1. Ecology
Download icon

An octopamine receptor confers selective toxicity of amitraz on honeybees and Varroa mites

  1. Lei Guo
  2. Xin-yu Fan
  3. Xiaomu Qiao
  4. Craig Montell
  5. Jia Huang  Is a corresponding author
  1. Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Insect Sciences, Zhejiang University, China
  2. Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, United States
Research Article
Cite this article as: eLife 2021;10:e68268 doi: 10.7554/eLife.68268
6 figures, 1 video, 1 table and 1 additional file

Figures

Figure 1 with 2 supplements
Amitraz and its main metabolite DPMF can activate Varroa multiple octopamine (OA) receptors in vitro.

(A) Phylogenetic tree of OA receptors from Varroa destructor, Apis mellifera, Rhipicephalus microplus, and Drosophila melanogaster. The values on the branches represent the bootstrap support. The candidate Varroa receptors are in bold. (B) The Isoleucine40 in the TM1 of Octβ2R, which is associated with amitraz resistance in the cattle tick Rhipicephalus microplus, is highly conserved in Arachnida and Insecta. (C, D) Dose-response curves of amitraz (C) and DPMF (D) against the Varroa OA receptors. EC50 values were calculated using log(agonist) versus response nonlinear fit, mean ± SEM, n = 3 trials, three replicates per trial.

Figure 1—figure supplement 1
Dose-response curves of octopamine (OA) against the indicated OA receptors.

EC50 was calculated using log(agonist) versus response nonlinear fit, mean ± SEM, n = 3 trials, three replicates per trial.

Figure 1—figure supplement 2
Effects of octopamine (OA) and amitraz on the Rhipicephalus microplus Octβ2R expressed in HEK293 cells.

Mean ± SEM, n = 3 trials.

Figure 2 with 1 supplement
Amitraz affects Drosophila aggression through Octβ2R.

(A) Preparation of test flies. In brief, group-housed male flies (~10–15) were fed 1% DMSO plus 5% sucrose (control) or 1 mM amitraz plus 5% sucrose for 2 hr (see Materials and methods). (B) The 8-well aggression arena used in this behavioral test. (C, D) Effects of 1 mM amitraz on the number of lunges (C) and latency to initiate fighting (D) in different octopamine (OA) receptor mutants and control flies. p values, Mann–Whitney U tests were performed to analyze statistically significant differences between treatment with 1% DMSO versus 1 mM amitraz in the indicated genotypes, mean ± SEM, n = 12–34.

Figure 2—figure supplement 1
Adult survival of flies reared on diets containing 1% DMSO or a range of amitraz concentrations.

In brief, 10 wild-type female flies were allowed to feed on diets with 1% DMSO or a series of amitraz concentrations ranging from 1 mM to 5 mM. Data are shown as mean ± SEM. n = 3 biological replicates.

Amitraz affects Drosophila locomotion through Octβ2R.

(A–G) Effects of amitraz on midline crossing activity in flies of the indicated genotypes. One 5–7-day-old female fly was gently introduced into each tube, which contained 1% DMSO (control), 100 µM amitraz or 1 mM amitraz added to the agarose-sucrose medium (2% agarose and 5% sucrose) at one end. The other end was sealed with a cotton plug. The tubes were placed in a Drosophila Activity Monitor System (see Materials and methods). Black and white bars represent the night and day periods of the 12:12 LD cycle. Yellow boxes indicate the 2-day window of daily crossing activity test. p values, one-way ANOVA and post hoc Bonferroni correction, mean ± SEM, n = 16–32.

Figure 4 with 2 supplements
The percentage of paralysis behavior, in which four octopamine (OA) receptor-expressing neurons were thermally hyperactivated with UAS-trpA1.

The following transgenes were used: oamb-Gal4> UAS-trpA1; oct-tyrRGal4> UAS-trpA1; octα2R-Gal4> UAS-trpA1; octβ2R-Gal4> UAS-trpA1. n = 50–100.

Figure 4—video 1
Thermogenetic activation of four octopamine (OA) receptor-expressing neurons using UAS-trpA1 induces paralysis behavior (related to Figure 4).

The following transgenes were used: oamb-Gal4>UAS-trpA1; oct-tyrRG4>UAS-trpA1; octα2R-Gal4>UAS-trpA1; octβ2R-Gal4>UAS-trpA1. The movie was speeded up 2×.

Figure 4—video 2
Thermogenetic activation of octβ2R-Gal4 neurons using UAS-trpA1 induces paralysis behavior (related to Figure 4).

The following transgenes were used: octβ2R-Gal4>UAS-trpA1.

Figure 5 with 4 supplements
Triple amino acids differences determine amitraz sensitivity in Octβ2R in vitro.

