Development of acaricide resistance in Pacific spider mite (<Emphasis Type="Italic">Tetranychus pacificus</Emphasis>) from California vineyards

In recent years, grape growers in California reported failures of acaricides against Tetranychus pacificus McGregor. We collected T. pacificus populations from four vineyards and tested them for resistance to bifenazate, propargite and pyridaben. In addition, we sequenced part of the cytochrome b gene of bifenazate-resistant and -susceptible T. pacificus to test for the presence of mutations reported to confer resistance to the congeneric T. urticae. None of the mutations conferring resistance to bifenazate in T. urticae were present in resistant T. pacificus. Resistance levels ranged from full susceptibility to statistically significant 11-fold resistance to pyridaben, sevenfold resistance to bifenazate and fourfold resistance to propargite compared to a susceptible population. Despite the relatively low levels of resistance detected, we estimated that under the conditions of our study the highest field rates of bifenazate and pyridaben application would cause less than 58 and 66% mortality of adult females in the most resistant populations, respectively. In contrast, field rates of propargite application would cause close to 100% mortality in the least susceptible population. These results highlight a potential link between resistance development and reduced field effectiveness for bifenazate and pyridaben. Finally, T. pacificus may be more tolerant to bifenazate and propargite than T. urticae, since the LC₅₀ values for the susceptible population of T. pacificus were several times higher than LC₅₀’s reported for susceptible T. urticae.     

Independently evolved and gene flow‐accelerated pesticide resistance in two‐spotted spider mites

Pest species are often able to develop resistance to pesticides used to control them, depending on how rapidly resistance can emerge within a population or spread from another resistant population. We examined the evolution of bifenazate resistance in China in the two‐spotted spider mite (TSSM) Tetranychus uticae Koch (Acari: Tetranychidae), one of the most resistant arthropods, by using bioassays, detection of mutations in the target cytb gene, and population genetic structure analysis using microsatellite markers. Bioassays showed variable levels of resistance to bifenazate. The cytb mutation G126S, which confers medium resistance in TSSM to bifenazate, had previously been detected prior to the application of bifenazate and was now widespread, suggesting likely resistance evolution from standing genetic variation. G126S was detected in geographically distant populations across different genetic clusters, pointing to the independent origin of this mutation in different TSSM populations. A novel A269V mutation linked to a low‐level resistance was detected in two southern populations. Widespread resistance associated with a high frequency of the G126S allele was found in four populations from the Beijing area which were not genetically differentiated. In this case, a high level of gene flows likely accelerated the development of resistance within this local region, as well as into an outlying region distant from Beijing. These findings, therefore, suggest patterns consistent with both local evolution of pesticide resistance as well as an impact of migration, helping to inform resistance management strategies in TSSM.     

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.

     

Mitochondrial Disease-related Mutation G167P in Cytochrome b of Rhodobacter capsulatus Cytochrome bc1 (S151P in Human) Affects the Equilibrium Distribution of [2Fe-2S] Cluster and Generation of Superoxide

Cytochrome bc₁ is one of the key enzymes of many bioenergetic systems. Its operation involves a large scale movement of a head domain of iron-sulfur protein (ISP-HD), which functionally connects the catalytic quinol oxidation Q_o site in cytochrome b with cytochrome c₁. The Q_o site under certain conditions can generate reactive oxygen species in the reaction scheme depending on the actual position of ISP-HD in respect to the Q_o site. Here, using a bacterial system, we show that mutation G167P in cytochrome b shifts the equilibrium distribution of ISP-HD toward positions remote from the Q_o site. This renders cytochrome bc₁ non-functional in vivo. This effect is remediated by addition of alanine insertions (1Ala and 2Ala) in the neck region of the ISP subunit. These insertions, which on their own shift the equilibrium distribution of ISP-HD in the opposite direction (i.e. toward the Q_o site), also act in this manner in the presence of G167P. Changes in the equilibrium distribution of ISP-HD in G167P lead to an increased propensity of cytochrome bc₁ to generate superoxide, which becomes evident when the concentration of quinone increases. This result corroborates the recently proposed model in which “semireverse” electron transfer back to the Q_o site, occurring when ISP-HD is remote from the site, favors reactive oxygen species production. G167P suggests possible molecular effects of S151P (corresponding in sequence to G167P) identified as a mitochondrial disease-related mutation in human cytochrome b. These effects may be valid for other human mutations that change the equilibrium distribution of ISP-HD in a manner similar to G167P.     

