Anastasia Tsagkarakou

The cys-loop ligand-gated ion channel gene family of Tetranychus urticae: implications for acaricide toxicology and a novel mutation associated with abamectin resistance

The cys-loop ligand-gated ion channel (cysLGIC) super family of Tetranychus urticae, the two-spotted spider mite, represents the largest arthropod cysLGIC super family described to date and the first characterised one within the group of chelicerates. Genome annotation, phylogenetic analysis and comparison of the cysLGIC subunits with their counterparts in insects reveals that the T. urticae genome encodes for a high number of glutamate- and histamine-gated chloride channel genes (GluCl and HisCl) compared to insects. Three orthologues of the insect γ-aminobutyric acid (GABA)-gated chloride channel gene Rdl were detected. Other cysLGIC groups, such as the nAChR subunits, are more conserved and have clear insect orthologues. Members of cysLGIC family mediate endogenous chemical neurotransmission and they are prime targets of insecticides. Implications for toxicology associated with the identity and specific features of T. urticae family members are discussed. We further reveal the accumulation of known and novel mutations in different GluCl channel subunits (Tu_GluCl1 and Tu_GluCl3) associated with abamectin resistance in T. urticae, and provide genetic evidence for their causality. Our study provides useful toxicological insights for the exploration of the T. urticae cysLGIC subunits as putative molecular targets for current and future chemical control strategies.

Current status of insecticide resistance in Q biotype Bemisia tabaci populations from Crete

BACKGROUND A major problem of crop protection in Crete, Greece, is the control of Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae) with chemical insecticides owing to the rapid development of resistance. The aim of this study was to investigate the establishment of resistance and the underlying mechanisms to major insecticide classes with classical bioassays and known biochemical resistance markers. RESULTS During a 2005-2007 survey, 53 Q biotype populations were collected. Application history records showed extensive use of neonicotinoids, organophosphates, carbamates and pyrethroids. High resistance levels were identified in the majority of populations (>80%) for imidacloprid (RF: 38-1958x) and alpha-cypermethrin (RF: 30-600x). Low resistance levels (RF < 12) were observed for pirimiphos-methyl. A strong correlation between resistance to imidacloprid and the number of applications with neonicotinoids was observed. Significant correlations were observed between COE and P450-dependent monoxygenase activity with resistance to alpha-cypermethrin and imidacloprid respectively. A propoxur-based AChE diagnostic test indicated that iAChE was widespread in most populations. Resistance levels for alpha-cypermethrin were increased when compared with a previous survey (2002-2003). Differentiation of LC(50) values between localities was observed for imidacloprid only. CONCLUSION Bemisia tabaci resistance evolved differently in each of the three insecticides studied. Imidacloprid resistance seems less established and less persistent than alpha-cypermethrin resistance. The low resistance levels for pirimiphos-methyl suggest absence of cross-resistance with other organophosphates or carbamates used.

Identification of pyrethroid resistance associated mutations in the para sodium channel of the two‐spotted spider mite Tetranychus urticae (Acari: Tetranychidae)

We investigated pyrethroid resistance mechanisms in Tetranychus urticae strains from Greece. Combined bioassay, biochemical and synergistic data indicated that although P450 mono‐oxygenase activities were associated with the trait, target site insensitivity was the major resistance component. A 3.3 kb cDNA fragment of the T. urticae para sodium channel gene encompassing segment 4 of domain II to segment 6 of domain IV was obtained by a degenerate PCR strategy. The T. urticae sequence showed highest identity (56%) to the scabies mite, Sarcoptes scabiei, and was phylogenetically classified within the divergent group of Arachnida. Comparison of resistant and susceptible strains identified the point mutation F1538I in segment 6 of domain III, which is known to confer strong resistance to pyrethroids, along with a second mutation (A1215D) in the intracellular linker connecting domains II and III with an unknown role. Three transcripts were identified corresponding to the k and l alternative exons. The mode of inheritance of resistance was confirmed as incompletely recessive, which is consistent with a target site mechanism for pyrethroids.

