The cys-loop ligand-gated ion channel gene family of Brugia malayi and Trichinella spiralis: a comparison with Caenorhabditis elegans

Nematode cys-loop ligand gated ion channels (CLGIC) mediate neurotransmission and are important targets for anthelmintics in parasitic nematodes. The CLGIC superfamily in nematodes includes ion channels gated by acetylcholine, γ-amino butyric acid (GABA), glutamate, glycine and 5-HT. The macrocyclic lactones and the nicotinic agonists are important groups of anthelmintics that target the glutamate gated chloride channels and the nicotinic acetylcholine receptors, respectively. The model organism Caenorhabditis elegans has the most diverse families of cys-loop LGIC known in any organism. Many parasitic nematodes have homologues of C. elegans receptors but to date no genome wide investigations have been done. The genome sequencing projects of Brugia malayi (clade III) and Trichinella spiralis (clade I) have allowed us to characterise the CLGIC families in these species. Although the main groups of CLGICs targeted by anthelmintics are represented in both the nematode genomes investigated here, the CLGIC family is much smaller in B. malayi and T. spiralis, suggesting that care must be taken when using C. elegans as a model organism for distantly related nematodes.     

The cys-loop ligand-gated ion channel gene superfamily of the nematode, Caenorhabditis elegans

The nematode, Caenorhabditis elegans, possesses the most extensive known superfamily of cys-loop ligand-gated ion channels (cys-loop LGICs) consisting of 102 subunit-encoding genes. Less than half of these genes have been functionally characterised which include cation-permeable channels gated by acetylcholine (ACh) and γ-aminobutyric acid (GABA) as well as anion-selective channels gated by ACh, GABA, glutamate and serotonin. Following the guidelines set for genetic nomenclature for C. elegans, we have designated unnamed subunits as lgc genes (ligand-gated ion channels of the cys-loop superfamily). Phylogenetic analysis shows that several of these lgc subunits form distinct groups which may represent novel cys-loop LGIC subtypes.     

The cys-loop ligand-gated ion channel superfamily of the honeybee, Apis mellifera

Members of the cys-loop ligand-gated ion channel (cys-loop LGIC) superfamily mediate neurotransmission in insects and are targets of successful insecticides. We have described the cys-loop LGIC superfamily of the honeybee, Apis mellifera, which is an important crop pollinator and a key model for social interaction. The honeybee superfamily consists of 21 genes, which is slightly smaller than that of Drosophila melanogaster comprising 23 genes. As with Drosophila, the honeybee possesses ion channels gated by acetylcholine, γ-amino butyric acid, glutamate and histamine as well as orthologs of the Drosophila pH-sensitive chloride channel (pHCl), CG8916, CG12344 and CG6927. Similar to Drosophila, honeybee cys-loop LGIC diversity is broadened by differential splicing which may also serve to generate species-specific receptor isoforms. These findings on Apis mellifera enhance our understanding of cys-loop LGIC functional genomics and provide a useful basis for the development of improved insecticides that spare a major beneficial insect species.Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. DQ667181–DQ667195.     

Loss of function mutation in the P2X7, a ligand-gated ion channel gene associated with hypertrophic cardiomyopathy

Purinergic Signalling - Hypertrophic cardiomyopathy (HCM) is an inherited heart failure condition, mostly found to have genetic abnormalities, and is a leading cause of sudden death in young...     

Monitoring of acaricide resistance of Tetranychus urticae (Acari: Tetranychidae) from Korean apple orchards

Tetranychus urticae populations were collected from ten commercial apple orchards and their susceptibilities to 12 acaricides were tested using a leaf disc bioassay. The resistance of each T. urticae population was reported as the LC₅₀ value, the resistance ratio (RR) and the slope of the probit–concentration regression. Cross resistances of T. urticae populations were estimated using the Spearman's rank correlation coefficient. Most local populations showed low resistance levels (RR ≤ 10). Development of resistance to METI and pyrethroid acaricides differed among local populations. The highest RR value (154.6) was found in the Uiseong population to tebufenpyrad. The Geochang population was highly resistant, especially to METI and pyrethroid acaricides. T. urticae populations collected from Suwon, Chungju, Yeongju and Geochang showed moderate resistance (10 < RR ≤ 40) to more than two acaricides. Resistance ratios to abamectin, chlorfenapyr, fenbutatin-oxide and milbemectin were low (RR ≤ 10) in all populations. The LC₅₀ values of abamectin, chlorfenapyr, fenbutatin-oxide and milbemectin ranged from 0.06 to 0.2 mg/l, from 0.67 to 3.38 mg/l, from 10.12 to 40.85 mg/l and from 0.47 to 3.01 mg/l, respectively. We discuss possible cross-resistance to acaricides using Spearman's rank correlation coefficient.     

