Role of esterase enzymes in monitoring for resistance of California red scale, Aonidiella aurantii (Homoptera: Diaspididae), to organophosphate and carbamate insecticides

Eighty-seven populations of California red scale, Aonidiella aurantii (Maskell), from the San Joaquin Valley of California were tested for insecticide resistance by using chlorpyrifos, methidathion, and/or carbaryl in a standard fruit-dip bioassay as well as for general esterase activity by using alpha-naphthyl acetate as a substrate in a colorimetric test. The percentage of individuals that survived a discriminating concentration of methidathion, chlorpyrifos, or carbaryl was significantly correlated with the percentage of individuals showing > 0.4 nmol of esterase activity per minute per microgram of protein in the colorimetric test. Scale survival of the organophosphates showed a higher correlation with esterase activity than survival of carbaryl. These results suggest that the colorimetric test of esterase activity is useful as an indicator of the frequency of organophosphate-resistant and, to a lesser extent, carbamate-resistant individuals in California red scale populations. The results of tests for activity and inhibition of acetylcholinesterase activity suggest that California red scale is using increased amounts of esterase enzymes, including acetylcholinesterase, to sequester organophosphate and carbamate insecticides, rather than modified acetylcholinesterase. Third instars collected from twigs, leaves, and fruit showed similar levels of esterase activity. The colorimetric test of esterase activity is a useful tool to detect organophosphate and carbamate resistance in San Joaquin Valley California red scale because of its speed of testing over a wide range of months, allowing for within-season decision making by citrus growers.     

Mutation in the ace‐1 gene of the tomato leaf miner (Tuta absoluta) associated with organophosphates resistance

The tomato leaf miner, Tuta absoluta (Lepidoptera: Gelechiidae), is a major invasive pest that has spread throughout many countries in the Mediterranean basin and parts of Asia over the last decade. The control of T. absoluta has relied heavily on the use of chemical insecticides, a strategy that has led to the evolution of resistance. In this study, biological and molecular methods were used to determine the susceptibility of five strains of T. absoluta to the organophosphate chlorpyrifos and to investigate the molecular mechanisms underlying resistance to this class of insecticides. High levels of resistance to chlorpyrifos were observed in all five strains tested. Cloning and sequencing of the gene encoding the organophosphate target site, ace‐1, of T. absoluta revealed the presence of an alanine to serine substitution at a position that has been previously linked with organophosphate resistance across a range of different insect and mite species. The presence of this mutation at high frequency in T. absoluta populations originating from various countries further supports the suggestion that the rapid expansion of this species is, in part, mediated by the resistance of this pest to chemical insecticides.     

Distribution and dynamics of esterase alleles in Culex pipiens complex in China

To investigate insecticide resistance and dynamic changes of carboxylesterase polymorphism in mosquitoes with time in the Culex pipiens complex (Diptera: Culicidae), nine field mosquito populations were collected in China. The resistance levels of fourth-instar larvae to organophosphate (dichlorvos, parathion, and chlorpyrifos), carbamate (fenobucarb and propoxur), and pyrethroid (permethrin, deltamethrin and tetramethrin) insecticides were determined by bioassay. Larvae had more resistance to organophosphate insecticides than to carbamate insecticides. A low but significant resistance was observed for carbamate insecticides. The resistance to pyrethroid insecticides varied from sensitive to high. Starch gel electrophoresis revealed the presence of the overproduced esterases B1, A2B2, A8B8, A9B9, B10 and A11B11. The frequency of each overproduced esterases varied depending on its regional localities. Compared with published surveys, the C. pipiens complex, which exhibited a high polymorphism of applied esterase alleles in China, showed dynamic evolution over time under local specific insecticide selection. The results are discussed in the context of recent alterations to insecticide campaigns, and in the evolution of resistance genes in Chinese C. pipiens populations.     

