ASTCC are specific to Apime-ASTC-R (means +/- SD from one representative experiment performed in triplicate). Base: calcium levels without peptide. Curves were obtained by fitting the data using nonlinear regression analysis in the GraphPad Prism software.
Fig 5.
Apime-ASTA-R and Apime-ASTC-R display constitutive inhibitory activity, down-regulating cAMP.
HEK cells transfected with Apime-ASTA-R or Apime-ASTC-R show lower levels of bioluminescence than mock-transfected cells (A). Bars represents mean+/-SD values of 8 transfections in duplicates (mock) or triplicates (Apime-AST-R). Forskolin analog NKH-477 has a reduced ability to increase bioluminescence levels in Apime-ASTA-R and Apime-ASTC-R transfected cells than in mock-transfected cells (B). Bars represent mean +/- SD values of 2 transfections in duplicates or triplicates. Application of increasing doses of Apime-ASTA on Apime-ASTA-R transfected cells induced a dose-dependent increase in bioluminescence (C). Application of increasing doses of Apime-ASTC on Apime-ASTC-R transfected cells did not change bioluminescence levels (D). Application of increasing doses of Apime-ASTCC on Apime-ASTC-R transfected cells increased bioluminescence levels at high concentrations (E). HEK cells transfected with luciferase reporter gene and an empty vector (mock-transfected) show a dose-dependent response to Apime-ASTCC (F). Bars represent mean +/- SD values of 3 transfections in triplicates.
Fig 6.
Apime-AstA-R and Apime-AstC-R mRNAs are expressed in the brain.
In situ hybridizations were performed with antisense probes specific for Apime-AstA-R (B, C, D) or Apime-AstC-R (F, G, H) on sections from pollen forager brains. Sense probes revealed no specific staining (A, E). Specific hybridization was detected in the optic lobe, and particularly between the lamina (la), medulla (me) and lobula (lo), as well as in the antennal lobe (D, H) and mushroom bodies (C, G). Scale bars: 200 mm (A-B, E-F) and 100 mm (C-D, G-H). Arrows indicate stained cell bodies.
Fig 7.
Binding of radiolabelled Apime-ASTA in vivo and in vitro.
125I-Apime-ASTA binding to bee brain crude membranes was displaced by increasing concentrations of non-labeled Apime-ASTA (A). Competitive binding with Apime-ASTA and increasing concentrations of a somatostatin homolog (SOM230) (B). Bars represent mean+/-SD (n = 3). Curves were obtained by fitting the data using nonlinear regression analysis in the GraphPad Prism software.
Fig 8.
Gene expression of allatostatins.
PCR was performed on cDNA samples obtained from different brain regions of adult worker bees: optic lobes (yellow in the bee brain illustration), ventral area (including antennal lobes in blue) and dorsal area (including mushroom bodies in red) of the central brain. (A) Electrophoresis on agarose gel of PCR fragments of Actin (used as a positive control 162 nt), Apime-AstA (120 bp), Apime-AstC (121 bp) and Apime-AstCC (105 bp, see S1 Table for primer sequences, original agarose gels are presented in S1 Fig). (B) qPCR was performed on dissected dorsal brain region from pollen foragers (n = 9, each sample run in triplicate) for the three allatostatin genes. Expression levels are normalized and relative to the geometric mean of Am18S and elf1α.
Fig 9.
Dose-dependent learning impairment induced by allatostatins.
Percentages of forager bees displaying conditioned proboscis extension responses (PER) in the third and last trial of conditioning, in honeybees injected with either vehicle alone (control bees, CT) or a given concentration of allatostatin A (A), C (B), or CC (C). For easier comparisons, learning performances of control bees are set at 100% and those of all other groups are relative to CT. N = 120–182 (A), N = 70–80 (B) N = 56–87 (C). Significant decreases of learning performance, as compared to those of saline-injected bees, are indicated (*: p<0.05, **:p<0.01, ***:p<0.001).
Fig 10.
Intact response to sucrose and odors after allatostatin treatment.
Responses elicited by stimulation of the antennae with increasing sucrose concentrations were tested following injection of saline (controls, CT) or either allatostatin (A). Overall, bees responded more to sucrose (solid line) than water (dashed line, GLM: 4.11±0.58, p<0.001), and the proportion of bees responding to sucrose increased with the concentration (GLM: 0.82±0.14, p<0.001) similarly in all groups, while the proportion of bees responding to water remained constant (GLM: -0.11±0.12, p = 0.34) (CT: N = 31, ASTA: N = 35 ASTC: N = 49, ASTCC: N = 29). Following olfactory appetitive conditioning, bees were tested for their capacity to generalize their learned responses to other odorants, as a way to assess their olfactory capacities (B). Allatostatin treatment had no impact on the response to any of the odors tested (CT: N = 73, ASTA: N = 66, ASTC: N = 75, ASTCC: N = 72). All bees were collected as they were leaving the hive, as for learning experiments.