Characterization and Expression Profiling of Neuropeptides and G-Protein-Coupled Receptors (GPCRs) for Neuropeptides in the Asian Citrus Psyllid, Diaphorina citri (Hemiptera: Psyllidae)

Abstract

Neuropeptides are endogenous active substances that widely exist in multicellular biological nerve tissue and participate in the function of the nervous system, and most of them act on neuropeptide receptors. In insects, neuropeptides and their receptors play important roles in controlling a multitude of physiological processes. In this project, we sequenced the transcriptome from twelve tissues of the Asian citrus psyllid, Diaphorina citri Kuwayama. A total of 40 candidate neuropeptide genes and 42 neuropeptide receptor genes were identified. Among the neuropeptide receptor genes, 35 of them belong to the A-family (or rhodopsin-like), four of them belong to the B-family (or secretin-like), and three of them are leucine-rich repeat-containing G-protein-coupled receptors. The expression profile of the 82 genes across developmental stages was determined by qRT-PCR. Our study provides the first investigation on the genes of neuropeptides and their receptors in D. citri, which may play key roles in regulating the physiology and behaviors of D. citri.

Keywords: Diaphorina citri; G-protein-coupled receptors; expression profiling; neuropeptides; phylogenetic tree; transcriptome.

Figure 1

Gene Ontology (GO) classifications of unigenes. The y-axis represents the number of the unigenes, the x-axis shows three categories and their subcategories.

Figure 2

Phylogenetic tree of the A-family neuropeptide GPCRs. The neighbor-joining trees were constructed using MEGA 6 software with 1000-fold bootstrap repetitions. The A-family neuropeptide GPCRs of D. citri are shown in red text. Dc, D. citri; Dm, D. melanogaster; Nl, N. lugens.

Figure 3

Phylogenetic tree of the B-family neuropeptide GPCRs. The neighbor-joining trees were constructed using MEGA 6 software with 1000-fold bootstrap repetitions. The B-family neuropeptide GPCRs of D. citri are shown in red text. Dc, D. citri; Dm, D. melanogaster; Nl, N. lugens; Bm, B. mori; Tc, T. castaneum.

Figure 4

Phylogenetic tree of leucine-rich repeat-containing GPCRs (LGRs). The neighbor-joining trees were constructed using MEGA 6 software with 1000-fold bootstrap repetitions. The LGRs of D. citri are shown in red text. Dc, D. citri; Dm, D. melanogaster; Nl, N. lugens; Bm, B. mori; Tc, T. castaneum. Ap, Acyrthosiphon pisum; Am, A. mellifera; tetur, Tetranychus urticae.

Figure 5

Expression profiles of neuropeptides in various tissues of D. citri based on FPKM values (fragments per kilobase per million reads). The mRNA levels, as represented by log2 (FPKM + 1) values, are shown in a heat map with colors ranging from blue (low expression) to red (high expression). MA, male antenna; MH, male head; MT, male thorax; ML, male leg; MAB, male abdomen; MAT, male abdomen terminal; FA, female antenna; FH, female head; FT, female thorax; FL, female leg; FAB, female abdomen; FAT, female abdomen terminal.

Figure 6

Expression profiles of neuropeptide receptors in various tissues of D. citri based on FPKM values. The mRNA levels, as represented by log2 (FPKM + 1) values, are shown in the heat map with colors ranging from blue (low expression) to red (high expression). MA, male antenna; MH, male head; MT, male thorax; ML, male leg; MAB, male abdomen; MAT, male abdomen terminal; FA, female antenna; FH, female head; FT, female thorax; FL, female leg; FAB, female abdomen; FAT, female abdomen terminal.

Figure 7

qRT-PCR results of neuropeptides throughout the D. citri life cycle. The y-axis represents the relative expression level and the x-axis the life cycle. The standard error is represented by the error bar and significant differences are represented by the different letters (p < 0.05). E, egg; N1-2, 1st- and 2nd-instar nymphs; N3, 3rd instar nymphs; N4, 4th instar nymphs; N5, 5th instar nymphs; 1D-A, the adults of one day after eclosion; 5D-A, five days after eclosion.

Figure 8

The gene expression level of neuropeptide receptors throughout the D. citri life cycle. The y-axis represents the relative expression level and the x-axis the life cycle. The standard error is represented by the error bar and significant differences are represented by the different letters (p < 0.05). E, egg; N1-2, 1st- and 2nd-instar nymphs; N3, 3rd instar nymphs; N4, 4th instar nymphs; N5, 5th instar nymphs; 1D-A, the adults of one day after eclosion; 5D-A, five days after eclosion.