TRPA1-induced repulsion behavior in the red flour beetle Tribolium castaneum


Nociception refers to the sensory perception of a noxious stimulus that may cause pain and/or harm, resulting in behavioral patterns of escape and repulsion.1. Several types of stimuli trigger nociceptive sensory transduction, for example heat, mechanical stimulation and chemicals.

Several types of membrane proteins, receptors and ion channels participate in the detection of stimuli. Transient receptor potential (TRP) channels are evolutionarily conserved cation channels between vertebrates and invertebrates2. TRP channels function as primary sensors for various types of information, including light, sound, temperature, taste, and chemical and physical stimuli. The TRP family is divided into seven subfamilies, namely TRPC, TRPA, TRPV, TRPN, TRPM, TRPP and TRML, which are based on the constituent sequence elements3.

The TRPA channel is largely conserved and contains several ankyrin repeats in the N-terminal region4. TRPA1 is activated by nociceptive thermal and irritant chemicals, such as harmful odorants and tastes. In mammals, TRPA1 is expressed in a subset of nociceptive neurons, and TRPA1 functions as a chemical nocisensor for many chemicals, such as pungent natural compounds, allyl isothiocyanate (AITC), menthol, and cinnamaldehyde.5.6. The TRPA chord family contains only one member, namely TRPA1, while Drosophila melanogaster contains four genes of the TRPA subfamily: TRPA1 who is the deal TRPA1 counterpart, water witch, Pyrexand painless. An additional gene from the TRPA subfamily, TRPA5is present in the beetle model insect, the red flour beetle Tribolium castaneum genome, and although Hymenoptera genomes lack TRPA1they contain TRPA5 and specific Hymenoptera APTR (HsTRPA) genesseven. In the other subgroups, the TRPM channel was initially named protein melastatin, because it was identified from melanocytes8.9; in addition, the mammalian TRPM8 channel has been reported to be activated by a chemical stimulus, namely menthol and its analoguesten. Although the TRPM subfamily consists of eight channels (TRPM1–8), only one TRPM channel gene is present in insectsseven. Interestingly, some natural ligands have been shown to activate multiple TRP channels; for example, menthol activates TRPA1 and TRPM8 in mammals11.12.

Plant-derived essential oils (EOs) are mixtures of volatile compounds used as insecticides, repellents and egg-laying deterrents because they contain environmentally sustainable ingredients produced by secondary metabolism.13.14. Among the essential oils, citronella oil, extracted from Cymbopogon species, is a potentially potent insect repellent against several species of mosquitoes, woodlice and beetles15,16,17,18. Among the chemical components, a monoterpene citronellal compound, 3,7-dimethyl-6-octenal, is one of the main compounds responsible for the insecticidal activity and the repellent behavior of T. castaneum16.19.

Previous studies have been conducted on the molecular recognition mechanism of citronellal activity, in which the insect TRPA1 is required to repel citronellal in two species of Diptera, D. melanogaster and Anopheles gambiae mosquito. Of note, citronellal activated both TRPA1s when heterologously expressed in Xenopus oocytes20.21.

Alternatively, host plant-derived odor perception is carried out by peripheral odorant receptors (ORs) that are expressed on the dendrites of olfactory sensory neurons (OSNs)22.23. Insect ORs form heteromeric cation channels with the obligate odorant receptor co-receptor (Orco), which functions not only as a measure of odor sensitivity, but also as a chaperone for OR localization and maintenance.24. Therefore, Orco knockdown and/or null mutations result in critical loss of olfactory function25.

In Drosophilatwo pathways are involved in the citronella response20. A channel is mediated by OR reception, because Orco the mutation resulted in impaired repellent activity; the other pathway is the G-protein-coupled phospholipase C signaling cascade, in which the TRPA1 channel functions downstream, and the TRPA1 isoform, namely TRPA1(A), caused a citronellal-enhanced taste aversion21.26. Additionally, the TRPA1 isoform, namely TRPA1s, in mosquito species such as A. gambiae and Culex pipiensis directly activated by citronellal20.27. Several TRPA1 isoforms have been described in other insect species, to date there are no reports of TRPA1 isoforms in T. castaneum.

With respect to TRPA1-mediated chemoreception, it has been reported that menthol, extracted from Mint species, evokes harmful rolling behavior Drosophila larvae, and that TRPA1 and TRPM interact genetically and are necessary for the manifestation of aversive behavior28. In vitro expression analysis showed that Drosophila TRPA1 was not activated by menthol6highlighting the ongoing debate over whether TRPA1 functions in menthol perception.

However, the repellency mechanism of citronellal against beetles has not yet been elucidated. Additionally, it remains unclear whether menthol stimulates the TRPA1-mediated repulsive behavior of T. castaneumalthough we have previously pointed out that I-menthol evokes TcTRPM-mediated contact repulsion behavior in T. castaneum29. The objective of the present study was to examine the repulsion behavior of T. castaneum to citronellal using RNAi-mediated inactivation of TcTRPA1; this aimed to determine whether TcTRPA1 participates in mediating the repulsion response. We further examined OR-mediated repellency behavior for citronellal and clarified the engagement of TcTRPA1 for menthol-derived repellant behavior in combination with TcTRPM.


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