A glutathione-S-transferase (TuGSTd05) associated with acaricide resistance in Tetranychus urticae directly metabolizes the complex II inhibitor cyflumetofen

Cyflumetofen is a recently introduced acaricide with a novel mode of action, acting as an inhibitor of complex II of mitochondrial electron transport chain. It is activated by hydrolysis and the resulting de-esterified metabolite is a much stronger inhibitor. Cyflumetofen represents a great addition for the control of mite species including Tetranychus urticae, a major agricultural pest, which has the ability to develop resistance to most classes of pesticides rapidly. A resistant strain (Tu008R) was recently described and synergism experiments pointed towards the involvement of GSTs. Here, we conducted genome-wide gene expression analysis, comparing Tu008R with its parental susceptible strain, and identified the delta GST TuGSTd05 as the prime resistance-conferring candidate. Docking analysis suggests that both cyflumetofen and its de-esterified metabolite are potential substrates for conjugation by TuGSTd05. Several amino acids were identified that might be involved in the interaction, with Y107 and N103 possibly having an important role. To further investigate interaction as well as the role of Y107 and N103 in vitro, we recombinantly expressed and kinetically characterized the wild type TuGSTd05, TuGSTd05 Y107F and TuGSTd05 N103L mutants. While cyflumetofen was not found to act as a strong inhibitor, the de-esterified metabolite showed strong affinity for TuGSTd05 (IC₅₀ = 4 μM), which could serve as a mechanism of rapid detoxification. Y107 and N103 might contribute to this interaction. HPLC-MS analysis provided solid indications that TuGSTd05 catalyzes the conjugation of ionized glutathione (GS⁻) to cyflumetofen and/or its de-esterified metabolite and the resulting metabolite and possible site of attack were identified.     

Geographical distribution and origin of acetylcholinesterase mutations conferring acaricide resistance in Tetranychus urticae populations from Turkey

Two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is a cosmopolitan pest species that can feed on more than 1000 host plant species. Historically, organophosphate (OP) and carbamate insecticides have been used to control this extremely polyphagous pest. However, its ability to develop acaricide resistance rapidly has led to failure in control. Mutations in acetylcholinesterase gene (ace), the target-site of OP and carbamate insecticides, have been reported to be one of the major mechanisms underlying this developing resistance. In this study, mutations previously associated with resistance (G119S, A201S, T280A, G328A, F331W/Y) in ace have been screened in 37 T. urticae populations collected across Turkey. All mutations were found in various populations, except G119S. Almost all populations had F331W/Y mutation (being fixed in 32 populations), whereas only two populations harboured A201S mutation, but not fixed. On the other hand, more than half of the populations contained T280A and G328A mutations. In addition, the presence of same haplotypes in populations originating from distinct geographic locations and a wide variety of ace haplotypes might indicate multiple origins of F331W and F331Y mutations; however, this needs further investigation. The results of area-wide screening showed that ace mutations are widely distributed among T. urticae populations. Therefore, the use of this group of insecticides should be limited or only rotational use might be regarded as a resistance management tool due to its different mode of action from other main acaricide groups in T. urticae control across Turkey.

     

A novel member of the ligand-gated chloride channel gene family from Haemonchus contortus

Ligand-gated chloride channels (LGCCs) are key components of the nervous system of parasitic nematodes and important targets for anthelmintics. Here, we describe the isolation and characterization of a novel member of the LGCC gene family (HcLGCC1) from the parasitic nematode Haemonchus contortus. Sequence analysis revealed that the channel subunit encoded by HcLGCC1 is anion selective and a member of a group of channels characterized as having two Cys-loops in the N-terminal ligand-binding domain. Although the overall function of HcLGCC1 is presently unknown, the gene may play a key role in the early developmental stages of the parasite. Further investigations into the function of LGCCs, such as HcLGCC1, in parasitic nematodes should have implications for the discovery of new anthelmintic targets.     

