Untangling a Gordian knot: the role of a GluCl3 I321T mutation in abamectin resistance in Tetranychus urticae

Abstract

BACKGROUND The cys‐loop ligand‐gated ion channels, including the glutamate‐gated chloride channel (GluCl) and GABA‐gated chloride channel (Rdl) are important targets for drugs and pesticides. The macrocyclic lactone abamectin primarily targets GluCl and is commonly used to control the spider mite Tetranychus urticae, an economically important crop pest. However, abamectin resistance has been reported for multiple T. urticae populations worldwide, and in several cases was associated with the mutations G314D in GluCl1 and G326E in GluCl3. Recently, an additional I321T mutation in GluCl3 was identified in several abamectin resistant T. urticae field populations. Here, we aim to functionally validate this mutation and determine its phenotypic strength. RESULTS The GluCl3 I321T mutation was introgressed into a T. urticae susceptible background by marker‐assisted backcrossing, revealing contrasting results in phenotypic strength, ranging from almost none to 50‐fold. Next, we used CRISPR‐Cas9 to introduce I321T, G314D and G326E in the orthologous Drosophila GluCl. 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 Xenopus oocytes revealed that the I321T mutation might reduce GluCl3 sensitivity to abamectin, but also suggested that all three T. urticae Rdls are affected by abamectin. CONCLUSION 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. © 2020 Society of Chemical Industry

Multiple TaqMan qPCR and droplet digital PCR (ddPCR) diagnostics for pesticide resistance monitoring and management, in the major agricultural pest Tetranychus urticae

“…The absence of H92R mutation does not support the contribution of this mechanism to the phenotype, pointing towards the participation of other mechanisms (e.g., metabolic resistance) 6 . Interestingly, although the specific population bears the I321T mutation that has been previously associated with moderate resistance levels to abamectin, 21 the current toxicity assays show susceptibility at the RD. Thus, Tu206 should, in fact, be considered low resistant, and not be treated with abamectin, as this could allow resistance to spread.…”

“…Target‐site mutations associated with different acaricidal/insecticidal subgroups that belong to different modes of action (MoA) in T. urticae have been discovered in genes encoding acetylcholinesterase ( Ace‐1 : G119S, F331W), associated with resistance to acetylcholinesterase inhibitors [organophosphates (OPs) and carbamates] (MoA: AChE inhibitors) 18 ; voltage‐gated sodium channel ( VGSC : L1024V, F1538I), associated with resistance to pyrethroids (MoA: Sodium channel modulators) 15,20 ; glutamate‐gated chloride channels ( GluCl1 : G314D, GluCl3 : G326E, I321T), associated with resistance to macrocyclic lactones (such as abamectin, MoA: GluCl allosteric modulators) 16,17,21,22 ; cytochrome b (cytb : among other mutations: G126S in combination with S141F in the cd1 helix, P262T in the ef helix), associated with bifenazate resistance (MoA: mitochondrial electron transport inhibitors, METI‐III) 23,24 ; chitin synthase 1 ( CHS1 : I1017F), which has been associated with resistance to mite growth inhibitors (MGIs) affecting CHS1 (MoA: MGIs, such as etoxazole), 19,25 and PSST subunit (H92R) associated with resistance to mitochondrial electron transport inhibitors (MoA: METI‐I, such as pyridaben) 14,26 …”

CRISPR/Cas9 modified An. gambiae carrying kdr mutation L1014F functionally validate its contribution in insecticide resistance and combined effect with metabolic enzymes

“…gambiae [ 22 ]. The use of CRISPR/Cas9 to study the effect of target site mutations in insecticide resistance has been predominantly performed using the model organism Drosophila melanogaster [ 16 , 23 – 25 ] and in one agricultural pest [ 26 ], thus our study reports, to the best of our knowledge, the first time this approach being used in a major insect vector of human disease pathogens. Several target-site resistance mutations have been described in these insects whose effect size needs to be clarified, but which could be assessed following the approach described here; these include mutations in acetylcholinesterase, the target of organophosphate and carbamate insecticides, the GABA-gated chloride channel that is associated with dieldrin resistance [ 27 ] and the chitin synthase that is associated with resistance to diflubenzuron and other benzoylurea insecticides inhibiting this enzyme [ 25 , 28 ].…”

Frequencies and mechanisms of pesticide resistance in Tetranychus urticae field populations in China

“…Target‐site mutations in resistance genes have been determined to be the main resistance mechanism (Ilias et al ., 2014). For example, T. urticae resistance to the widely used pesticide abamectin involves mutations in the abamectin target, the glutamate‐gated chloride channel gene (GluCl) (Kwon et al ., 2010a; Dermauw et al ., 2012; Xue et al ., 2021). Similarly, resistance of the mite to pyrethroids and OPs results from mutations in target sites of the genes that regulate the voltage‐gated sodium channel (VGSC) and that encode acetylcholinesterase (Ace), respectively (Kwon et al ., 2009; Tsagkarakou et al ., 2009; Khajehali et al ., 2010).…”