Directed evolution predicts cytochrome b G37V target site modification as probable adaptive mechanism towards the QiI fungicide fenpicoxamid in Zymoseptoria tritici

Acquired resistance is a threat to antifungal efficacy in medicine and agriculture. The diversity of possible resistance mechanisms and highly adaptive traits of pathogens make it difficult to predict evolutionary outcomes of treatments. We used directed evolution as an approach to assess the resistance risk to the new fungicide fenpicoxamid in the wheat pathogenic fungus Zymoseptoria tritici. Fenpicoxamid inhibits complex III of the respiratory chain at the ubiquinone reduction site (Qi site) of the mitochondrially encoded cytochrome b, a different site than the widely used strobilurins which inhibit the same complex at the ubiquinol oxidation site (Q_o site). We identified the G37V change within the cytochrome b Q_i site as the most likely resistance mechanism to be selected in Z. tritici. This change triggered high fenpicoxamid resistance and halved the enzymatic activity of cytochrome b, despite no significant penalty for in vitro growth. We identified negative cross-resistance between isolates harbouring G37V or G143A, a Q_o site change previously selected by strobilurins. Double mutants were less resistant to both QiIs and quinone outside inhibitors compared to single mutants. This work is a proof of concept that experimental evolution can be used to predict adaptation to fungicides and provides new perspectives for the management of QiIs.     

Phenotypic- and Genotypic-Resistance Detection for Adaptive Resistance Management in Tetranychus urticae Koch

Rapid resistance detection is necessary for the adaptive management of acaricide-resistant populations of Tetranychus urticae. Detection of phenotypic and genotypic resistance was conducted by employing residual contact vial bioassay (RCV) and quantitative sequencing (QS) methods, respectively. RCV was useful for detecting the acaricide resistance levels of T. urticae, particularly for on-site resistance detection; however, it was only applicable for rapid-acting acaricides (12 out of 19 tested acaricides). QS was effective for determining the frequencies of resistance alleles on a population basis, which corresponded to 12 nonsynonymous point mutations associated with target-site resistance to five types of acaricides [organophosphates (monocrotophos, pirimiphos-methyl, dimethoate and chlorpyrifos), pyrethroids (fenpropathrin and bifenthrin), abamectin, bifenazate and etoxazole]. Most field-collected mites exhibited high levels of multiple resistance, as determined by RCV and QS data, suggesting the seriousness of their current acaricide resistance status in rose cultivation areas in Korea. The correlation analyses revealed moderate to high levels of positive relationships between the resistance allele frequencies and the actual resistance levels in only five of the acaricides evaluated, which limits the general application of allele frequency as a direct indicator for estimating actual resistance levels. Nevertheless, the resistance allele frequency data alone allowed for the evaluation of the genetic resistance potential and background of test mite populations. The combined use of RCV and QS provides basic information on resistance levels, which is essential for choosing appropriate acaricides for the management of resistant T. urticae.     

Roles in inhibitor recognition and quinol oxidation of the amino acid side chains at positions of cytb providing resistance to Qo-inhibitors of the bc1 complex from Rhodobacter capsulatus