Acetylcholinesterase point mutations in European strains of Tetranychus urticae (Acari: Tetranychidae) resistant to organophosphates

BACKGROUND In Tetranychus urticae Koch, acetylcholinesterase insensitivity is often involved in organophosphate (OP) and carbamate (CARB) resistance. By combining toxicological, biochemical and molecular data from three reference laboratory and three OP selected strains (OP strains), the AChE1 mutations associated with resistance in T. urticae were characterised. RESULTS The resistance ratios of the OP strains varied from 9 to 43 for pirimiphos-methyl, from 78 to 586 for chlorpyrifos, from 8 to 333 for methomyl and from 137 to 4164 for dimethoate. The insecticide concentration needed to inhibit 50% of the AChE1 activity was, in the OP strains, at least 2.7, 55, 58 and 31 times higher for the OP pirimiphos-methyl, chlorpyrifos oxon, paraoxon and omethoate respectively, and 87 times higher for the CARB carbaryl. By comparing the AChE1 sequence, four amino acid substitutions were detected in the OP strains: (1) F331W (Torpedo numbering) in all the three OP strains; (2) T280A found in the three OP strains but not in all clones; (3) G328A, found in two OP strains; (4) A201S found in only one OP strain. CONCLUSIONS Four AChE1 mutations were found in resistant strains of T. urticae, and three of them, F331W, G328A and A201S, are possibly involved in resistance to OP and CARB insecticides. Among them, F331W is probably the most important and the most common in T. urticae. It can be easily detected by the diagnostic PCR-RLFP assay developed in this study.

Gene flow among Tetranychus urticae (Acari: Tetranychidae) populations in Greece

Polymorphism of four enzymatic loci has been examined in 27 populations of Tetranychus urticae in relation to their geographical distribution and to two ecological parameters: open field vs. greenhouse habitats, and species of the colonized host plant. Genetic differentiation was significantly correlated to geographical distance in both types of habitat. Mite density and distribution of infested plants appear to be important factors for the population structure of T. urticae. In open field, T. urticae specimens from citrus trees were genetically more similar to other ‘citrus’ samples collected in different localities than they were from mites collected in the same locality on other plant species.

Mechanisms of resistance to organophosphates in Tetranychus urticae (Acari: Tetranychidae) from Greece.

We investigated the mechanisms conferring resistance to methyl-parathion (44-fold) and to methomyl (8-fold) in Tetranychus urticae from Greece by studying the effect of synergists on the resistance and the kinetic characteristics of various enzymes in a resistant strain (RLAB) and a susceptible reference strain (SAMB). It is shown that S,S,S-tributyl phosphorotrithioate, a synergist that inhibits esterases and glutathione S-transferases, and piperonyl butoxide, a synergist that inhibits cytochrome P450 mediated monooxygenases, did not affect the level of methyl-parathion or methomyl resistance in RLAB and that resistance ratios to both insecticides did not change significantly in the presence of either synergist. Isoelectric focusing of esterase allozymes on single mites revealed no differences in staining intensity and glutathione S-transferase activity was not significantly different in the two strains. The activity of two cytochrome P450 monooxygenase groups was compared. No significant difference of 7-ethoxyresorufin-O-diethylase activity was observed between strains that were two-fold higher in RLAB than in SAMB. The kinetic characteristics of acetylcholinesterase, the target enzyme of organophosphates and carbamates, revealed that acetylcholinesterase in RLAB was less sensitive to inhibition by paraoxon and methomyl in comparison with SAMB. I(50), the inhibitor concentration inducing 50% decrease of acetylcholinesterase activity was greater (119- and 50-fold with paraoxon and methomyl, respectively) and the bimolecular constant k(i) was lower (39- and 47-fold with paraoxon and methomyl, respectively) in RLAB compared to SAMB.

