Citation
- Authors: Finetti L. et al.
- Year: 2021
- Journal: J Exp Biol 224 jeb238816
- Applications: in vitro / DNA / jetOPTIMUS
- Cell type: HEK-293
Description: Human embryonic kidney Fibroblast
Known as: HEK293, 293
Method
Transient expression of HhTAR1 in HEK 293 cells
HEK 293 cells were grown at 37°C and 5% CO2 in DMEM, high glucose supplemented with 10% FBS. To prevent bacterial contamination, penicillin (100 U ml−1) and streptomycin (0.1 mg ml−1) were added to the medium.
The cells were transiently transfected with pcDNA 3.1 (+)/HhTAR1 in T75 cell culture flasks using JetOPTIMUS (Polyplus-Transfection, New York, NY, USA), following the manufacturer's protocol.
Cells were incubated in the transfection medium for 24 h under normal cell growth conditions before their use in the calcium mobilization assay.
Abstract
In insects, tyramine receptor 1 (TAR1) has been shown to control several physiological functions, including olfaction. We investigated the molecular and functional profile of the Halyomorpha halys type 1 tyramine receptor gene (HhTAR1) and its role in olfactory functions of this pest. Molecular and pharmacological analyses confirmed that the HhTAR1 gene codes for a true TAR1. RT-qPCR analysis revealed that HhTAR1 is expressed mostly in adult brain and antennae as well as in early development stages (eggs, 1st and 2nd instar nymphs). In particular, among the antennomeres that compose a typical H. halys antenna, HhTAR1 was more expressed in flagellomeres. Scanning electron microscopy investigation revealed the type and distribution of sensilla on adult H. halys antennae: both flagellomeres appear rich in trichoid and grooved sensilla, known to be associated with olfactory functions. Through an RNAi approach, topically delivered HhTAR1 dsRNA induced a 50% downregulation in gene expression after 24 h in H. halys 2nd instar nymphs. An innovative behavioural assay revealed that HhTAR1 RNAi-silenced 2nd instar nymphs were less susceptible to the alarm pheromone component (E)-2 decenal as compared with controls. These results provide critical information concerning the role of TAR1 in olfaction regulation, especially alarm pheromone reception, in H. halys. Furthermore, considering the emerging role of TAR1 as target of biopesticides, this work opens the way for further investigation on innovative methods for controlling H. halys.