Multiple combinations of RDL subunits diversify the repertoire of GABA receptors in the honey bee parasite Varroa destructor

Abstract

Edited by Roger J. Colbran In insects, ␥-aminobutyric acid (GABA) is the major inhibitory neurotransmitter, and GABA-gated ion channels are the target of different classes of insecticides, including fipronil. We report here the cloning of six subunits (four RDL, one LCCH3, and one GRD) that constitute the repertoire of the GABA-gated ion channel family of the Varroa mite (Varroa destructor), a honey bee ectoparasite. We also isolated a truncated GRD subunit with a premature stop codon. We found that when expressed in Xenopus laevis oocytes, three of the four RDL subunits (VdesRDL1, VdesRDL2, and VdesRDL3) formed functional, homomultimeric anionic receptors, whereas GRD and LCCH3 produced heteromultimeric cationic receptors. These receptors displayed specific sensitivities toward GABA and fipronil, and VdesRDL1 was the most resistant to the insecticide. We identified specific residues in the VdesRDL1 pore-lining region that explain its high resistance to fipronil. VdesRDL4 did not form a functional receptor when expressed alone, but it assembled with VdesRDL1 to form a heteromultimeric receptor with properties distinct from those of the VdesRDL1 homomultimeric receptor. Moreover, VdesRDL1 physically interacted with VdesRDL3, generating a heteromultimeric receptor combining properties of both subunits. On the other hand, we did not detect any functional interaction between VdesLCCH3 and the VdesRDL subunits, an observation that differed from what was previously reported for Drosophila melanogaster. In conclusion, this study provides insights relevant to improve our understanding of the precise role of GABAergic signaling in insects and new tools for the development of Varroa mitespecific insecticidal agents that do not harm honey bees.

Distinct molecular targets for macrocyclic lactone and isoxazoline insecticides in the human louse: new prospects for the treatment of pediculosis

“…These results indicate that Phh-RDL forms a chloride permeable GABA-gated channel that does not permeate sodium ions, consistently with the pore selectivity filter sequence ( Fig. 2 ) and RDL channels from other species [51, 52]. In summary, these findings demonstrate that two novel functional homomeric receptors from the human body louse, Phh-GluCl and Phh-RDL, with respectively high affinity to glutamate or GABA were reconstituted in Xenopus oocytes.…”

“…4C-D). As previously shown in the literature, RDL and GluCls are permeable to chloride anions [35,37,[48][49][50][51]. Here, the TM2 amino acid sequences of Phh-RDL and Phh-GluCl are respectively 100% identical with RDL and GluCls from other insect species such as A. mellifera, D. melanogaster, T. castaneum (Fig.…”

Heterogeneous expression of GABA receptor‐like subunits LCCH3 and GRD reveals functional diversity of GABA receptors in the honeybee Apis mellifera

“…In addition, the arthropod phylum presents a large molecular diversity of GABA receptor subunit genes. For example, Varroa destructor , the honeybee mite, has six genes encoding GABA receptor subunits (four RDL, one GRD, and one LCCH3) (Ménard et al, 2018), while Acyrthosiphon pisum , the pea aphid, has only two genes encoding RDL subunits (Del Villar & Jones, 2018). These molecular and pharmacological diversities must lead to species‐specific physiological perturbations induced by GABA receptor‐targeting insecticides that probably act with different mechanisms and at distinct time windows.…”

“…Xenopus oocytes were prepared by collagenase (Sigma) digestion at a concentration of 1 mg·ml −1 in calcium‐free solution for 2 h at room temperature, then incubated at 19°C under gentle agitation in ND96 solution (in mM): NaCI 96, KCl 2, MgCl 2 2, CaCl 2 1.8, HEPES 5, pH 7.4 with NaOH, and supplemented with 50 mg·ml −1 gentamicin. Stage V and stage VI oocytes were injected with 20–40 nl of an aqueous solution of in vitro transcribed cRNAs of Am GABA receptor subunits, at 1 μg·μl −1 , with a stoichiometry 1:1 as previously described (Ménard et al, 2018). Macroscopic currents were recorded 1 to 3 days after injection with the two‐electrode voltage‐clamp technique, in a so‐called ND‐Herg recording solution containing (in mM) NaCl (96), KCl (3), CaCl 2 (0.5), MgCl 2 (1), and HEPES (5), with pH adjusted to 7.4 with NaOH.…”

A unique primary structure of RDL (resistant to dieldrin) confers resistance to GABA‐gated chloride channel blockers in the two‐spotted spider mite Tetranychus urticae Koch

“…Nakao et al (2011) reported that an RDL from a fipronil-resistant strain of the small brown planthopper Laodelphax striatellus has Asn at the 2′ position. Among Acari, the VdRDL1 of the parasitic mite Varroa destructor is insensitive to fipronil and has Ser at the 2′ position and Met at the 6′ position in the M2 segment (Price and Lummis, 2014;Ménard et al, 2018). Although few reports to date have characterized the M2 segment of arthropod RDLs, the uniqueness of H305 and I309 in TuRDL needs to be examined further, particularly by comparing RDLs from acariform mites.…”