A novel mtDNA ND6 gene mutation associated with LHON in a Caucasian family

Leber's hereditary optic neuropathy (LHON) is a frequent cause of inherited blindness. A routine screening for common mtDNA mutations constitutes an important first in its diagnosis. However, a substantial number of LHON patients do not harbor known variants, both pointing to the genetic heterogeneity of LHON and bringing into question its genetic diagnosis. We report a familial case that exhibited typical features of LHON but lacked any of the common mutations. Genetic analysis revealed a novel pathogenic defect in the ND6 gene at 14279A that was not detected in any haplogroup-matched controls screened for it, nor has it been previously reported. This mutation causes a substantial conformational change in the secondary structure of the polypeptide matrix coil and may explain the LHON expression. Thus, it expands the spectrum of deleterious changes affecting ND6-encoding subunit and further highlights the functional significance of this gene, providing additional clues to the disease pathogenesis.     

A mutation in the PSST homologue of complex I (NADH:ubiquinone oxidoreductase) from Tetranychus urticae is associated with resistance to METI acaricides

The acaricidal compounds pyridaben, tebufenpyrad and fenpyroximate are frequently used in the control of phytophagous mites such as Tetranychus urticae, and are referred to as Mitochondrial Electron Transport Inhibitors, acting at the quinone binding pocket of complex I (METI-I acaricides). Because of their very frequent use, resistance evolved fast more than 20 years ago, and is currently wide-spread. Increased activity of P450 monooxygenases has been often associated with resistance, but target-site based resistance mechanisms were never reported.Here, we report the discovery of a mutation (H92R) in the PSST homologue of complex I in METI-I resistant T. urticae strains. The position of the mutation was studied using the high-resolution crystal structure of Thermus thermophilus, and was located in a stretch of amino acids previously photo-affinity labeled by fenpyroximate. Selection experiments with a strain segregating for the mutant allele, together with marker-assisted back-crossing of the mutation in a susceptible background, confirmed the involvement of the mutation in METI-I resistance. Additionally, an independent genetic mapping approach; QTL analysis identified the genomic region of pyridaben resistance, which included the PSST gene. Last, we used CRISPR-Cas9 genome editing tools to introduce the mutation in the Drosophila PSST homologue.     

Dirofilaria immitis: identification of a novel ligand-gated ion channel-related polypeptide

We have isolated a cDNA from Dirofilaria immitis that encodes a predicted ion channel subunit, Di-LGR-1. Secondary structure predictions and database searches reveal that Di-LGR-1 is distantly related to ligand-gated anion channels, such as the GABA(A) receptors, though there are marked differences in the sequences of the putative channel forming regions. Di-LGR-1 has 52% sequence identity to the Caenorhabditis elegans predicted polypeptide, T27A1.4: neighbour-joining trees show that these two polypeptides are the most divergent members of the nematode ligand-gated anion channel family. No close homologues are present in vertebrates, suggesting that their function may be specific to nematodes. RNAi experiments using a fragment of T27A1.4 with C. elegans failed to reveal any obvious phenotype, so the function of these channels remains unknown.     

Common binding sites for cholesterol and neurosteroids on a pentameric ligand-gated ion channel

Cholesterol is an essential component of cell membranes, and is required for mammalian pentameric ligand-gated ion channel (pLGIC) function. Computational studies suggest direct interactions between cholesterol and pLGICs but experimental evidence identifying specific binding sites is limited. In this study, we mapped cholesterol binding to Gloeobacter ligand-gated ion channel (GLIC), a model pLGIC chosen for its high level of expression, existing crystal structure, and previous use as a prototypic pLGIC. Using two cholesterol analogue photolabeling reagents with the photoreactive moiety on opposite ends of the sterol, we identified two cholesterol binding sites: an intersubunit site between TM3 and TM1 of adjacent subunits and an intrasubunit site between TM1 and TM4. In both the inter- and intrasubunit sites, cholesterol is oriented such that the 3‑OH group points toward the center of the transmembrane domains rather than toward either the cytosolic or extracellular surfaces. We then compared this binding to that of the cholesterol metabolite, allopregnanolone, a neurosteroid that allosterically modulates pLGICs. The same binding pockets were identified for allopregnanolone and cholesterol, but the binding orientation of the two ligands was markedly different, with the 3‑OH group of allopregnanolone pointing to the intra- and extracellular termini of the transmembrane domains rather than to their centers. We also found that cholesterol increases, whereas allopregnanolone decreases the thermal stability of GLIC. These data indicate that cholesterol and neurosteroids bind to common hydrophobic pockets in the model pLGIC, GLIC, but that their effects depend on the orientation and specific molecular interactions unique to each sterol.     

The selectivity filter of a ligand-gated ion channel. The helix-M2 model of the ion channel of the nicotinic acetylcholine receptor

Evidence from electrophysiology and biochemistry supports the hypothesis that the ion channel of the nicotinic acetylcholine receptor is formed by homologous amino acid sequences of all receptor subunits, called helices M2. A model of the ion channel is proposed and the selectivity filter is described as a ring of negatively-charged amino acid side chains [(1988) Nature 335, 645-648] which may undergo conformational changes upon permeation of the cation.     