Resurgence of the cotton bollworm Helicoverpa armigera in northern Greece associated with insecticide resistance

Helicoverpa armigera has been controlled effectively with chemical insecticides in the major cotton crop production areas of northern Greece for many years. However, a resurgence of the pest was observed in 2010, which significantly affected crop production. During a 4‐year survey (2007 – 2010), we examined the insecticide resistance status of H. armigera populations from two major and representative cotton production areas in northern Greece against seven insecticides (chlorpyrifos, diazinon, methomyl, alpha‐cypermethrin, cypermethrin, gamma‐cyhalothrin and endosulfan). Full dose‐response bioassays on third instar larvae were performed by topical application. Lethal doses at 50% were estimated by probit analysis and resistance factors (RF) were calculated, compared to a susceptible laboratory reference strain. Resistance levels were relatively moderate until 2009, with resistance ratios below 10‐fold for organophosphates and carbamates and up to 16‐fold for the pyrethroid alpha‐cypermethrin. However, resistance rose to 46‐ and 81‐fold for chlorpyrifos and alpha‐cypermethrin, respectively in 2010, when the resurgence of the pest was observed. None of the known pyrethroid resistance mutations were found in the pyrethroid‐resistant insects. The possible association between resistance and H. armigera resurgence in Greece is discussed.     

Could resistance to insecticides in Plutella xylostella (Lep., Plutellidae) be overcome by insecticide mixtures?

Abstract: To investigate if synergism occurs between pyrethroids, organophosphates and new insecticides, we tested representatives of these compounds (bifenthrin, chlorpyrifos, spinosad, indoxacarb and emamectin) against the diamondback moth (Plutella xylostella). Larvicidal activity of these insecticides was assessed separately and together on a susceptible strain (Lab‐UK) of P. xylostella as well as a field population collected from Multan. The field population showed significant resistance to chlorpyrifos (331 100‐fold), bifenthrin (45 200‐fold), emamectin (1800‐fold), spinosad (11‐fold) and indoxacarb (5600‐fold) when compared with the Lab‐UK population. When insecticides were mixed based on LC₅₀ and tested at serial concentrations against Lab‐UK, significant synergy (CI < 1) occurred between bifenthrin, spinosad and emamectin. In contrast, the interaction between bifenthrin and indoxacarb was additive (CI = 1). The toxicity of bifenthrin against the field population increased significantly (P < 0.01) when combined with spinosad, emamectin and indoxacarb. Synergistic effects could be attributed to the complementary modes of action by these insecticide classes acting on different components of nerve impulse transmission (which are not identical for bifenthrin and indoxacarb either). However, chlorpyrifos/bifenthrin mixture was not significantly different either from bifenthrin or chlorpyrifos alone, indicating an additive affect. In combination with spinosad and emamectin, tested against the resistant field population, the toxicity of chlorpyrifos increased significantly and even more so with indoxacarb. Mixtures could also give rise to multiple resistance that may extend across other chemical classes and thus become difficult to manage. Therefore, alternative strategies such as mosaics or rotations should be considered. That is, though synergistic effects have been found, this should not be followed up as a strategy to manage resistant field populations.     

Establishing the role of detoxifying enzymes in field‐evolved resistance to various insecticides in the brown planthopper (Nilaparvata lugens) in South India

The brown planthopper (BPH), Nilaparvata lugens (Stål), is one of the major pests of rice throughout Asia. Extensive use of insecticides for suppressing N. lugens has resulted in the development of insecticide resistance leading to frequent control failures in the field. The aim of the present study was to evaluate resistance in the field populations of N. lugens from major rice growing states of South India to various insecticides. We also determined the activity of detoxifying enzymes (esterases [ESTs], glutathione S‐transferases [GSTs], and mixed‐function oxidases [MFOs]). Moderate levels of resistance were detected in the field populations to acephate, thiamethoxam and buprofezin (resistance factors 1.05–20.92 fold, 4.52–14.99 fold, and 1.00–18.09 fold, respectively) as compared with susceptible strain while there were low levels of resistance to imidacloprid (resistance factor 1.23–6.70 fold) and complete sensitivity to etofenoprox (resistance factor 1.05–1.66 fold). EST activities in the field populations were 1.06 to 3.09 times higher than the susceptible strain while for GST and MFO the ratios varied from 1.29 to 3.41 and 1.03 to 1.76, respectively. The EST activity was found to be correlated to acephate resistance (r = 0.999, P ≥ 0.001). The high selection pressure of organophosphate, neonicotinoid, and insect growth regulator (IGR) in the field is likely to be contributing for resistance in BPH to multiple insecticides, leading to control failures. The results obtained will be beneficial to IPM recommendations for the use of effective insecticides against BPH.     

