Identification and expression profiles of neuropeptides and their G protein-coupled receptors in the rice stem borer Chilo suppressalis

Abstract

In insects, neuropeptides play important roles in the regulation of multiple physiological processes by binding to their corresponding receptors, which are primarily G protein-coupled receptors (GPCRs). The genes encoding neuropeptides and their associated GPCRs in the rice stem borer Chilo suppressalis were identified by a transcriptomic analysis and were used to identify potential targets for the disruption of physiological processes and the protection of crops. Forty-three candidate genes were found to encode the neuropeptide precursors for all known insect neuropeptides except for arginine-vasopressin-like peptide (AVLP), CNMamide, neuropeptide-like precursors 2-4 (NPLP2-4), and proctolin. In addition, novel alternative splicing variants of three neuropeptide genes (allatostatin CC, CCHamide 1, and short neuropeptide F) are reported for the first time, and 51 putative neuropeptide GPCRs were identified. Phylogenetic analyses demonstrated that 44 of these GPCRs belong to the A-family (or rhodopsin-like), 5 belong to the B-family (or secretin-like), and 2 are leucine-rich repeat-containing GPCRs. These GPCRs and their likely ligands were also described. qRT-PCR analyses revealed the expression profiles of the neuropeptide precursors and GPCR genes in various tissues of C. suppressalis. Our study provides fundamental information that may further our understanding of neuropeptidergic signaling systems in Lepidoptera and aid in the design of peptidomimetics, pseudopeptides or small molecules capable of disrupting the physiological processes regulated by these signaling molecules and their receptors.

Figure 1

Protein alignment of the SIFamide (A) and IMFamide (B) precursor sequences from C. suppressalis (Cs), B. mori (Bm), N. lugens (Nl), T. castaneum (Tc), A. mellifera (Am), D. melanogaster (Dm), H. armigera (Ha), and D. plexippus (Dp). Identities are highlighted in dark red, and similarities are indicated by gray. The dashed boxes indicate the signal peptides, the solid red boxes indicate the mature peptides, and the solid blue boxes indicate the conserved residues between SIFamide and IMFamide. The red asterisks mark the conserved cysteine residues, and the red arrow indicates the one substitution at the N-terminal of SIFamide.

Figure 2. Phylogenetic tree of the A-family neuropeptide GPCRs from C. suppressalis (Cs), B. mori (Bm), and D. melanogaster (Dm).

Neighbor-joining trees were constructed using MEGA 5 software with 1000-fold bootstrap re-sampling. The numbers at the nodes of the branches represent the level of bootstrap support for each branch.

Figure 3. Phylogenetic tree of the B-family neuropeptide GPCRs from C. suppressalis (Cs), B. mori (Bm), N. lugens (Nl), and D. melanogaster (Dm).

Neighbor-joining trees were constructed using MEGA 5 software with 1000-fold bootstrap re-sampling. The numbers at the nodes of the branches represent the level of bootstrap support for each branch.

Figure 4. Phylogenetic tree of the leucine-rich repeat-containing GPCRs from C. suppressalis (Cs), B. mori (Bm), N. lugens (Nl), D. melanogaster (Dm), T. castaneum (Tc), A. mellifera (Am), A. pisum (Ap), Pediculus humanus corporis (Pc), Lymnaea stagnalis (Ls), and Tetranychus urticae (tetur).

Neighbor-joining trees were constructed using MEGA 5 software with 1000-fold bootstrap re-sampling. The numbers at the nodes of the branches represent the level of bootstrap support for each branch.

Figure 5. qRT-PCR results showing the relative expression levels of the neuropeptides in various tissues of C. suppressalis.

Standard errors are represented by the error bars, and significant differences are represented by the different letters above each bar (p < 0.05).

Figure 6. qRT-PCR results showing the relative expression levels of G protein-coupled receptors for the neuropeptides in various tissues of C. suppressalis.

Standard errors are represented by the error bars, and significant differences are represented by the different letters above each bar (p < 0.05). Neuropeptide receptor A1-A35 are abbreviated as A1-A35, respectively; Neuropeptide receptor B1-B4 are abbreviated as B1-B4, respectively.