Cell-free expression, purification and characterization of Drosophila melanogaster odorant receptor OR42a and its co-receptor.

Olfactory receptors (OR), a group of classic membrane proteins, plays a vital role in insect reproduction and acclimatization. Deciphering the molecular mechanism of insect olfaction could enhance pest control and environmental protection. Studies on ORs have faced a major bottleneck due to the notoriously strong hydrophobicity of ORs, which results in difficult expression in heterologous cell systems. Here, we demonstrated that insect ORs could be functionally produced using the E. coli cell-free protein synthesis system (CFPS), in which the highest yield of total ORs is 350 µg per 1 ml reaction. We tested the effects of detergent types and concentrations on soluble expression of ORs. The ORs showed a classic α-helical infrared spectrum. Quartz crystal microbalance (QCM) was used to demonstrate that ORs fold correctly and respond to their ligands. This is the first report that insect OR42a could be functionally produced in vitro. This approach may facilitate the development of biomimetic olfactory biosensors and may also be utilized for drug positioning and development, environmental protection and agriculture.

Variant Ionotropic Receptors are Expressed in the Antennae of Anopheles sinensis (Diptera: Culicidae).

Mosquitoes transmit many harmful diseases that seriously threaten public health. The mosquito's olfactory system is of great significance for host selection. Inotropic receptors (IRs) and olfactory receptors (ORs) have been demonstrated to be capable of odorant molecular recognition. Analyzing the molecular principles of mosquito olfaction facilitates the development of prevention and therapy techniques. Advances in the understanding of IRs have been seriously inadequate compared to those of ORs. Here, we provide evidence that 35 Anopheles sinensis IR (AsIR) genes are expressed, 7 of which are in the antennae and 2 have expression levels that are upregulated with a blood meal. A homologous analysis of the sequences showed that AsIRs are a subfamily of ionotropic glutamate receptors (iGLURs). This is the first that time IRs have been identified in Anopheles sinensis in vitro. The ultrastructure of the antennae supports the theory that diverse sensilla are distributed in the antennae. The results here may facilitate the revelation of the regulation mechanism in AsIRs, which could mitigate the transmission of diseases by mosquitoes.

The antenna transcriptome changes in mosquito Anopheles sinensis, pre- and post- blood meal.

Antenna is the main chemosensory organ in mosquitoes. Characterization of the transcriptional changes after blood meal, especially those related to chemoreception, may help to explain mosquito blood sucking behavior and to identify novel targets for mosquito control. Anopheles sinensis is an Asiatic mosquito species which transmits malaria and lymphatic filariasis. However, studies on chemosensory biology in female An. sinensis are quite lacking. Here we report a transcriptome analysis of An. sinensis female antennae pre- and post- blood meal. We created six An. sinensis antenna RNA-seq libraries, three from females without blood meal and three from females five hours after a blood meal. Illumina sequencing was conducted to analyze the transcriptome differences between the two groups. In total, the sequenced fragments created 21,643 genes, 1,828 of them were novel. 12,861 of these genes were considered to be expressed (FPKM >1.0) in at least one of the two groups, with 12,159 genes expressed in both groups. 548 genes were differentially expressed in the blood-fed group, with 331 genes up-regulated and 217 genes down-regulated. GO enrichment analysis of the differentially expressed genes suggested that there were no statistically over represented GO terms among down-regulated genes in blood-fed mosquitoes, while the enriched GO terms of the up-regulated genes occurred mainly in metabolic process. For the chemosensory gene families, a subtle distinction in the expression levels can be observed according to our statistical analysis. However, the firstly comprehensive identification of these chemosensory gene families in An. sinensis antennae will help to characterize the precise function of these proteins in odor recognition in mosquitoes. This study provides a first global view in the changes of transcript accumulation elicited by blood meal in An. sinensis female antennae.

Olfactory Ionotropic Receptors in Mosquito Aedes albopictus (Diptera: Culicidae).

Ionotropic glutamate receptors (iGluRs) are a conserved family of ligand-gated ion channels that primarily function to mediate neuronal communication at synapses. A variant subfamily of iGluRs, the ionotropic receptors (IRs), was recently identified in insects and proved with the function in odorant recognition. Ionotropic receptors participate in a distinct olfactory signaling pathway that is independent of olfactory receptors activity. In the present study, we identify 102 putative IR genes, dubbed as AalbIr genes, in mosquito Aedes albopictus (Skuse) by in silico comparative sequence analysis. Among AalbIr genes, 19 show expression in the female antenna by RT-PCR. These putative olfactory AalbIRs share four conservative hydrophobic domains of amino acids, similar to the transmembrane and ion channel pore regions found in conventional iGluRs. To determine the potential function of these olfactory AalbIRs in host-seeking, we compared their transcript expression levels in the antennae of blood-fed females with that of non-blood-fed females by quantitative real-time RT-PCR. Three AalbIr genes showed downregulation when the mosquito finished a bloodmeal. These results may help to improve our understanding of the IR-mediated olfactory signaling in mosquitoes.