The substitutions M140I, F144S and L, G152S, T163A and V333A in cytochrome b of the ubiquinol-cytochrome c oxidoreductase (bc₁ complex) from Rhodobacter capsulatus provide resistance to the quinol oxidation (Q_o inhibitors myxothiazol, mucidin and stigmatellin. Site-directed mutagenesis with degenerate primers was used to define the role of these positions in inhibitor recognition and quinol oxidation, and a collection of various substitutions at each of these positions was obtained. The effects of these mutations on quinol oxidation, nature and level of inhibitor resistance, prosthetic group incorporation and assembly of the complex were analysed. Most of these mutations, unlike those at position 158 reported earlier, yielded functional bc₁, complexes able to support the photosynthetic growth of R. capsulatus. However, they perturbed steady-state quinol oxidation and inhibitor recognition indicating that they are important for the function of the Q_o site. In particular, the presence of a methyl group on the β-carbon (He and Val residues) at position 140, the absence of an aromatic ring (Phe, Tyr and Trp residues) at position 144 and the loss of residues with small side chains (Gly and Ala) at position 152 correlated with resistance to myxothiazol. On the other hand, no myxothiazol resistance was observed with the substitutions at positions 163 and 333 suggesting that they affected solely the recognition of stigmatellin. Five substitutions, M140R, F144H and R, G152P and T163R, yielded photosynthesis-deficient mutants with assembled but impaired bc₁ complexes. Unexpectedly, two substitutions at position 163 (T to F or P) yielded mutants lacking the three subunits of     

Whole-genome sequencing to detect mutations associated with resistance to insecticides and Bt proteins in Spodoptera frugiperda

The fall armyworm (FAW), Spodoptera frugiperda, is a major pest native to the Americas that has recently invaded the Old World. Point mutations in the target-site proteins acetylcholinesterase-1 (ace-1), voltage-gated sodium channel (VGSC) and ryanodine receptor (RyR) have been identified in S. frugiperda as major resistance mechanisms to organophosphate, pyrethroid and diamide insecticides respectively. Mutations in the adenosine triphosphate-binding cassette transporter C2 gene (ABCC2) have also been identified to confer resistance to Cry1F protein. In this study, we applied a whole-genome sequencing (WGS) approach to identify point mutations in the target-site genes in 150 FAW individuals collected from China, Malawi, Uganda and Brazil. This approach revealed three amino acid substitutions (A201S, G227A and F290V) of S. frugiperda ace-1, which are known to be associated with organophosphate resistance. The Brazilian population had all three ace-1 point mutations and the 227A allele (mean frequency = 0.54) was the most common. Populations from China, Malawi and Uganda harbored two of the three ace-1 point mutations (A201S and F290V) with the 290V allele (0.47–0.58) as the dominant allele. Point mutations in VGSC (T929I, L932F and L1014F) and RyR (I4790M and G4946E) were not detected in any of the 150 individuals. A novel 12-bp insertion mutation in exon 15 of the ABCC2 gene was identified in some of the Brazilian individuals but absent in the invasive populations. Our results not only demonstrate robustness of the WGS-based genomic approach for detection of resistance mutations, but also provide insights for improvement of resistance management tactics in S. frugiperda.     

Resistance status of the carmine spider mite,Tetranychus cinnabarinus and the twospotted spider mite,Tetranychus urticae to selected acaricides on strawberries

The carmine spider mite, Tetranychus cinnabarinus (Boisduval) and the twospotted spider mite, Tetranychus urticae Koch, are serious pests of strawberries and many other horticultural crops. Control of these pests has been heavily dependent upon chemical acaricides. Objectives of this study were to determine the resistance status of these two pest species to commonly used acaricides on strawberries in a year‐round intensive horticultural production region. LC₉₀ of abamectin for adult carmine spider mites was 4% whereas that for adult twospotted spider mites was 24% of the top label rate. LC_90s of spiromesifen, etoxazole, hexythiazox and bifenazate were 0.5%, 0.5%, 1.4% and 83% of their respective highest label rates for carmine spider mite eggs, 0.7%, 2.7%, 12.1% and 347% of their respective highest label rates for the nymphs. LC_90s of spiromesifen, etoxazole, hexythiazox and bifenazate were 4.6%, 11.1%, 310% and 62% of their respective highest label rates for twospotted spider mite eggs, 3%, 13%, 432,214% and 15% of their respective highest label rates for the nymphs. Our results suggest that T. cinnabarinus have developed resistance to bifenazate and that the T. urticae have developed resistance to hexythiazox. These results strongly emphasize the need to develop resistance management strategies in the region.     