Multiple resistance and biochemical mechanisms of pyridaben resistance in Tetranychus urticae (Acari: Tetranychidae)

A field colony of Tetranychus urticae (Koch) (Acari: Tetranychidae) resistant to pyridaben was selected with pyridaben successively for 20 generations to produce the PR-20 strain. Resistance and multiple resistance levels of the PR-20 strain to 15 acaricides were determined using a spray bioassay. The PR-20 strain was extremely resistant to pyridaben (resistance ratio [RR] = 240]. The strain exhibited extremely strong resistance to fenpyroximate (RR=373) and acrinathrin (RR=329) and strong resistance to benzoximate (RR=84). An RR = 10-40 was observed with abamectin, fenazaquin, fenbutatin oxide, fenpropathrin, and tebufenpyrad. The PR-20 strain showed low levels of resistance (RR <10) to azocyclotin, bromopropylate, chlorfenapyr, dicofol, milbemectin, and propargite. Synergist experiments with different metabolic inhibitors revealed that piperonyl butoxide (PBO), a mixed function oxidase (MFO) inhibitor, had the greatest effect on pyridaben resistance. PBO significantly caused pyridaben resistance in the PR-20 strain to drop to the full susceptibility level of the susceptible (S) strain. However, there was no significant difference in MFO activities measured using a model substrate between the S and PR-20 strains. These results suggest that use of certain acaricides with little multiple resistance or PBO will be useful for the management of pyridaben resistance in the field.     

二斑叶螨对七种杀螨剂的抗药性测定及其机理研究

室内测定了相对敏感种群(S)和抗性种群(R)对常用7种杀螨剂的敏感性,并测定了羧酸酯酶、谷胱甘肽S-转移酶和乙酰胆碱酯酶3种酶的比活力。结果表明:二斑叶螨Tetranychus urticae Koch R种群已对甲氰菊酯和哒螨灵产生了抗性,抗性倍数分别为5.45和105.47。其中,甲氰菊酯对雌成螨的毒力最低(>3000mg/L),已远远超过田间推荐剂量,不宜继续使用。酶活测定结果表明:谷胱甘肽S-转移酶解毒活性的提高是二斑叶螨对甲氰菊酯产生抗性的原因之一;二斑叶螨对哒螨灵抗性的增强可能与羧酸酯酶有关。     

Pre-adult development of Phytoseiulus persimilis on diets of Tetranychus urticae and Tetranychus lintearius: implications for the biological control of Ulex europaeus

Predation by the phytoseiid mite, Phytoseiulus persimilis, is considered a major threat to the effectiveness of biological control of gorse, Ulex europaeus, using Tetranychus lintearius. To assess this threat and to determine if the impact of P. persimilis on T. lintearius populations is comparable to its impact on T. urticae populations, its development and predator : prey generation time ratios were assessed. The pre-adult mortality and development time of two populations of P. persimilis fed on two diets, T. urticae and T. lintearius, were determined at two temperatures, 14 and 24°C. There were no significant differences in either mortality or development time between the two populations of P. persimilis at these temperatures. There is therefore no evidence that the two tested populations of P. persimilis are behaving as different strains. Similarly, diet had no significant effect on either mortality or development time at these temperatures. At 14°C the mortality of P. persimilis was significantly higher and development was significantly longer than at 24°C. Using pre-adult development as a surrogate for generation times, predator : prey generation time ratios were calculated between P. persimilis and both T. urticae and T. lintearius using data from this and other studies. The predator : prey generation time ratios between P. persimilis and T. lintearius were lower than those between P. persimilis and T. urticae. These results indicate that the impact of P. persimilis on T. lintearius populations is likely to be comparable to its impact on T. urticae populations. This provides further evidence that predation by P. persimilis is having a deleterious effect on T. lintearius populations and therefore reducing its effectiveness as a biological control agent for gorse.     

酯酶基因扩增及突变与昆虫抗药性

有机磷和氨基甲酸酯类杀虫剂的大量使用导致昆虫对其产生抗药性。酯酶是昆虫体内重要的解毒代谢酶,酯酶基因表达量上升和点突变使其代谢或结合杀虫剂的能力增强是昆虫对常用农药产生抗药性的2个重要原因。文章概述昆虫酯酶基因扩增及突变所导致的抗药性,进一步分析了酯酶突变对蛋白结构和功能的影响。