Determination of the resistance to organophosphate, carbamate, and pyrethroid insecticides in Panamanian Anopheles albimanus (Diptera: Culicidae) mosquitoes

Introduction. The susceptibility of Anopheles albimanus to organophosphates, carbamates and pyrethroid insecticides was unknown in the Panama communities of Aguas Claras, Pintupo and Puente Bayano, located in the Amerindian Reservation of Madungandi. This region is considered a malaria transmission area, where An. albimanus is the main vector. Objective. The resistance to organophosphate insecticides, carbamates and pyrethroids was evaluated in field populations of the Anopheles albimanus in Panama. Materials and methods. Progeny of An. albimanus collected in three localities in the indigenous Madugandi region were exposed to bioassays of susceptibility to organophosphate insecticides (fenitrothion, malathion and chlorpyrifos), the carbamate (propoxur) and pyrethroids (deltamethrin, lambdacyhalothrin, cyfluthrin and cypermethrin). The protocols were in accordance with those established for adult mosquitoes by World Health Organization. Results. The three strains of the An. albimanus were resistant to the pyrethroid insecticides deltamethrin, lambdacyhalothrin, cyfluthrin and cypermethrin. Susceptibility remained for the organophosphate insecticides fenitrothion, malathion, chlorpyrifos, and the carbamate insecticide propoxur. Conclusion. The results provided important information to the vector control program, contributing to the application of new strategies on the use of insecticides, and thereby lengthening the life of the insecticide in use.     

Organophosphate and pyrethroid resistances in the tomato leaf miner Tuta absoluta (Lepidoptera: Gelechiidae) from Iran

The tomato leafminer, Tuta absoluta (Meyrich) (Lepidoptera: Gelechiidae), is a serious pest of tomato crops worldwide. The intensive use of chemical pesticides to control it has led to the selection of resistant populations. This study investigated the resistance of T. absoluta populations to pyrethroid and the organophosphate insecticides from ten regions of Iran. The resistance ratios at LC₅₀ for chlorpyrifos and diazinon varied among populations from 4.3 to 12 and from 1.4 to 9.0, respectively. The resistance ratios of the pyrethroids cypermethrin, deltamethrin and permethrin varied from 1.3 to 3.7, 2.7 to 13 and 1.2 to 4.3, respectively. Inclusion of synergists in toxicological bioassays and the variation observed in the activity of esterases, glutathione S‐transferase and cytochrome P450‐dependent monooxygenase suggest the existence of metabolically based resistance. Esterase and P450 biochemical assays were positively correlated with deltamethrin, and cypermethrin tolerance and diazinon tolerance correlated with esterase activity. The genes encoding the organophosphate and pyrethroid target sites acetylcholinesterase (ace1) and sodium channel (kdr) were partly sequenced. The genotyping revealed mutations in high frequencies in all populations leading to an A201S substitution in ace1 and three substitutions in the sodium channel gene L1014F, M918T, T929I. In summary, our results indicate the presence of organophosphate and pyrethroid resistance in Iranian T. absoluta populations with involvement of both detoxification enzymes and target site alterations. Most likely the populations of T. absoluta imported to Iran were resistant upon arrival.     

A point mutation in the acetylcholinesterase-1 gene is associated with chlorpyrifos resistance in the plant bug Apolygus lucorum

Control of Chinese Apolygus lucorum relies heavily on organophosphate insecticides. Here we describe resistance to the organophosphate chlorpyrifos in an A. lucorum strain, BZ-R, which was developed from a field-collected strain (BZ) by selection with chlorpyrifos in the laboratory. BZ-R showed 21–58 fold resistance to chlorpyrifos compared with the laboratory reference strain LSF and another susceptible strain, BZ-S, derived from BZ. BZ-R also showed several fold resistance to two other organophosphates and a carbamate. No synergism of chlorpyrifos by metabolic enzyme inhibitors nor any increase in detoxifying enzyme activities were observed in BZ-R. No sequence differences in acetylcholinesterase-2 were found to be associated with the resistance but the frequency of an alanine to serine substitution at position 216 of acetylcholinesterase-1 was 100% in BZ-R, ∼21–23% in SLF and BZ, and 0% in BZ-S. A single generation treatment of chlorpyrifos on the BZ strain also increased its frequency of the serine substitution to 64%. Recombinantly expressed acetylcholinesterase-1 carrying the serine substitution was about five fold less sensitive to inhibition by chlorpyrifos oxon than the wild-type enzyme. Quantitative real-time PCR found no differences in ace1 or ace2 expression levels among the strains tested. Thus the chlorpyrifos resistance is strongly associated with the serine substituted acetylcholinesterase-1. An equivalent substitution has been found to confer resistance to many organophosphate and carbamate insecticides in four other insect species.     

