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. 2015 Mar 12:9:84.
doi: 10.3389/fnins.2015.00084. eCollection 2015.

Molecular cloning and characterization of the allatotropin precursor and receptor in the desert locust, Schistocerca gregaria

Els Lismont  1 , Rut Vleugels  1 , Elisabeth Marchal  2 , Liesbeth Badisco  1 , Pieter Van Wielendaele  1 , Cynthia Lenaerts  1 , Sven Zels  1 , Stephen S Tobe  3 , Jozef Vanden Broeck  1 , Heleen Verlinden  1
Affiliations

Molecular cloning and characterization of the allatotropin precursor and receptor in the desert locust, Schistocerca gregaria

Els Lismont et al. Front Neurosci. .

Abstract

Allatotropins (ATs) are pleiotropic neuropeptides initially isolated from the tobacco hornworm, Manduca sexta. In 2008, the first receptor for AT-like peptides (ATR) was characterized in Bombyx mori. Since then, ATRs have also been characterized in M. sexta, Tribolium castaneum, Aedes aegypti and Bombus terrestris. These receptors show sequence similarity to vertebrate orexin (ORX) receptors. When generating an EST-database of the desert locust (Schistocerca gregaria) central nervous system, we found cDNA sequences encoding the Schgr-AT precursor and a fragment of its putative receptor. This receptor cDNA has now been completed and functionally expressed in mammalian cell lines. Activation of this receptor, designated as Schgr-ATR, by Schgr-AT caused an increase in intracellular calcium ions, as well as cyclic AMP (cAMP), with an EC50 value in the nanomolar range. In addition, the transcript distribution of both the Schgr-AT precursor and Schgr-ATR was investigated by means of quantitative real-time PCR. Moreover, we found more evidence for the myotropic and allatostimulatory actions of Schgr-AT in the desert locust. These data are discussed and situated in a broader context by comparison with literature data on AT and ATR in insects.

Keywords: GPCR; insect; juvenile hormone; motility; neuropeptide; orexin; peptide.

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Figures

Figure 1
Figure 1
Amino acid sequence of the Schgr-ATR (GenBank acc. no. JN543509) and homologous receptors from Manduca sexta (GenBank acc. no. ADX66344) and Tribolium castaneum (GenBank acc. no. XP_973738). The amino acid position is indicated at the right. Identical residues between the aligned sequences are highlighted in black, and conservatively substituted residues in gray. Dashes indicate gaps that were introduced to maximize homologies. Putative transmembrane regions (TM1-TM7) are indicated by gray bars. The position of the W (here changed to Y) (•) that is usually conserved in many rhodopsin-like GPCRs and the DRW motif (▲▲▲) are labeled.
Figure 2
Figure 2
Precursor sequence of Schgr-AT. The sequence of AT is highlighted in green. The predicted signal peptide sequence is shown in orange and the recognition sites for proteolytic processing of the proneuropeptide are shown in blue. The G-residue predicted to be transformed into the C-terminal amide is shown in yellow.
Figure 3
Figure 3
Neighbor-joining tree of insect ATR-like receptors in dendrogram display with representative branch length. Phylogenetic and molecular evolutionary analyses were conducted by using MEGA version 6. The FMRFamide-receptor of D. melanogaster (GenBank acc. no. AAF47700) was used as an outgroup to root the tree. Proteins marked with an asterisk were functionally characterized. Bootstrap-support values are based on 1000 replicates and are indicated on the nodes. The other GenBank accession numbers are: S. gregaria ATR (JN543509), M. sexta ATR (ADX66344), T. castaneum ATR (XP_973738), B. terrestris ATR (XP_003402490), A. mellifera ATR (XP_001120335), M. rotundata ATR (XP_003708421), N. vitripennis ATR (XP_008217710), R. prolixus ATR (AHE41431), A. aegypti ATR (AEN03789), B. mori neuropeptide A5 and A16 receptor (NP_001127740 and NP_001127714), and D. plexippus ATR (EHJ74388).
Figure 4
Figure 4
Dose-response curve for bioluminescence responses induced in (A) CHO-WTA11-Schgr-ATR cells, (B) CHO-PAM28-Schgr-ATR cells, (C) HEK293- Schgr -ATR cells. In all cell lines, the bioluminescence was measured in two independent transfections in triplicate and data are given in percentage (±S.D.) of the maximal response. The zero response level corresponds to treatment with BSA buffer only.
Figure 5
Figure 5
Graphic representation of the transcript levels of the precursor of Schgr-AT and the Schgr-ATR measured in sexually mature S. gregaria in two experiments. The data represent mean values ± S.E.M. of three independent tissue samples run in duplicate, normalized relative to Actin and GAPDH transcript levels. Abbreviations used: Br, brain; OL, optic lobes; CC, corpora cardiaca; CA, corpora allata; PG, prothoracic gland; SOG, suboesophageal ganglion; SG, salivary gland; TG1, prothoracic ganglion; TG2, mesothoracic ganglion; TG3, metathoracic ganglion; Gon, gonads; FB, fat body; Muscle, flight muscle; FG, foregut; MT, Malpighian tubules; MG, midgut; HG, hindgut; AG, male accessory glands, AbG, abdominal ganglia. The first three abdominal ganglia (1–3) are fused to the metathoracic ganglion (TG3). On the left the results of the first qRT-PCR study are depicted. The data of the gregarious and solitarious animals are represented together. On the right the results of the second qRT-PCR study are depicted.
Figure 6
Figure 6
Myotropic activity of Schgr-AT on the midgut of Schistocerca gregaria. Arrows indicate the administration of Schgr-AT. (A) and (B) represent two independent measurements on the same midgut.
Figure 7
Figure 7
The effect of Schgr-AT on the rate of JH biosynthesis in the CA as measured in an in vitro radio-chemical assay (RCA). The bars represent averages ± S.E.M. of 8 control and 9 Schgr-AT treated, individual CA dissected from vitellogenic adult females. Significant differences (p < 0.05) are indicated by an asterisk (*).
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