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The macrocyclic lactone abamectin primarily targets GluCl and is commonly used to control the spider mite <jats:italic>Tetranychus urticae<\/jats:italic>, an economically important crop pest. However, abamectin resistance has been reported for multiple <jats:italic>T. urticae<\/jats:italic> populations worldwide, and in several cases was associated with the mutations G314D in <jats:italic>GluCl1<\/jats:italic> and G326E in <jats:italic>GluCl3<\/jats:italic>. Recently, an additional I321T mutation in <jats:italic>GluCl3<\/jats:italic> was identified in several abamectin resistant <jats:italic>T. urticae<\/jats:italic> field populations. Here, we aim to functionally validate this mutation and determine its phenotypic strength.<\/jats:p><\/jats:sec><jats:sec><jats:title>RESULTS<\/jats:title><jats:p>The <jats:italic>GluCl3<\/jats:italic> I321T mutation was introgressed into a <jats:italic>T. urticae<\/jats:italic> susceptible background by marker\u2010assisted backcrossing, revealing contrasting results in phenotypic strength, ranging from almost none to 50\u2010fold. Next, we used CRISPR\u2010Cas9 to introduce I321T, G314D and G326E in the orthologous <jats:italic>Drosophila GluCl<\/jats:italic>. Genome modified flies expressing GluCl I321T were threefold less susceptible to abamectin, while CRISPRed GluCl G314D and G326E flies were lethal. Last, functional analysis in <jats:italic>Xenopus<\/jats:italic> oocytes revealed that the I321T mutation might reduce GluCl3 sensitivity to abamectin, but also suggested that all three <jats:italic>T. urticae<\/jats:italic> Rdls are affected by abamectin.<\/jats:p><\/jats:sec><jats:sec><jats:title>CONCLUSION<\/jats:title><jats:p>Three different techniques were used to characterize the role of I321T in GluCl3 in abamectin resistance and, combining all results, our analysis suggests that the I321T mutation has a complex role in abamectin resistance. Given the reported subtle effect, additional synergistic factors in resistance warrant more investigation. \u00a9 2020 Society of Chemical Industry<\/jats:p><\/jats:sec>","DOI":"10.1002\/ps.6215","type":"journal-article","created":{"date-parts":[[2020,12,7]],"date-time":"2020-12-07T09:10:04Z","timestamp":1607332204000},"page":"1581-1593","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":53,"title":["Untangling a <scp>G<\/scp>ordian knot: the role of a <scp>GluCl3 I321T<\/scp> mutation in abamectin resistance in <i>Tetranychus urticae<\/i>"],"prefix":"10.1002","volume":"77","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4842-6516","authenticated-orcid":false,"given":"Wenxin","family":"Xue","sequence":"first","affiliation":[{"name":"Department of Plants and Crops, Faculty of Bioscience Engineering Ghent University  Ghent 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