Effects of temperature on fitness costs in chlorpyrifos‐resistant brown planthopper,Nilaparvata lugens (Hemiptera: Delphacidae)

Insecticide resistance is inevitable if an insecticide is widely used to control insect pests. Fortunately, the resistance‐associated fitness costs often give chances to manage resistances. In most cases, the fitness cost in resistant insects is often evaluated under laboratory conditions for insect development, which limits its practical application in pest control in the field. In a laboratory population R9 with 253‐fold resistance to chlorpyrifos after nine‐generation selection with chlorpyrifos, the relative fitness was only 0.206 under laboratory conditions (25°C, humidity 70%–80% and 16 h light/8 h dark photoperiod), when compared to S9, a susceptible counterpart (resistance ratio = 2.25‐fold) from the same origin as R9 but without any selection with insecticides. Temperatures varied the resistance‐associated fitness costs, with enhanced costs at high temperatures and reduced costs at low temperatures, such as 0.174 at 32°C and 0.527 at 18°C. The copulation rate and fecundity were two key factors for the reduced costs at low temperatures. Another finding was that R9 individuals needed much more time to recover from heat shock than that of S9, but R9 and S9 individuals were similarly sensitive to cold shock. The low fitness cost at low temperatures would increase the overwintering population, which might further increase risks of rapid development and widespread distribution of chlorpyrifos resistance in Nilaparvata lugens.     

Monitoring insecticide resistance in Australian Frankliniella occidentalis Pergande (Thysanoptera: Thripidae) detects fipronil and spinosad resistance

Abstract  Insecticide resistance monitoring using a Potter precision spray tower with discriminating concentration and log dose probability techniques underpins the Australian insecticide management strategy for Frankliniella occidentalis Pergande. Abamectin, acephate, chlorpyrifos, dichlorvos, dimethoate, endosulfan, fipronil, malathion, methamidophos methidathion, methiocarb, methomyl, pyrazophos and spinosad are recommended for use against F. occidentalis but abamectin, methiocarb and pyrazophos are the only chemicals where insecticide resistance has not been detected. Although not registered, chlorfenapyr was effective against F. occidentalis and should be pursued for that purpose. In contrast, chlorpyrifos, dichlorvos and malathion resistance were detected at low to moderate levels throughout the study period putting their sustainable use for F. occidentalis control in doubt . Although it appears that acephate, dimethoate, endosulfan, fipronil, methamidophos, methidathion and spinosad remain effective, some populations contained a small percentage of thrips that survived exposure to a concentration that killed 100% of the susceptible strain. Subsequent laboratory selection of one such population separately with fipronil and spinosad caused an increase in resistance to these insecticides. These products must now be considered at risk. This is the first report of fipronil or spinosad resistance in populations of F. occidentalis.     

Insecticide resistance in populations of Tuta absoluta (Lepidoptera: Gelechiidae)

1 Control failures of insecticides used against the tomato leafminer Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) in Brazil led to the investigation of the possible occurrence of resistance of this insect pest to abamectin, cartap, methamidophos and permethrin. 2 The insect populations were collected from seven sites in the states of Minas Gerais, Rio de Janeiro, and São Paulo. These populations were subjected to concentration–mortality bioassays using insecticide‐impregnated filter papers. 3 We were unable to obtain a single population which provided a susceptibility standard for all insecticides tested. Therefore, the resistance levels were estimated in relation to the most susceptible population to each insecticide. Resistance to abamectin and cartap were observed in all populations when compared with the susceptible standard population, with resistance ratios ranging from 5.2‐ to 9.4‐fold and from 2.2‐ to 21.9‐fold for abamectin and cartap, respectively. Resistance to permethrin was observed in five populations with resistance ratios ranging from 1.9‐ to 6.6‐fold, whereas resistance to methamidophos was observed in four populations with resistance ratios ranging from 2.6‐ to 4.2‐fold. 4 The long period and high frequency of use of these insecticides against this insect pest suggest that the evolution of insecticide resistance on them has been relatively slow. Alternatively, the phenomenon might be widespread among Brazilian populations of T. absoluta making the finding of suitable standard susceptible populations difficult and leading to an underestimation of the insecticide resistance levels in this pest. 5 Higher levels of resistance to abamectin, cartap and permethrin are correlated with greater use of these compounds by growers. This finding suggests that local variation in insecticide use was an important cause of variation in susceptibility.     