Determination of acaricide resistance allele frequencies in field populations of Tetranychus urticae using quantitative sequencing

Resistances to monocrotophos, fenpropathrin and abamectin in Tetranychus urticae are primarily conferred by reduced sensitivities of respective target sites [i.e., acetylcholinesterase (TuAChE), voltage-sensitive sodium channel (TuVSSC) and glutamate-gated chloride channel (TuGluCl)], which are due to point mutations (G228S and F439W in TuAChE; L1022V in TuVSSC; G323D in TuGluCl). As a population-based genotyping technique, a quantitative sequencing (QS) protocol was developed for the determination of the resistance-associated mutation frequencies in T. urticae. Standard prediction equations revealed high correlation coefficients (r² = 0.993–0.999), demonstrating that the resistant nucleotide signal ratio is highly proportional to the resistance allele frequencies. The lower and higher detection limits for the four resistance mutations were 3.7–13.1% (7.8 ± 3.3%) and 89.4–97.3% (93.3 ± 3.2%), respectively, suggesting that QS can be employed as a preliminary monitoring tool for the detection of resistance allele frequencies, which ranged approximately 7.8–93.3% at the 95% confidence level. The QS was successfully employed for the determination of resistance allele frequencies in 26 T. urticae populations. The two TuAChE mutations responsible for monocrotophos resistance were almost saturated in most field populations. The TuVSSC L1022V mutation tentatively associated with fenpropathrin resistance was also found in 9 field populations. However, the TuGluCl G323D mutation conferring abamectin resistance was found only in one field population, suggesting that abamectin resistance is not yet widespread. The QS protocol, as an alternative to traditional bioassays, will greatly facilitate resistance monitoring of T. urticae.     

Geographic spread,genetics and functional characteristics of ryanodine receptor based target-site resistance to diamide insecticides in diamondback moth,Plutella xylostella

Anthranilic diamides and flubendiamide belong to a new chemical class of insecticides acting as conformation sensitive activators of the insect ryanodine receptor (RyR). These compounds control a diverse range of different herbivorous insects including diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), a notorious global pest on cruciferous crops, which recently developed resistance due to target-site mutations located in the trans-membrane domain of the Plutella RyR. In the present study we further investigated the genetics and functional implications of a RyR G4946E target-site mutation we recently identified in a Philippine diamondback moth strain (Sudlon). Strain Sudlon is homozygous for the G4946E mutation and has been maintained under laboratory conditions without selection pressure for almost four years, and still exhibit stable resistance ratios of >2000-fold to all commercial diamides. Its F1 progeny resulting from reciprocal crosses with a susceptible strain (BCS-S) revealed no maternal effects and a diamide susceptible phenotype, suggesting an autosomally almost recessive mode of inheritance. Subsequent back-crosses indicate a near monogenic nature of the diamide resistance in strain Sudlon. Radioligand binding studies with Plutella thoracic microsomal membrane preparations provided direct evidence for the dramatic functional implications of the RyR G4946E mutation on both diamide specific binding and its concentration dependent modulation of [³H]ryanodine binding. Computational modelling based on a cryo-EM structure of rabbit RyR1 suggests that Plutella G4946E is located in trans-membrane helix S4 close to S4–S5 linker domain supposed to be involved in the modulation of the voltage sensor, and another recently described mutation, I4790M in helix S2 approx. 13 Å opposite of G4946E. Genotyping by pyrosequencing revealed the presence of the RyR G4946E mutation in larvae collected in 2013/14 in regions of ten different countries where diamide insecticides largely failed to control diamondback moth populations. Thus, our study highlights the global importance of the G4946E RyR target-site mutation, which as a mechanism on its own, confers high-level resistance to diamide insecticides in diamondback moth.     