Assessment of the Bemisia tabaci CYP6CM1vQ transcript and protein levels in laboratory and field-derived imidacloprid-resistant insects and cross-metabolism potential of the recombinant enzyme

Abstract  Over‐expression of the cytochrome P450 CYP6CM1 gene has been associated with imidacloprid resistance in a number of Q and B biotype Bemisia tabaci laboratory strains from distinct geographical origins worldwide. We recently demonstrated that the Q biotype version of the CYP6CM1 protein (CYP6CM1vQ) is capable of metabolizing imidacloprid. Here, we show that the levels of BtCYP6CM1vQ were also elevated in laboratory‐resistant strains and field‐derived populations, with variable imidacloprid resistance levels, collected in Crete. High levels of CYP6CM1vQ transcripts were also determined in survivors of a heterogeneous field population, after exposure to discriminating imidacloprid dosage. Using peptide antibody‐based detection assays, we demonstrated that in line with transcriptional data, the CYP6CM1vQ protein levels were higher in imidacloprid‐resistant insects, which further implicates the gene as the causal factor of resistance. Finally, assessment of the cross‐metabolism potential of CYP6CM1vQ against additional neonicotinoid molecules used for B. tabaci control revealed that clothianidin and thiacloprid, but not acetamiprid or thiamethoxam, are metabolized by the recombinant enzyme in vitro.

Mechanisms of Acaricide Resistance in the Two-Spotted Spider Mite Tetranychus urticae

The subclass Acari, comprising mites and ticks, is one of the largest and biologically most diverse groups within the class of the Arachnida, which also includes scorpions, spiders and harvestmen. They are distributed worldwide and have successfully colonised a wide range of terrestrial and aquatic habitats. The majority of mite species living on the aerial parts of higher plants feed mainly on microflora or predate on other small arthropods living on plants. However, mite species within the order of the Prostigmata, belonging to the families of the Tetranychidae (spider mites), Tenuipalpidae (false spider mites), Tarsonemidae (tarsonemid mites) and the superfamily of the Eriophyoidea (gall and rust mites) are able to use their specialized needle-like mouthparts to feed on the plant cells and tissues. Their feeding activity can lead to severe losses in field and protected crops (Walter and Proctor 1999; Evans 1992).

QUANTITATIVE VARIATION AND SELECTION OF ESTERASE GENE AMPLIFICATION IN CULEX PIPIENS

Although descriptions of evolutionary mechanisms are common in the literature, very few studies focus on the possible evolution of the adaptive genes themselves, i.e. their quantitative and qualitative changes. Evolution of insecticide resistance in Culex pipiens is a suitable model for studying such processes. In this species, organophosphorous insecticide resistance can be achieved through the overproduction of esterases that sequester the insecticide, and this overproduction can be caused by gene amplification. It is generally assumed, but never verified, that esterase activity, and therefore resistance, is monotonically related to gene amplification. We have analysed resistance, esterase activity and gene amplification in different laboratory strains and natural populations in order to detect variability and to infer effects of selection on these factors. We have shown that resistance, esterase activity and amplification covary, that insecticide selection is able to increase amplification levels, and that a fitness cost is probably attached to the amplification in laboratory strains, related to the level of amplification. The importance of variation in gene amplification level is discussed and some evolutionary implications are proposed.

Genetic Differentiation in Tetranychus Urticae (Acari: Tetranychidae) from greenhouses in France

The polymorphism of 4 enzymatic systems of 21 Tetranychus urticae samples collected in southern France was analyzed in order to ascertain genetic differentiation within and between greenhouses. Genetic structure was investigated in relation to mite density, geographic distribution of greenhouses and the colonized host plant. We found that mite density and distribution of infested plants influence gene flow within greenhouses. Between greenhouses isolation by distance was significant. Differentiation did not appear to be associated with the colonized plant species.