Trade‐off between thermal tolerance and insecticide resistance in Plutella xylostella

Fitness costs associated with resistance to insecticides have been well documented, usually at normal temperature conditions, in many insect species. In this study, using chlorpyrifos‐resistant homozygote (R_R) and chlorpyrifos‐susceptible homozygote (S_S) of resistance ace1 allele of Plutella xylostella (DBM), we confirmed firstly that high temperature experience in pupal stage influenced phenotype of wing venation in insecticide‐resistant and insecticide‐susceptible Plutella xylostella, and S_S DBM showed significantly higher thermal tolerance and lower damages of wing veins under heat stress than R_R DBM. As compared to S_S DBM, R_R DBM displayed significantly lower AChE sensitivity to chlorpyrifos, higher basal GSTs activity and P450 production at 25°C, but higher inhibitions on the enzyme activities and P450 production as well as reduced resistance to chlorpyrifos under heat stress. Furthermore, R_R DBM displayed significantly higher basal expressions of hsp69s, hsp72s, hsp20, hsp90, Apaf‐1, and caspase‐7 at 25°C, but lower induced expressions of hsps and higher induced expressions of Apaf‐1, caspase‐9, and caspase‐7 under heat stress. These results suggest that fitness costs of chlorpyrifos resistance in DBM may partly attribute to excess consumption of energy caused by over production of detoxification enzymes and hsps when the proteins are less demanded at conducive environments but reduced expressions when they are highly demanded by the insects to combat environmental stresses, or to excess expressions of apoptotic genes under heat stress, which results in higher apoptosis. The evolutionary and ecological implications of these findings at global warming are discussed.     

Characterization of mosquito CYP6P7 and CYP6AA3: differences in substrate preference and kinetic properties

Cytochrome P450 monooxygenases are involved in insecticide resistance in insects. We previously observed an increase in CYP6P7 and CYP6AA3 mRNA expression in Anopheles minimus mosquitoes during the selection for deltamethrin resistance in the laboratory. CYP6AA3 has been shown to metabolize deltamethrin, while no information is known for CYP6P7. In this study, CYP6P7 was heterologously expressed in the Spodoptera frugiperda (Sf9) insect cells via baculovirus‐mediated expression system. The expressed CYP6P7 protein was used for exploitation of its enzymatic activity against insecticides after reconstitution with the An. minimus NADPH‐cytochrome P450 reductase enzyme in vitro. The ability of CYP6P7 to metabolize pyrethroids and insecticides in the organophosphate and carbamate groups was compared with CYP6AA3. The results revealed that both CYP6P7 and CYP6AA3 proteins could metabolize permethrin, cypermethrin, and deltamethrin pyrethroid insecticides, but showed the absence of activity against bioallethrin (pyrethroid), chlorpyrifos (organophosphate), and propoxur (carbamate). CYP6P7 had limited capacity in metabolizing λ‐cyhalothrin (pyrethroid), while CYP6AA3 displayed activity toward λ‐cyhalothrin. Kinetic properties suggested that CYP6AA3 had higher efficiency in metabolizing type I than type II pyrethroids, while catalytic efficiency of CYP6P7 toward both types was not significantly different. Their kinetic parameters in insecticide metabolism and preliminary inhibition studies by test compounds in the flavonoid, furanocoumarin, and methylenedioxyphenyl groups elucidated that CYP6P7 had different enzyme properties compared with CYP6AA3. © 2011 Wiley Periodicals, Inc.     