Spider mite resistance to miticides in South Carolina strawberry and implications for improved integrated pest management

Tetranychus urticae Koch (Acari: Tetranychidae), twospotted spider mite, is a major secondary pest of strawberry and can cause significant yield loss. Tetranychus urticae is typically controlled using miticides, which has led to rapid resistance development. In South Carolina (USA), extension agents and growers have reported field failures of miticides (inadequate pest suppression), but resistance has not been quantitatively determined. In 2018, we determined the level of miticide resistance of six T. urticae populations found on strawberry across South Carolina. We examined efficacy of all miticides registered for use on US strawberry by conducting an initial diagnostic bioassay at 20% of the maximum labeled field rate. Any population?×?active ingredient combination resulting in?<?55% mortality was identified as ‘potentially resistant’ and concentration–response bioassays were then conducted to calculate LC₅₀ values for an individual population. These values were compared with those of a known-susceptible laboratory population to calculate resistance ratios (RR). Our results indicate that examined South Carolina populations of T. urticae from strawberry were highly resistant to bifenthrin (RR?=?100–60,000) and there was reduced susceptibility to fenbutatin-oxide (RR?=?25–123). The 'Sardinia' population had decreased abamectin susceptibility (RR?=?25). No resistance to hexythiazox, etoxazole, acequinocyl, bifenazate, fenpyroximate, spiromesifen, or cyflumetofen was found. Based on available data, it appears that miticide resistance is not a likely cause of field failures and issues related to application error and coverage should be investigated. Overall, this work supports the need to reduce the use of broad-spectrum pesticides and older products, in favor of newer miticide chemistries due to resistance issues.

     

The redlegged earth mite draft genome provides new insights into pesticide resistance evolution and demography in its invasive Australian range

Genomic data provide valuable insights into pest management issues such as resistance evolution, historical patterns of pest invasions and ongoing population dynamics. We assembled the first reference genome for the redlegged earth mite, Halotydeus destructor (Tucker, 1925), to investigate adaptation to pesticide pressures and demography in its invasive Australian range using whole-genome pool-seq data from regionally distributed populations. Our reference genome comprises 132 autosomal contigs, with a total length of 48.90 Mb. We observed a large complex of ace genes, which has presumably evolved from a long history of organophosphate selection in H. destructor and may contribute towards organophosphate resistance through copy number variation, target-site mutations and structural variants. In the putative ancestral H. destructor ace gene, we identified three target-site mutations (G119S, A201S and F331Y) segregating in organophosphate-resistant populations. Additionally, we identified two new para sodium channel gene mutations (L925I and F1020Y) that may contribute to pyrethroid resistance. Regional structuring observed in population genomic analyses indicates that gene flow in H. destructor does not homogenize populations across large geographic distances. However, our demographic analyses were equivocal on the magnitude of gene flow; the short invasion history of H. destructor makes it difficult to distinguish scenarios of complete isolation vs. ongoing migration. Nonetheless, we identified clear signatures of reduced genetic diversity and smaller inferred effective population sizes in eastern vs. western populations, which is consistent with the stepping-stone invasion pathway of this pest in Australia. These new insights will inform development of diagnostic genetic markers of resistance, further investigation into the multifaceted organophosphate resistance mechanism and predictive modelling of resistance evolution and spread.     

The overexpression of acetylcholinesterase compensates for the reduced catalytic activity caused by resistance-conferring mutations in Tetranychus urticae

