Characterisation and tissue distribution of the PISCF allatostatin receptor in the red flour beetle, Tribolium castaneum

Abstract

The insect PISCF/allatostatins (ASTs) are pleiotropic peptides that are involved in the regulation of juvenile hormone biosynthesis, are myoinhibitory on the gut and the heart, and suppress feeding in various insects, but their roles in beetles are poorly understood. To provide further insight into the significance of PISCF/ASTs in beetles, the PISCF/AST receptor from Tribolium castaneum has been characterised and its tissue distribution determined. The biological activity of the T. castaneum PISCF/AST (Trica-AS) was also investigated. The Trica-AS receptor shows high sequence homology to other insect PISCF/AST receptors, which are related to the mammalian somatostatin/opioid receptors, a family of G protein-coupled receptors. The Trica-AS receptor was activated in a dose-dependent manner by both Trica-AS and T. castaneum allatostatin double C (Trica-ASTCC) as well as Manduca sexta-allatostatin (Manse-AS). Other allatoregulatory peptides (a FLG/AST, a MIP/AST and an allatotropin) and somatostatin(14) were inactive on this receptor. Receptor transcript levels in tissues, determined by qRT-PCR, were highest in the head and the gut, with variable amounts in the fat body and reproductive organs. There were measurable differences in receptor levels of the head, fat body and reproductive organs between males and females. There was also a widespread distribution of Trica-AS in various tissues of T. castaneum. The Trica-AS peptide precursor was most abundant in the head and there was a significant difference between levels in the heads and reproductive organs of males and females. Whole mount immunocytochemistry localised Trica-AS in the median and lateral neurosecretory cells of the brain, in the corpus cardiacum and throughout the ventral nerve cord. The peptide was also present in midgut neurosecretory cells, but no immunostaining was detected in the reproductive organs or Malpighian tubules. The widespread distribution of both Trica-AS and its receptor suggest this peptide may have multiple roles in beetles. However, Trica-AS had no effect on the spontaneous contractions of the gut or ovaries of T. castaneum but this peptide did stimulate the release of proteases from the anterior midgut of another beetle, Tenebrio molitor. The activation of the Trica-AS receptor by Trica-ASTCC implies a physiological role for this peptide in beetles, which remains to be identified.

Genomic and peptidomic analyses of the neuropeptides from the emerging pest, Drosophila suzukii

“…Furthermore, a peptide with homology to PISCF/AST, first identified by Veenstra [51] and called allatostatin CC (ASTCC), has also been identified. Audsley et al [6] showed that the Tribolium castaneum ASTCC activated the PISCF/AST receptor in this insect suggesting that these two peptides may have similar functions, but its role in fruit flies has not been determined.…”

“…Myosuppressin (TDVDHVFLRFamide) was also identified by its monoisotopic mass (1247.6) and the monoisotopic mass 1430.7 is in agreement with pyrokinin (PK) 1 [2][3][4][5][6][7][8][9][10][11][12][13][14][15] (GPSASSGLWFGPRLamide). Additional mass ions present did not correspond to the masses of any predicted peptide shown in Table 1, and remain unassigned.…”

“…All three peptides were detected in HPLC fractions of D. suzukii brain extracts ( Figure 2) and their sequences were confirmed by fragmentation of their parent ions. In addition, a mass ion corresponding to PK1 [2][3][4][5][6][7][8][9][10][11][12][13][14][15] was also detected and sequenced (Table 1). …”

More than two decades of research on insect neuropeptide GPCRs: an overview

“…In Diptera two AST-C receptors have been characterized for Drosophila using Xenopus oocytes (Kreienkamp et al, 2002) and for Aedes using HEK cells (Mayoral et al, 2010). Only one AST-C receptor was found to be present in Bombyx (Yamanaka et al, 2008) and in Tribolium (Audsley et al, 2012). Activation of the Bombyx AST-C receptor elicits an increase in intracellular cAMP levels (Yamanaka et al, 2008), while the Tribolium receptor was deorphanized in HEK cells, inducing a Ca 2+ response (Audsley et al, 2012).…”

Functional activity of allatotropin and allatostatin in the pupal stage of a holometablous insect, Tribolium castaneum (Coleoptera, Tenebrionidae)

“…Audsley et al [8] have recently characterized the AS type C receptor in T. castaneum to provide further insight into the pleiotropic role of the peptide. Characterization and quantification of the T. castaneum AT-like receptor showed a prominent sexual dimorphism with significantly higher transcript levels in the male fat body and reproductive system [42].…”