Behavioural and insecticidal effects of organophosphate‐, carbamate‐ and pyrethroid‐treated mosquito nets against African malaria vectors

Three insecticides – the pyrethroid deltamethrin, the carbamate carbosulfan and the organophosphate chlorpyrifos‐methyl – were tested on mosquito nets in experimental huts to determine their potential for introduction as malaria control measures. Their behavioural effects and efficacy were examined in Anopheles gambiae Giles s.s. (Diptera: Culicidae) and Anopheles funestus Giles s.s. in Muheza, Tanzania, and in Anopheles arabiensis Patton and Culex quinquefasciatus Say in Moshi, Tanzania. A standardized dosage of 25 mg/m² plus high dosages of carbosulfan (50 mg/m², 100 mg/m² and 200 mg/m²) and chlorpyrifos‐methyl (100 mg/m²) were used to compare the three types of insecticide. At 25 mg/m², the rank order of the insecticides for insecticide‐induced mortality in wild An. gambiae and An. funestus was, respectively, carbosulfan (88%, 86%) > deltamethrin (79%, 78%) > chlorpyrifos‐methyl (35%, 53%). The rank order of the insecticides for blood‐feeding inhibition (reduction in the number of blood‐fed mosquitoes compared with control) in wild An. gambiae and An. funestus was deltamethrin > chlorpyrifos‐methyl > carbosulfan. Carbosulfan was particularly toxic to endophilic anophelines at 200 mg/m², killing 100% of An. gambiae and 98% of An. funestus that entered the huts. It was less effective against the more exophilic An. arabiensis (67% mortality) and carbamate‐resistant Cx quinquefasciatus (36% mortality). Carbosulfan deterred anophelines from entering huts, but did not deter carbamate‐resistant Cx quinquefasciatus. Deltamethrin reduced the proportion of insects engaged in blood‐feeding, probably as a consequence of contact irritancy, whereas carbosulfan seemed to provide personal protection through deterred entry or perhaps a spatial repellent action. Any deployment of carbosulfan as an individual treatment on nets should be carried out on a large scale to reduce the risk of diverting mosquitoes to unprotected individuals. Chlorpyrifos‐methyl was inferior to deltamethrin in terms of mortality and blood‐feeding inhibition and would be better deployed on a net in combination with a pyrethroid to control insecticide‐resistant mosquitoes.     

Baseline susceptibility of Planococcus ficus (Hemiptera: Pseudococcidae) from California to select insecticides

Between 2006 and 2008, 20 populations of Planococcus ficus (Signoret), from Coachella and San Joaquin Valleys of California were measured in the laboratory for susceptibility to buprofezin, chlorpyrifos, dimethoate, methomyl, and imidacloprid. Toxicity was assessed using a petri dish bioassay technique for contact insecticides and by a systemic uptake technique for imidacloprid. Mixed life stages were tested for susceptibility to all insecticides except for buprofezin, which was measured against early and late instars (first, second, and third). Dose-response regression lines from the mortality data established LC50 and LC99 values by both techniques. Responses of populations from the two geographical locations to all five insecticides varied, in some cases significantly. Variations in susceptibility to each insecticide among sample sites showed a sevenfold difference for buprofezin, 11-fold to chlorpyrifos, ninefold to dimethoate, 24-fold to methomyl, and 8.5-fold to imidacloprid. In spite of susceptibility differences between populations, baseline toxicity data revealed that all five insecticides were quite effective based on low LC50s. Chlorpyrifos was the most toxic compound to Planococcus ficus populations as shown by lowest LC50s. Buprofezin was toxic to all immature stages but was more potent to first instars. The highest LC99 estimated by probit analysis of the bioassay data of all 20 populations for each compound was selected as a candidate discriminating dose for use in future resistance monitoring efforts. Establishment of baseline data and development of resistance monitoring tools such as bioassay methods and discriminating doses are essential elements of a sustainable management program for Planococcus ficus.     

Assessment of esterase gene expression as a risk marker for insecticide resistance in Florida Culex nigripalpus (Diptera: Culicidae)

Esterases are enzymatic proteins known to play a role in insecticide resistance formation. To further our understanding of the development of insecticide resistance, we tested the gene expression level of a gene implicated in insecticide resistance (Temsha est‐1) from Culex nigripalpus Theobald (Diptera: Culicidae) in field mosquitoes. We found that the level of expression of TE‐1 differed depending on the frequency of exposure to organophosphate insecticide through expression studies. Temsha est‐1 cDNA is 1,808 base pairs and fully sequenced with up to 96% nucleotide sequence identity to esterase B genes of other mosquito species. The genes from five different species, including TE‐1, were closely related by genetic distance and phylogenetic analysis. Differential expression of this gene that is correlated to differences in susceptibility towards organophosphate would provide the ability to use Temsha est‐1 as an indicator of the formation of tolerance/resistance. This would greatly enhance mosquito control efforts by allowing targeted application of insecticides to mosquito populations that are most susceptible. Also, it would provide resistance information so that a rational design could be used for insecticide rotation schedules.     