The mutations (G228S, A391T and F439W) and duplication of the acetylcholinesterase (AChE) gene (Tuace) are involved in monocrotophos resistance in the two-spotted spider mites, Tetranychus urticae (Kwon et al., 2010a, Kwon et al., 2010b). The overexpression of T. urticae AChE (TuAChE) as a result of Tuace duplication was confirmed in several field-collected populations by Western blotting using an AChE-specific antibody. To investigate the effects of each mutation on the insensitivity and fitness cost of AChE, eight variants of TuAChE were expressed in vitro using the baculovirus expression system. Kinetic analysis revealed that the G228S and F439W mutations confer approximately 26-fold and 99-fold increases in the insensitivity to monocrotophos, respectively, whereas the insensitivity increased over 1165-fold in the AChE with double mutations. Nevertheless, the presence of these mutations reduced the catalytic efficiency of AChE significantly. In particular, the TuAChE having both mutations together exhibited a 17.8～27.1-fold reduced catalytic efficiency, suggesting an apparent fitness cost in the monocrotophos-resistant mites. The A391T mutation did not change the kinetic properties of either the substrate or inhibitor when present alone but mitigated the negative impacts of the F439 mutation. To simulate the catalytic activity of the overexpressed TuAChE in two T. urticae strains (approximately 6 copies for AD strain vs. 2 copies for PyriF strain), appropriate TuAChE variants were combined to make up the desired AChE copies and mutation frequencies, and their enzyme kinetics were determined. The reconstituted 6-copy and 2-copy TuAChEs exhibited catalytic efficiency levels comparable to those of a single-copy wildtype TuAChE, suggesting that, if mutations are present, multiple copies of AChE are required to restore a normal level of catalytic activity in the monocrotophos-resistant mites. In summary, the present study provides clear evidence that Tuace duplication resulted in the proportional overexpression of AChE, which was necessary to compensate for the reduced catalytic activity of AChE caused by mutations.     

Distribution and Frequency of kdr Mutations within Anopheles gambiae s.l. Populations and First Report of the Ace.1G119S Mutation in Anopheles arabiensis from Burkina Faso (West Africa)

An entomological survey was carried out at 15 sites dispersed throughout the three eco-climatic regions of Burkina Faso (West Africa) in order to assess the current distribution and frequency of mutations that confer resistance to insecticides in An. gambiae s.l. populations in the country. Both knockdown (kdr) resistance mutation variants (L1014F and L1014S), that confer resistance to pyrethroid insecticides, were identified concomitant with the ace-1 G119S mutation confirming the presence of multiple resistance mechanisms in the An. gambiae complex in Burkina Faso. Compared to the last survey, the frequency of the L1014F kdr mutation appears to have remained largely stable and relatively high in all species. In contrast, the distribution and frequency of the L1014S mutation has increased significantly in An. gambiae s.l. across much of the country. Furthermore we report, for the first time, the identification of the ace.1 G116S mutation in An. arabiensis populations collected at 8 sites. This mutation, which confers resistance to organophosphate and carbamate insecticides, has been reported previously only in the An. gambiae S and M molecular forms. This finding is significant as organophosphates and carbamates are used in indoor residual sprays (IRS) to control malaria vectors as complementary strategies to the use of pyrethroid impregnated bednets. The occurrence of the three target-site resistance mutations in both An. gambiae molecular forms and now An. arabiensis has significant implications for the control of malaria vector populations in Burkina Faso and for resistance management strategies based on the rotation of insecticides with different modes of action.     

The IleL229 → Met mutation impairs the quinone binding to the QB-pocket in reaction centers of Rhodobacter sphaeroides

A spontaneous mutant (R/89) of photosynthetic purple bacterium Rhodobacter sphaeroides R-26 was selected for resistance to 200 M atrazin. It showed increased resistance to interquinone electron transfer inhibitors of o-phenanthroline (resistance factor, RF=20) in UQ_o reconstituted isolated reaction centers and terbutryne in reaction centers (RF=55) and in chromatophores (RF=85). The amino acid sequence of the Q_B binding protein of the photosynthetic reaction center (the L subunit) was determined by sequencing the corresponding pufL gene and a single mutation was found (Ile^L229 Met). The changed amino acid of the mutant strain is in van der Waals contact with the secondary quinone Q_B. The binding and redox properties of Q_B in the mutant were characterized by kinetic (charge recombination) and multiple turnover (cytochrome oxidation and semiquinone oscillation) assays of the reaction center. The free energy for stabilization of Q_AQ_B ^– with respect to Q_A ^–Q_B was G_AB=–60 meV and 0 meV in reaction centers and G_AB=–85 meV and –46 meV in chromatophores of R-26 and R/89 strains at pH 8, respectively. The dissociation constants of the quinone UQ_o and semiquinone UQ_o ^– in reaction centers from R-26 and R/89 showed significant and different pH dependence. The observed changes in binding and redox properties of quinones are interpreted in terms of differential effects (electrostatics and mesomerism) of mutation on the oxidized and reduced states of Q_B.Abbreviations BChl bacteriochlorophyll - Ile isoleucine - Met methionin - P primary donor - Q_A primary quinone acceptor - Q_B secondary quinone acceptor - RC reaction center protein - UQ_o 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50 This work is dedicated to the memory of Randall Ross Stein (1954–1994) and is, in a small way, a testament to the impact which Randy's ideas have had on the development of the field of competitive herbicide binding.     

