Identification of Chemosensory Genes Based on the Antennal Transcriptomic Analysis of Plagiodera versicolora

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Abstract

Insects can sense surrounding chemical signals by their accurate chemosensory systems. This system plays a vital role in the life history of insects. Several gene families participate in chemosensory processes, including odorant receptors (ORs), ionotropic receptors (IRs), gustatory receptors (GRs), chemosensory proteins (CSPs), odorant binding proteins (OBPs), and sensory neuron membrane proteins (SNMPs). Plagiodera versicolora (Coleoptera: Chrysomelidae), is a leaf-eating forest pest found in salicaceous trees worldwide. In this study, a transcriptome analysis of male and female adult antennae in P. versicolora individuals was conducted, which identified a total of 98 candidate chemosensory genes including 40 ORs, 7 IRs, 13 GRs, 10 CSPs, 24 OBPs, and 4 SNMPs. Subsequently, the tissue expression profiles of 15 P. versicolora OBPs (PverOBPs) and 39 ORs (PverORs) were conducted by quantitative real-time PCR. The data showed that almost all PverOBPs and PverORs were highly expressed in the male and female antennae. In addition, several OBPs and ORs (PverOBP10, PverOBP12, PverOBP18, PverOR24, and PverOR35) had higher expression levels in female antennae than those in the male antennae, indicating that these genes may be taking part in some female-specific behaviors, such as find mates, oviposition site, etc. This study deeply promotes further understanding of the chemosensory system and functional studies of the chemoreception genes in P. versicolora.

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Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

Kenneth Livak, Thomas D. Schmittgen (2001)

The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).

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Insect olfactory receptors are heteromeric ligand-gated ion channels.

Koji Sato, Maurizio Pellegrino, Takao Nakagawa … (2008)

In insects, each olfactory sensory neuron expresses between one and three ligand-binding members of the olfactory receptor (OR) gene family, along with the highly conserved and broadly expressed Or83b co-receptor. The functional insect OR consists of a heteromeric complex of unknown stoichiometry but comprising at least one variable odorant-binding subunit and one constant Or83b family subunit. Insect ORs lack homology to G-protein-coupled chemosensory receptors in vertebrates and possess a distinct seven-transmembrane topology with the amino terminus located intracellularly. Here we provide evidence that heteromeric insect ORs comprise a new class of ligand-activated non-selective cation channels. Heterologous cells expressing silkmoth, fruitfly or mosquito heteromeric OR complexes showed extracellular Ca2+ influx and cation-non-selective ion conductance on stimulation with odorant. Odour-evoked OR currents are independent of known G-protein-coupled second messenger pathways. The fast response kinetics and OR-subunit-dependent K+ ion selectivity of the insect OR complex support the hypothesis that the complex between OR and Or83b itself confers channel activity. Direct evidence for odorant-gated channels was obtained by outside-out patch-clamp recording of Xenopus oocyte and HEK293T cell membranes expressing insect OR complexes. The ligand-gated ion channel formed by an insect OR complex seems to be the basis for a unique strategy that insects have acquired to respond to the olfactory environment.

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Drosophila odorant receptors are both ligand-gated and cyclic-nucleotide-activated cation channels.

Dieter Wicher, Ronny Schäfer, René Bauernfeind … (2008)

From worm to man, many odorant signals are perceived by the binding of volatile ligands to odorant receptors that belong to the G-protein-coupled receptor (GPCR) family. They couple to heterotrimeric G-proteins, most of which induce cAMP production. This second messenger then activates cyclic-nucleotide-gated ion channels to depolarize the olfactory receptor neuron, thus providing a signal for further neuronal processing. Recent findings, however, have challenged this concept of odorant signal transduction in insects, because their odorant receptors, which lack any sequence similarity to other GPCRs, are composed of conventional odorant receptors (for example, Or22a), dimerized with a ubiquitously expressed chaperone protein, such as Or83b in Drosophila. Or83b has a structure akin to GPCRs, but has an inverted orientation in the plasma membrane. However, G proteins are expressed in insect olfactory receptor neurons, and olfactory perception is modified by mutations affecting the cAMP transduction pathway. Here we show that application of odorants to mammalian cells co-expressing Or22a and Or83b results in non-selective cation currents activated by means of an ionotropic and a metabotropic pathway, and a subsequent increase in the intracellular Ca(2+) concentration. Expression of Or83b alone leads to functional ion channels not directly responding to odorants, but being directly activated by intracellular cAMP or cGMP. Insect odorant receptors thus form ligand-gated channels as well as complexes of odorant-sensing units and cyclic-nucleotide-activated non-selective cation channels. Thereby, they provide rapid and transient as well as sensitive and prolonged odorant signalling.