Cytochrome P450 MA expression in insecticide-resistant German cockroaches (Dictyoptera: Blattellidae).

Cytochrome P450 monooxygenases are detoxification enzymes commonly involved in insecticide resistance by insects. Recently, an overexpressed form of this enzyme, P450 MA, was purified from an insecticide-resistant strain of German cockroach, Blattella germanica (L.), and polyclonal antisera (anti-P450 MA) was produced. To test hypotheses that the overexpressed condition of P450 MA has evolved in > 1 geographic location and that P450 MA might be involved in insecticide resistance to specific insecticides, investigations were conducted using 4 insecticide-resistant and 1 susceptible German cockroach strains. In western blots that used anti-P450 MA antiserum as a probe, substantial differences in expression of P450 MA were observed. Strains showing the highest P450 MA expression had both the highest tolerance to the organophosphate insecticide chlorpyrifos and cytochrome P450-mediated demethylation activity. Results support the hypothesis that cytochrome P450 MA is potentially overexpressed in insecticide-resistant populations on a global scale.     

Biotype and status of insecticide resistance of whitefly Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) in Alhassa oasis,Eastern Province of Saudi Arabia.

The tobacco whitefly, Bemisia tabaci, is one of the major insects infesting vegetable plants. This pest is well known in Alhassa oasis, Saudi Arabia; which is historically agricultural land cultivated with date palm trees and different vegetables. A molecular key based on the sequence of the mitochondrial cytochrome oxidase gene CO1 was used for the identification of strains of the tobacco whitefly Bemisia tabaci collected from farms located in four areas of the Alhassa oasis; Northern, Southern, Eastern and Western. Only one biotype (B‐biotype) of B. tabaci was reported in the oasis. Resistance of B. tabaci was tested against eight insecticides, the results showed moderate to low levels of resistance to the tested insecticides. However, the resistance to neonicotinoid insecticides was low and established at 1.3 fold to both Imidacloprid and Acetamiprid. In addition, medium levels of resistance were detected to the insect growth regulator Pyriproxyfen (30 fold), and the pyrethroid Deltamethrin (30 fold), Bifenthrin (24 fold) and Cypermethrin (13 fold). A medium level of resistance was also detected to the carbamate insecticide Carbosulfan and was 40 fold of the laboratory strains. A low level of resistance to the organophosphorus insecticide was detected to Phenthoate (11 fold). However, these results reflect that the farmers were less dependent on the use of insecticides to control B. tabaci in the oasis and they may be implementing other environmentally sound techniques to keep the pest below the damage threshold.     

Functional and genetic characteristics of Chlorantraniliprole resistance in the diamondback moth,Plutella xylostella (Lepidoptera: Plutellidae)

The diamondback moth (Plutella xylostella) is a globally distributed and important economic pest, and it has developed resistance to all conventional insecticide classes used in the field. Chlorantraniliprole is a new chemical class of insecticide that acts as a conformation‐sensitive activator of the insect ryanodine receptor (RyR). In the present study, a field strain (16.3‐fold resistance to chlorantraniliprole) was collected in Korea and lab‐selected with chlorantraniliprole for more than one year. The resulting strain presented 2,157‐fold resistance to chlorantraniliprole. A point mutation (G4946E) in the RyR gene was observed at a high frequency in the resistant strain. Enzyme assays indicated that glutathione S‐transferase (GST) and P450 activity in the resistant strain were 2.4‐ and 1.96‐times higher than that of the susceptible strain, respectively. The expression of the RyR, GST (sigma, omega, and zeta) and CYP321E1 gene was higher in the resistant strain than in the susceptible strain. The F₁ progeny resulting from reciprocal crosses did not reveal maternal effects or a diamide‐susceptible phenotype, which suggests an autosomal nearly recessive mode of inheritance. In addition, we surveyed the susceptibility to 13 insecticides (3 diamides, 2 synthetic pyrethroids, 2 spinosyns, 1 organophosphate, 1 oxadiazine, 1 avermectin, and 3 others) in the chlorantraniliprole‐resistant strain. The resistant strain exhibited high cross‐resistance to flubendiamide (5,910 fold) and showed no cross‐resistance to spinetoram, spinosad, indoxacarb, and metaflumizone. These results can serve as an important basis for guiding the use of insecticides in the field.