(A, B) The predicted ligand-binding domain of amitraz in the Varroa (A) and the honeybee (B) Octβ2R. Amitraz and three amino acids mutated in this study are shown. (C) Superposition of the predicted ligand-binding domain of the honeybee (golden cartoon) and the Varroa (blue cartoon) Octβ2R structures. (D) Amino acid substitution in the ligand-binding domain (TM5-TM7) of Octβ2R in representative species from Arachnida and Hymenoptera. The predicted amino acids involved in the binding of amitraz are indicated in red. Three amino acids (E208, I335, I350) highlighted in green are conserved among species of bees. EL: extracellular loop; TM: transmembrane domain. (E, F) Dose-response curve of amitraz (E) and DPMF (F) against the indicated octopamine (OA) receptors. EC50 was calculated using log(agonist) versus response nonlinear fit, mean ± SEM, n = 3–4 trials, three replicates per trial.

Figure 5—figure supplement 1
Dose-response curves of octopamine (OA) against the indicated OA receptors.

EC50 values were calculated using log (agonist) versus response nonlinear fit, mean ± SEM, n = 3 trials, three replicates per trial.

Figure 5—figure supplement 2
Adult survival when reared on sucrose solution containing 5 mM amitraz and 1 mM detoxicative enzyme inhibitor.

Abbreviations: PBO: piperonyl butoxide; DEF: S,S,S-tributylphosphorotrithioate; DEM: diethyl maleate. One way ANOVA and post hoc Mann-Whitney U tests, mean ± SEM, n = 3-9 biological replicates. * p<0.05; ** p<0.01.

Figure 5—figure supplement 3
Dose-response curves of OA, amitraz, and DPMF against the fly (A-C) and honeybee (D-F) OA receptors.

EC50 values were calculated using log (agonist) versus response nonlinear fit, mean ± SEM, n = 1-3 trials, 3 replicates per trial.

Figure 5—figure supplement 4
Effects of 2,4-dimethylaniline (DMA) and 2,4-dimethylformanilide (DMF) on VdOctβ2R in vitro.

Mean ± SEM, n = 5 trials.

Transgenic flies expressing Octβ2R variants show different sensitivities to amitraz.

(A, B) Number of lunges (A) and latencies before initiating fighting (B) in the Octβ2R null mutant expressing VdOctβ2R, AmOctβ2R, or AmOctβ2RV3X. Changes were compared to the AmOctβ2R flies. AmOctβ2RE208V, I335T, I350V, abbreviated to AmOctβ2RV3X. Genotype: elav-gal4/UAS-XXOctβ2R; octβ2Rf05679/octβ2Rf05679. p values, Kruskal–Wallis and post hoc Mann–Whitney U tests, mean ± SEM, n = 18–24. (C–E) Midline crossing activity in Octβ2R null mutants expressing VdOctβ2R, AmOctβ2R, or the AmOctβ2RV3X. (F) Daily crossing activities exhibited by Octβ2R null mutants expressing VdOctβ2R, AmOctβ2R, or the AmOctβ2RV3X. Changes were compared to the AmOctβ2R flies. Genotype: elav-gal4/UAS-XXOctβ2R; octβ2Rf05679/octβ2Rf05679. p values, two-way ANOVA and post hoc Bonferroni correction, mean ± SEM, n = 16.

Videos

Video 1
Silencing of octα2R-Gal4 neurons using UAS-Shibirets decreases activity.