Point mutations in the Drosophila sodium channel gene para associated with resistance to DDT and pyrethroid insecticides

The gene para in Drosophila melanogaster encodes an α subunit of voltage-activated sodium channels, the presumed site of action of DDT and pyrethroid insecticides. We used an existing collection of Drosophila para mutants to examine the molecular basis of target-site resistance to pyrethroids and DDT. Six out of thirteen mutants tested were associated with a largely dominant, 10- to 30-fold increase in DDT resistance. The amino acid lesions associated with these alleles defined four sites in the sodium channel polypeptide where a mutational change can cause resistance: within the intracellular loop between S4 and S5 in homology domains I and III, within the pore region of homology domain III, and within S6 in homology domain III. Some of these sites are analogous with those defined by knockdown resistance (kdr) and super-kdr resistance-associated mutations in houseflies and other insects, but are located in different homologous units of the channel polypeptide. We find a striking synergism in resistance levels with particular heterozygous combinations of para alleles that appears to mimic the super-kdr double mutant housefly phenotype. Our results indicate that the alleles analyzed from natural populations represent only a subset of mutations that can confer resistance. The implications for the binding site of pyrethroids and mechanisms of target-site insensitivity are discussed. Received: 9 May 1997 / Accepted: 21 July 1997     

How rapid are the internal reactions of the ubiquinol:cytochrome c 2 oxidoreductase?

The temperature dependence of the partial reactions leading to turn-over of the UQH₂:cyt c ₂ oxidoreductase of Rhodobacter sphaeroides have been studied. The redox properties of the cytochrome components show a weak temperature dependence over the range 280–330 K, with coefficients of about 1 m V per degree; our results suggest that the other components show similar dependencies, so that no significant change in the gradient of standard free-energy between components occurs over this temperature range. The rates of the reactions of the high potential chain (the Rieske iron sulfur center, cytochromes c ₁ and c ₂, reaction center primary donor) show a weak temperature dependence, indicating an activation energy < 8 kJ per mole for electron transfer in this chain. The oxidation of ubiquinol at the Q_z-site of the complex showed a strong temperature dependence, with an activation energy of about 32 kJ mole^–1. The electron transfer from cytochrome b-566 to cytochrome b-561 was not rate determining at any temperature, and did not contribute to the energy barrier. The activation energy of 32 kJ mole^–1 for quinol oxidation was the same for all states of the quinone pool (fully oxidized, partially reduced, or fully reduced before the flash). We suggest that the activation barrier is in the reaction by which ubiquinol at the catalytic site is oxidized to semiquinone. The most economical scheme for this reaction would have the semiquinone intermediate at the energy level indicated by the activation barrier. We discuss the plausibility of this simple model, and the values for rate constants, stability constant, the redox potentials of the intermediate couples, and the binding constant for the semiquinone, which are pertinent to the mechanism of the ubiquinol oxidizing site.Abbreviations (BChl)₂ P870, primary donor of the photochemical reaction center - b/c ₁ complex ubiquinol: cytochrome c ₂ oxidoreductase - cyt b _H cytochrome b-561 or higher potential cytochrome b - cyt b _L cytochrome b-566, or low potential cytochrome b - cyt c ₁, cyt c ₂, cyt c _t cytochromes c ₁ and c ₂, and total cytochrome c (cyt c ₁ and cyt c ₂) - Fe.S Rieske-type iron sulfur center, Q - QH₂ ubiquinone, ubiquinol - Q_z, Q_zH₂, Q_z ^– ubiquinone, ubiquinol, and semiquinone anion of ubiquinone, bound at quinol oxidizing site - Q_z-site ubiquinol oxidizing site (also called Q_o-(outside) - Q_o (Oxidizing) - Q_P (Positive proton potential) site) - Q_c-site uubiquinone reductase site (also called the Q_i-(inside) - Q_R (Reducing), or - Q_N (Negative proton potential) site) - UHDBT 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazol     

