Electrical signals are transmitted from cell to cell by means of transmitting substances that are released from one cell and activate special receptors on the next cell. A very important neurotransmitter is acetylcholine and the receptors are called acetylcholine receptors. There are several variants of these, the variant that transmits signals between nerve cells or between nerve - and muscle cells is called nicotinic acetylcholine receptors because they are also activated by nicotine. This receptor is composed of five different proteins. One of the most widely used groups of insecticides in the world, the neonicotinoids, activate these receptors in the same way as nicotine, but with a longer duration of effect. This group of insecticides are of significant concern because they can pose adverse effects on beneficial insects, e.g. bees. They are directly toxic, but can in lower doses have other undesirable effects, such as impacting their ability to navigate. It has been difficult to study these effects at the receptor level because no one has previously been able to express a complete receptor from insects and other arthropods in an experimental model system. However, our group recently managed this from an important parasite on fish, salmon lice, by cloning the necessary genes into frog eggs, and we could then make accurate measurements of the effect neonicotinoids have on this receptor from salmon lice. It was necessary to simultaneously express three auxiliary proteins for the receptor to function. In this project, we will build on these results. Although this type of receptor has significant similarities between different arthropods, they are not identical. Therefore, the receptor must be expressed for each species to be studied. The goal is to do this for two insects (bees and mosquitoes), as well as spider mites and ticks. In the project, the exact spatial location of the five separate subunits that make up the receptor will also be determined.
Nicotinic acetylcholine receptors (nAChRs) mediate chemical neurotransmission in the nervous system and at neuromuscular junctions in vertebrates. The natural transmitter is acetylcholine, but they are also the direct targets for neonicotinoids, some of the worlds largest selling insecticides. These chemicals are widely used against ectoparasites, arthropod vectors and agricultural pests. There are significant concerns regarding adverse effects of neonicotinoids on beneficial insects, and some neonicotinoids are now banned for out-door use in many countries. In-depth studies on the function of these receptors in arthropods are surprisingly scarce. Until 2020, no research groups had managed to express functional nAChRs in an ex-vivo system, using genes coding for both a- and ß-subunits from the target arthropod species. Co-expression of nAChR a-subunits from insects and ß-subunits from vertebrates (mainly chicken) had been the only feasible way of obtaining functional receptors with both subunit types in Xenopus laevis oocytes. In 2020, two groups independently overcame this hurdle, one being the applicant here. We managed to express two nAChRs from the marine arthropod Lepeophtheirus salmonis into Xenopus laevis oocytes using genes from this species only. In this project, these results will be used to attempt cloning of nAChRs from other arthropods like bees, mites, ticks, and mosquitos and test their sensitivity towards natural transmitters, antiparasitic agents, pesticides and other compounds. Thus, the present project aims to lead to a greatly improved way of obtaining nAChRs from arthropods, with a switch from arthropod-vertebrate chimeric nAChRs to arthropod-specific native nAChRs. These nAChRs will further serve as tools for fundamental research on the function of the arthropod nervous system and serve as elegant models to determine the effect of various existing and novel compounds on these receptors from parasites, pests, and non-target organisms.