The following transgenes were used: octα2R-Gal4> UAS-Shibirets. The movie was speeded up 2×.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Drosophila melanogaster)Canton-SShanghai Institute of Biochemistry and Cell BiologyCat#BCF47
Genetic reagent (Drosophila melanogaster)w1118Bloomington Drosophila Stock CenterCat#5905RRID:BDSC_5905
Genetic reagent (Drosophila melanogaster)elav-Gal4Bloomington Drosophila Stock CenterCat#8765RRID:BDSC_8765
Genetic reagent (Drosophila melanogaster)octβ2R-DfBloomington Drosophila Stock CenterCat#56254RRID:BDSC_56254
Genetic reagent (Drosophila melanogaster)octβ3RMB04794Bloomington Drosophila Stock CenterCat#24819RRID:BDSC_24819
Genetic reagent (Drosophila melanogaster)octβ1RKoon and Budnik, 2012
Genetic reagent (Drosophila melanogaster)octβ2Rf05679Lim et al., 2014Cat#18896RRID:BDSC_18896
Genetic reagent (Drosophila melanogaster)oambdelDeng et al., 2019
Genetic reagent (Drosophila melanogaster)oamb-Gal4Zhou et al., 2012
Genetic reagent (Drosophila melanogaster)octα2RattpDeng et al., 2019
Genetic reagent (Drosophila melanogaster)octα2R-Gal4Deng et al., 2019
Genetic reagent (Drosophila melanogaster)octβ2R-Gal4Deng et al., 2019
Genetic reagent (Drosophila melanogaster)UAS-trpA1Hamada et al., 2008
Genetic reagent (Drosophila melanogaster)UAS-ShibiretsKitamoto, 2001
Genetic reagent (Drosophila melanogaster)oct-tyrRGal4This paperMutant allele; Materials and methods, ‘Fly strains’
Genetic reagent (Drosophila melanogaster)UAS-VdOctβ2RThis paperMutant allele; Materials and methods, ‘Fly strains’
Genetic reagent (Drosophila melanogaster)UAS-AmOctβ2RThis paperMutant allele; Materials and methods, ‘Fly strains’
Genetic reagent (Drosophila melanogaster)UAS-AmOctβ2RV3XThis paperMutant allele; Materials and methods, ‘Fly strains’
Chemical compound, drugAmitrazSigmaCat#45323
Chemical compound, drugN2-(2,4-Dimethylphenyl)-N1-methyformamidineSigmaCat#BP641
Chemical compound, drug2,4-DimethylanilineSigmaCat#301493
Chemical compound, drug(±)-Octopamine hydrochlorideSigmaCat#68631
Chemical compound, drug2,4-DimethylformanilideAccuStandardCat#P-1100S-CN
Chemical compound, drugPiperonyl butoxideAladdinCat#P113864
Chemical compound, drugS,S,S-TributylphosphorotrithioateAladdinCat#T114221
Chemical compound, drugDiethyl maleateAladdinCat#D104017
Chemical compound, drugPoly-D-lysineSigmaCat#P0296
Chemical compound, drugSucroseSinopharmCat#10021418
Chemical compound, drugAgaroseSinopharmCat#63005518
Cell linesHEK 293The Cell Bank of Type Culture Collection of Chinese Academy of SciencesCat#GNHu43https://www.cellbank.org.cn/
Recombinant DNA reagentPlasmid: pcDNA3.1-VdOAMBThis paperSee ‘Construction of expression plasmids’
Recombinant DNA reagentPlasmid: pcDNA3.1-VdOctβ2RThis paperSee ‘Construction of expression plasmids’
Recombinant DNA reagentPlasmid: pcDNA3.1-VdOctβ2RI40FThis paperSee ‘Construction of expression plasmids’
Recombinant DNA reagentPlasmid: pcDNA3.1-VdOctα2RThis paperSee ‘Construction of expression plasmids’
Recombinant DNA reagentPlasmid: pcDNA3.1-VdOct-tyrRThis paperSee ‘Construction of expression plasmids’
Recombinant DNA reagentPlasmid: pcDNA3.1-AmOAMBThis paperSee ‘Construction of expression plasmids’
Recombinant DNA reagentPlasmid: pcDNA3.1-AmOctβ2RThis paperSee ‘Construction of expression plasmids’
Recombinant DNA reagentPlasmid: pcDNA3.1-AmOctβ2RV3XThis paperSee ‘Construction of expression plasmids’
Recombinant DNA reagentPlasmid: pcDNA3.1-AmOctα2RThis paperSee ‘Construction of expression plasmids’
Recombinant DNA reagentPlasmid: pcDNA3.1-AmOct-tyrRThis paperSee ‘Construction of expression plasmids’
Recombinant DNA reagentPlasmid: pcDNA3.1-DmOAMBThis paperSee ‘Construction of expression plasmids’
Recombinant DNA reagentPlasmid: pcDNA3.1-DmOctβ2RThis paperSee ‘Construction of expression plasmids’
Recombinant DNA reagentPlasmid: pcDNA3.1-DmOctα2RThis paperSee ‘Construction of expression plasmids’
Recombinant DNA reagentPlasmid: pcDNA3.1-DmOct-tyrRThis paperSee ‘Construction of expression plasmids’
Recombinant DNA reagentPlasmid: pcDNA3.1-RmOctβ2RThis paperSee ‘Construction of expression plasmids’
Recombinant DNA reagentPlasmid: pcDNA3.1-RmOctβ2RI61FThis paperSee ‘Construction of expression plasmids’
SoftwareSoftMax Pro software (v. 7.1.2.0)Molecular Deviceshttps://www.moleculardevices.com/
SoftwareMolecular Operating Environments (MOE, 2015.10)Chemical Computing Grouphttps://www.chemcomp.com/
SoftwarePrism 7.0GraphPadGraphPad Prism, RRID:SCR_002798
OtherDMEM mediaThermoFisher ScientificCat#10566016
OtherLipofectamine 2000ThermoFisher ScientificCat#11668019
Other96 well polystyrene microplatesThermoFisher ScientificCat#165305
Other0.25% Trypsin-EDTAThermoFisher ScientificCat#25200072
OtherFura 2-AM and Pluronic F-127Dojindo Molecular TechnologiesCat#F025

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)