Genomics, transcriptomics, and peptidomics of neuropeptides and protein hormones in the red flour beetle Tribolium castaneum

Abstract

Neuropeptides and protein hormones are ancient molecules that mediate cell-to-cell communication. The whole genome sequence from the red flour beetle Tribolium castaneum, along with those from other insect species, provides an opportunity to study the evolution of the genes encoding neuropeptide and protein hormones. We identified 41 of these genes in the Tribolium genome by using a combination of bioinformatic and peptidomic approaches. These genes encode >80 mature neuropeptides and protein hormones, 49 peptides of which were experimentally identified by peptidomics of the central nervous system and other neuroendocrine organs. Twenty-three genes have orthologs in Drosophila melanogaster: Sixteen genes in five different groups are likely the result of recent gene expansions during beetle evolution. These five groups contain peptides related to antidiuretic factor-b (ADF-b), CRF-like diuretic hormone (DH37 and DH47 of Tribolium), adipokinetic hormone (AKH), eclosion hormone, and insulin-like peptide. In addition, we found a gene encoding an arginine-vasopressin-like (AVPL) peptide and one for its receptor. Both genes occur only in Tribolium and not in other holometabolous insects with a sequenced genome. The presence of many additional osmoregulatory peptides in Tribolium agrees well with its ability to live in very dry surroundings. In contrast to these extra genes, there are at least nine neuropeptide genes missing in Tribolium, including the genes encoding the prepropeptides for corazonin, kinin, and allatostatin-A. The cognate receptor genes for these three peptides also appear to be absent in the Tribolium genome. Our analysis of Tribolium indicates that, during insect evolution, genes for neuropeptides and protein hormones are often duplicated or lost.

Figure 1.

Representative MALDI-TOF mass spectrum of a single anterodorsal ganglionic sheath preparation of abdominal ganglion-2 (neurohemal release site of CAPA-peptides) of an adult Tribolium. The direct mass spectrometric screening of nervous tissues may provide reproducible semiquantitative data. Note the different abundances of the expressed capa peptides. CAPA-PVK-1, which is present with a number of truncated/extended forms, has a lower signal intensity than CAPA-PVK-2; CAPA-pyrokinin is not detectable at all. Lower panel shows the annotated sequence with thick gray lines for the peptides determined in the mass spectrometric analysis as shown in the upper panel. Underlines are for putative mature peptides containing C-terminal -PRX motifs, and the letters with gray backgrounds are canonical signals for amidations and/or basic cleavages. Putative signal peptide is boxed.

Figure 2.

Structure of typical preproinsulin-like peptides (A) and sequence alignment of insulin-like peptide from Tribolium castaneum, Apis mellifera, Anopheles gambiae, Drosophila melanogaster, and one Bombyx mori Bombyxin (BBXA1, P33718) (B–D). Conserved cysteines are marked by “C” on the top of putative A- and B-chains (double lines, =) with canonical dibasic cleavage sites as vertical bars (|) and unknown cleavage sites as question marks (?). Tribolium and Apis sequences are from their genome database, Drosophila genes are from FlyBase, and Anopheles sequences are from Riehle et al. (2002).

Figure 3.

Sequence alignment of insect neuroparsin and related sequences. The sequences are from Schistocerca gregaria neuroparsin precursor 1 (AC38869), Locusta migratoria neuroparsin A precursor (P10776), Apis mellifera queen brain-selective protein-1 (Q1T786), Aedes aegypti ovarian ecdysteroidogenic hormone I (AAD00823), and Mus musculus insulin-like growth factor binding protein 5 protease (Mm IGFBP). Strictly conserved cysteines are marked with asterisks (*) on the top of the sequence alignment.

Figure 4.

Sequence alignment for the arginine-vasopressin (AVP) and oxytocin prepropeptide family. Sequences in the box are for mature peptides with canonical amidation and dibasic sequences (#). Strictly conserved 16 cysteines are marked with asterisks (*) on the top of the sequence alignment. GenBank accession nos. for the sequences are AAB86629, AAL92860, BAA01736, BAD12146, ABN79655, BAA36458, BAC82436, BAC82435, and Q00945.

Figure 5.

Structure of the genomic region containing five clustered genes for Tribolium castaneum ADF-b1 to -5 (GLEAN_09955–09959; upper panel) and sequence alignment of the five ADF-b with Tenebrio molitor ADF-b (P83109) and cuticle protein TmPCP9.2 (Baernholdt and Anderson 1998). The putative mature peptides located at the carboxy termini are indicated by a box. The conserved intron position is marked on the top of the sequence alignment. Note that all five ADF-b genes are predicted to have small introns at the homologous position in the signal peptides with phase 0.

Figure 6.

Sequence alignment and phylogenetic relationships of insect corticotropin releasing factor (CRF)-like diuretic hormones (DH). The hypothetical tree implies possible evolutionary relationships among the DHs from different species of insects. Sequences are from Hyles_DH30 (P82015), Manduca_DH30 (P24858), Tenebrio_DH37 (P56618), Tribolium_DH37 (GLEAN_02243), Manduca_DH41 (AAB59200.1), Hyles_DH41 (P82014), Tenebrio_DH47 (P56619), Tribolium_DH47, Schistocerca_DH44 (P67801), Musca_DH44 (P67800), Drosophila_DH44 (NP_649922.2), Zootermopsis_DH46 (P82707), Periplaneta_DH46 (P41538), Diploptera_DH46 (P82373), Locusta_DH46 (AAB19827.1), Apis_DH43 (CAI45289.1), and Acheta_DH46 (P23834). The three Bombyx DHs were from the GenBank genome database. Amino acid numbers are not indicated because many have only mature peptide sequences in the GenBank.

Figure 7.

Sequence alignment of adipokinetic hormones (AKH). The mature peptides are in the box and followed by the glycine required for amidation and dibasic cleavage sites. Conserved cysteines are marked by asterisks (*) on the top of the alignment. Note that the middle of the sequence is omitted. Sequences are from Anopheles_AKH1 (ABD43194.1), Aedes_AKH1(EAT35885.1), Locusta_AKH3 (P19872), Periplaneta_AKH (AAV41425.1), Blaberus_AKH (Q17128), Drosophila_AKH (NP_523918.1), Manduca_AKH (P67788), Apis_AKH (from Hummon et al. 2006), Locusta_AKH2 (P08379), Locusta_AKH1 (P55319), Anopheles_AKH2 (ABD60145.1), Aedes_AKH2 (EAT37004.1), and Locusta_AKH4 (Siegert 1999).

Figure 8.

Sequence alignment of eclosion hormones (EH). The six conserved cysteines are marked by asterisks (*) on the top of the alignment. Sequences are from Manduca_EH (P11919), Helicoverpa_EH (AAV69026.1), Ostrinia_EH (ABG66962.1), Bombyx_EH (P25331), Apis_EH (XP_001122120.1), Aedes_EH (EAT41662.1), Romalea_EH (AAD28480.1), and Drosophila_EH (CAA51050.1).