Ryanodine receptor point mutations confer diamide insecticide resistance in tomato leafminer,Tuta absoluta (Lepidoptera: Gelechiidae)

Insect ryanodine receptors (RyR) are the molecular target-site for the recently introduced diamide insecticides. Diamides are particularly active on Lepidoptera pests, including tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae). High levels of diamide resistance were recently described in some European populations of T. absoluta, however, the mechanisms of resistance remained unknown. In this study the molecular basis of diamide resistance was investigated in a diamide resistant strain from Italy (IT-GELA-SD4), and additional resistant field populations collected in Greece, Spain and Brazil. The genetics of resistance was investigated by reciprocally crossing strain IT-GELA-SD4 with a susceptible strain and revealed an autosomal incompletely recessive mode of inheritance. To investigate the possible role of target-site mutations as known from diamondback moth (Plutella xylostella), we sequenced respective domains of the RyR gene of T. absoluta. Genotyping of individuals of IT-GELA-SD4 and field-collected strains showing different levels of diamide resistance revealed the presence of G4903E and I4746M RyR target-site mutations. These amino acid substitutions correspond to those recently described for diamide resistant diamondback moth, i.e. G4946E and I4790M. We also detected two novel mutations, G4903V and I4746T, in some of the resistant T. absoluta strains. Radioligand binding studies with thoracic membrane preparations of the IT-GELA-SD4 strain provided functional evidence that these mutations alter the affinity of the RyR to diamides. In combination with previous work on P. xylostella our study highlights the importance of position G4903 (G4946 in P. xylostella) of the insect RyR in defining sensitivity to diamides. The discovery of diamide resistance mutations in T. absoluta populations of diverse geographic origin has serious implications for the efficacy of diamides under applied conditions. The implementation of appropriate resistance management strategies is strongly advised to delay the further spread of resistance.     

Evaluation of Resistance Pattern to Fenpyroximate and Pyridaben in Tetranychus Urticae collected from Ggreenhouses and Apple Orchards using Lethal Concentration-slope Relationship

This study aimed to monitor the present and future developments of the resistance of Tetranychus urticae Koch to fenpyroximate and pyridaben, using the relationship of the LC₅₀ and slope of the concentration-mortality line in a probit model, for the provision of reliable resistance management tactics. Tetranychus urticae populations were collected from 16 commercial greenhouses, where various crops were cultivated, as well as from 10 apple orchards throughout Korea. The resistance to fenpyroximate and pyridaben of each population was estimated by calculating the median lethal concentration (LC₅₀), resistance ratio (RR) and slope of the concentration-mortality regression. Most of the greenhouse populations exhibited moderate levels of resistance, whereas the apple orchard populations showed only low levels, indicating that T. urticae populations in greenhouses were more strongly selected than those in apple orchards. Four population groups were established based on either the habitats (greenhouse and apple orchard) or acaricides (fenpyroximate and pyridaben). To test the hypothesis, “the slope is greatest at low and high levels of resistance,” the slopes were regressed as a function of the LC₅₀, and fitted to a polynomial regression. The polynomial regression model explained this relationship well for the four population groups (p < 0.05), indicating that the development of resistance toward fenpyroximate or pyridaben was consistent with the gradient. A laboratory selection study agreed with the results from both acaricide field populations. These results suggest that the gradient was a good indicator of the susceptibility of T. urticae to genetic variations, which was related to the LC₅₀. The application of these findings is also discussed in relation to the resistance management of T. urticae.