De novo assembly and characterization of central nervous system transcriptome reveals neurotransmitter signaling systems in the rice striped stem borer, Chilo suppressalis

Abstract

Background: Neurotransmitter signaling systems play crucial roles in multiple physiological and behavioral processes in insects. Genome wide analyses of de novo transcriptome sequencing and gene specific expression profiling provide rich resources for studying neurotransmitter signaling pathways. The rice striped stem borer, Chilo suppressalis is a destructive rice pest in China and other Asian countries. The characterization of genes involved in neurotransmitter biosynthesis and transport could identify potential targets for disruption of the neurochemical communication and for crop protection.

Results: Here we report de novo sequencing of the C. suppressalis central nervous system transcriptome, identification and expression profiles of genes putatively involved in neurotransmitter biosynthesis, packaging, and recycling/degradation. A total of 54,411 unigenes were obtained from the transcriptome analysis. Among these unigenes, we have identified 32 unigenes (31 are full length genes), which encode 21 enzymes and 11 transporters putatively associated with biogenic aminergic signaling, acetylcholinergic signaling, glutamatergic signaling and GABAergic signaling. RT-PCR and qRT-PCR results indicated that 12 enzymes were highly expressed in the central nervous system and all the transporters were expressed at significantly high levels in the central nervous system. In addition, the transcript abundances of enzymes and transporters in the central nervous system were validated by qRT-PCR. The high expression levels of these genes suggest their important roles in the central nervous system.

Conclusions: Our study identified genes potentially involved in neurotransmitter biosynthesis and transport in C. suppressalis and these genes could serve as targets to interfere with neurotransmitter production. This study presents an opportunity for the development of specific and environmentally safe insecticides for pest control.

Fig. 1

Length distribution of transcripts and unigenes in the C. suppressalis central nervous system transcriptome assembly

Fig. 2

The number of unigenes annotated in public database searched

Fig. 3

Characteristics of similarity search of unigenes against Nr database. (a) E-value distribution of BLAST hits for each unigene with a cut off E-value of 1.0E-5. (b) Similarity distribution of the top BLAST hits for each unigene. (c) Species distribution of the top BLAST hits for each unigene in Nr database

Fig. 4

Distribution of unigenes among the KEGG pathways. (a) The top ten KEGG pathways with highest numbers of unigenes in C. suppressalis central nervous system. (b) The KEGG pathways in the pathway ‘Nervous system’. Abbreviation for pathways: Purine metabolism (PuM), Ribosome (Rib), Protein processing in endoplasmic reticulum (PPER), Spliceosome (Spl), Pyrimidine metabolism (PyM), RNA transport (RNAT), Cell cycle (CC), MAPK signaling pathway (MAPK), Endocytosis (Endo), Oxidative phosphorylation (OXP); Dopaminergic synapse (DopS), Glutamatergic synapse (GluS), Neurotrophin signaling pathway (NSP), Cholinergic synapse (ChS), Synaptic vesicle cycle (SVC), Long-term potentiation(LtP), Retrograde endocannabinoid signaling (RES), GABAergic synapse (GABAS), Serotonergic synapse (SerS), Long-term depression (LtD)

Fig. 5

The phylogenetic analysis of enzymes involved in the biosynthesis pathway of biogenic amines in insects. Included are C. suppressalis (Cs), B. mori (Bm), T. castaneum (Tc), D. melanogaster (Dm), and Apis mellifera (Am). The accession numbers of the squences are available in Additional file 11. 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

Fig. 6

The phylogenetic analysis of enzymes involved in the biosynthesis pathway of acetylcholine, glutamate, and GABA in insects. Included are C. suppressalis (Cs), B. mori (Bm), T. castaneum (Tc), and D. melanogaster (Dm). The accession numbers of the squences are available in Additional file 11. 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

Fig. 7

The phylogenetic analysis of transporters involved in neurotransmitter signaling systems in insects. Included are C. suppressalis (Cs), T. ni (Tn), B. mori (Bm), T. castaneum (Tc), C. floridanus (Cf), and D. melanogaster (Dm). The accession numbers of the squences are available in Additional file 11. 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

Fig. 8

qRT-PCR results showing the relative transcript abundance of the unigenes encoding enzymes and transporters involved in the neurotransmitter signaling systems in the central nervous system of C. suppressalis

Fig. 9

The biosynthesis pathway of neurotransmitters in insects. a The biosynthesis pathway of dopamine. b The biosynthesis pathway of tyramine and octopamine. c The biosynthesis pathway of serotonin. (d) The biosynthesis pathway of histamine. e The biosynthesis pathway of acetylcholine. f The biosynthesis pathway of glutamate. g The biosynthesis pathway of GABA

Fig. 10

RT-PCR results showing the relative expression of the C. suppressalis neurotransmitter-related genes in various tissues. a The relative expression of the enzymes involved in the biosynthesis pathway of biogenic amines in various tissues; b The relative expression of the enzymes involved in the biosynthesis pathway of acetylcholine, glutamate, and GABA in various tissues; c The relative expression of the transporters involved in the neurotransmitter signaling systems in various tissues. EF-1 was used as internal reference gene to test the integrity of each cDNA templates; the similar intensity of EF-1 bands between various tissues indicate the use of equal template concentrations. CNS, central nervous system; FB, fat body; Gut, gut; HC, hemocytes

Fig. 11

qRT-PCR results showing the relative expression levels of the enzymes involved in the biosynthesis pathway of biogenic amines in various tissues in C. suppressalis. The standard error is represented by the error bar, and the different letters above each bar denote significant differences (p < 0.05)

Fig. 12

qRT-PCR results showing the relative expression levels of the enzymes involved in the biosynthesis pathway of acetylcholine, glutamate, and GABA in various tissues in C. suppressalis. The standard error is represented by the error bar, and the different letters above each bar denote significant differences (p < 0.05)

Fig. 13

qRT-PCR results showing the relative expression levels of the transporters involved in the neurotransmitter signaling systems in various tissues in C. suppressalis. The standard error is represented by the error bar, and the different letters above each bar denote significant differences (p < 0.05)

Fig. 14

Putative neurotransmitter signaling pathways in insects. ACh, acetylcholine; Ch, choline; ChT, choline transporter; VAChT, vesicular acetylcholine transporter; nAChR, nicotinic acetylcholine receptor; mAChR, muscarinic acetylcholine receptor; DA, dopamine; OA, octopamine; TA, tyramine; 5HT, serotonin; HA, histamine; MA, monoamine; MAT, monoamine transporter; VMAT, vesicular monoamine transporter; DAR, dopamine receptor; OAR, octopamine receptor; TAR, tyramine receptor; 5HTR, serotonin receptor; HAR, histamine receptor; Glu, glutamate; Gln, glutamine; EAAT, excitatory amino acid transporter; VGluT, vesicular glutamate transporter; GluR, glutamate receptor; GABA, γ-aminobutyric acid; GAT, GABA transporter; VGAT, vesicular GABA transporter; GABAR, GABA receptor; AC, adenylyl cyclase; DAG, diacylglycerol; IP3, 1,4,5-trisphosphate; PKA, protein kinase A; PKC: protein kinase C; PLC, phospholipase C; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate