Monitoring pyrethroid resistance in field collected Blattella germanica Linn. (Dictyoptera: Blattellidae) in Indonesia

The German cockroach, Blattella germanica , is a major and the most common pest in public areas in Indonesia. Although intensive control measures have been carried out to control the populations of this pest, results have been far from successful, which is believed to be because of its resistance to insecticides. A standard World Health Organization (WHO) glass jar test was carried out to determine the resistance level of this insect to pyrethroid insecticides, the most commonly used insecticides for cockroach control in Indonesia. A susceptible S1 strain collected from Tembagapura Papua was compared with four strains collected from Bandung, West Java: strain S2, from a local restaurant; strain S3, from the Bandung train station; and strains S4 and S5, from two different hotels. All strains showed low resistance to the pyrethroid, except the S5 strain, which had a Resistance Ratio (RR)₅₀ of 95 for permethrin. The addition of piperonyl butoxide (PBO) suggests that the detoxifying enzyme mixed function oxidases (MFO) played an important role in the development of resistance to permethrin in the S5 strain, suggested by the high Synergist Ratio (SR) of 70.4. However, the low level of resistance to cypermethrin was not affected by PBO, suggesting that other mechanisms of pyrethroid resistance are involved. Our study is the first report of German cockroach resistance to permethrin in Indonesia, and the findings can be used in formulating potential strategies for cockroach resistance management.     

Relationship between the para-homologous sodium channel point mutation (g --> c at nucleotide 2979) and knockdown resistance in the German cockroach using multiplex polymerase chain reaction to discern genotype

Extensive use of pyrethroid insecticides for urban pest control has led to widespread pyrethroid resistance in the German cockroach. A mutation at nucleotide position 2979 (G to C, causing a leucine to phenylalanine change) in the S6 transmembrane segment of domain II of the para-homologous voltage-gated sodium channel has been previously identified in knockdown-resistant cockroaches and demonstrated by site-directed mutagenesis to reduce channel sensitivity to pyrethroids. In a recent survey, 83% of pyrethroid-resistant German cockroach populations were found to possess this mutation. A German cockroach strain with a low incidence of the L993F mutation was subjected to selection pressure with cypermethrin and subsequently evaluated over several generations for the knockdown resistance phenotype. Correspondingly, we determined the genotype of individual cockroaches of each population at the 2979 position of the para-homologous gene. Genotype was discerned by development of a polymerase chain reaction method that employed a mismatched primer-template set. A direct relationship was observed between mean knockdown time and the presence of the kdr mutation. Furthermore, individuals homozygous for the kdr mutation exhibited a significantly higher mean knockdown time than heterozygotes or wildtype cockroaches. This is the first report demonstrating the progressive expression of the kdr allele in response to insecticide selection pressure.     

Monitoring for insecticide resistance in field-collected populations of Blattella germanica (Blattaria: Blattellidae)

Seven field-collected populations of German cockroach, Blattella germanica (L.) (ICN, DNH, CHN, DGU, BSN, GSN, and GWJ), were tested for susceptibility to eight different insecticides by a topical application method, and were compared to an insecticide-susceptible KSS strain. Marked regional variations of insecticide susceptibility were observed. Extremely high to high levels of resistance were observed in bifenthrin [resistance ratio (RR), 46–159], deltamethrin (RR, 61–160), and esfenvalerate [RR, 70–270; except for BSN females (RR, 20) and GSN females (RR, 24)]. Low to moderate levels of resistance were observed in cypermethrin [RR, 16–29; except for DGU females (RR, 88) and BSN females (RR, 41)], permethrin [RR, 11–39; except for DNH females (RR, 110) and BSN females (RR, 44)], chlorpyrifos [RR, 2–13; except for GSN and GWJ females (RR, ca. 140)], chlorpyrifos-methyl (RR, 2–8), and fenthion (RR, 8–17). All of the colonies were susceptible to one or more of the insecticides examined. These results indicate that careful selection and rotational use of these insecticides should result in continued satisfactory control against field populations of German cockroach.     

Insecticide resistance and cross-resistance in the house fly (Diptera: Muscidae)

A house fly strain, ALHF, was collected from a poultry farm in Alabama after a control failure with permethrin, and further selected in the laboratory with permethrin for five generations. The level of resistance to permethrin in ALHF was increased rapidly from an initial 260-fold to 1,800-fold after selection. Incomplete suppression of permethrin resistance by piperonyl butoxide (PBO) and S,S,S,-tributylphosphorotrithioate (DEF) reveals that P450 monooxygenase- and hydrolase-mediated detoxication, and one or more additional mechanisms are involved in resistance to permethrin. The ALHF strain showed a great ability to develop resistance or cross-resistance to different insecticides within and outside the pyrethroid group including some relatively new insecticides. Resistance to beta-cypermethrin, cypermethrin, deltamethrin, and propoxur (2,400-4,200-, 10,000-, and > 290-fold, respectively, compared with a susceptible strain, aabys) in ALHF house flies was partially or mostly suppressed by PBO and DEF, indicating that P450 monooxygenases and hydrolases are involved in resistance to these insecticides. Partial reduction in resistance with PBO and DEF implies that multiresistance mechanisms are responsible for resistance. Fifteen- and more than fourfold resistance and cross-resistance to chlorpyrifos and imidacloprid, respectively, were not effected by PBO or DEF, indicating that P450 monooxygenases and hydrolases are not involved in resistance to these two insecticides. Forty-nine-fold cross-resistance to fipronil was mostly suppressed by PBO and DEF, revealing that monooxygenases are a major mechanism of cross-resistance to fipronil. Multiresistance mechanisms in the ALHF house fly strain, however, do not confer cross-resistance to spinosad, a novel insecticide derived from the bacterium Saccharopolyspora spinosa. Thus, we propose that spinosad be used as a potential insecticide against house fly pests, especially resistant flies.     

Musca domestica L D.

Field strains of the house fly (Musca domestica L. Diptera: Muscidae) were collected in April and September 2002 from cow farms (Antalya, Izmir) and garbage dumps (Adana, Ankara, Istanbul, Sanliurfa) in Turkey. The resistance levels of first to fifth generation offspring were evaluated against six insecticides (cypermethrin, cyphenothrin, deltamethrin, permethrin, resmethrin, fenitrothion). Resistance levels for pyrethroid group insecticides ranged from 23.27 (permethrin-Istanbul fall strain) to 633.09 (cypermethrin-Izmir spring strain) and for fenitrothion ranged from 5.78 (Istanbul fall strain) to 51.04 (Antalya spring strain). Our results showed that pyrethroid resistance was high and changed from spring to fall in relation to usage and application frequencies of these compounds at the study sites. Although fenitrothion resistance levels were determined to be lower than pyrethroids, these levels were still high and led to control failure. Flies from cow farms were more resistant than those from garbage dumps, but resistance levels for Sanliurfa and Adana strains were also high in relation to usage of different insecticides for agricultural purposes. Although resistance levels against different pyrethroids decreased from spring to fall, these levels still indicated the presence of a strong selective pressure on the populations.     

The status and seasonal changes of organophosphate and pyrethroid resistance in Turkish populations of the house fly, Musca domestica L. (Diptera: Muscidae).

Field strains of the house fly (Musca domestica L. Diptera: Muscidae) were collected in April and September 2002 from cow farms (Antalya, Izmir) and garbage dumps (Adana, Ankara, Istanbul, Sanliurfa) in Turkey. The resistance levels of first to fifth generation offspring were evaluated against six insecticides (cypermethrin, cyphenothrin, deltamethrin, permethrin, resmethrin, fenitrothion). Resistance levels for pyrethroid group insecticides ranged from 23.27 (permethrin-Istanbul fall strain) to 633.09 (cypermethrin-Izmir spring strain) and for fenitrothion ranged from 5.78 (Istanbul fall strain) to 51.04 (Antalya spring strain). Our results showed that pyrethroid resistance was high and changed from spring to fall in relation to usage and application frequencies of these compounds at the study sites. Although fenitrothion resistance levels were determined to be lower than pyrethroids, these levels were still high and led to control failure. Flies from cow farms were more resistant than those from garbage dumps, but resistance levels for Sanliurfa and Adana strains were also high in relation to usage of different insecticides for agricultural purposes. Although resistance levels against different pyrethroids decreased from spring to fall, these levels still indicated the presence of a strong selective pressure on the populations.     

The joint toxicity of type I, II, and nonester pyrethroid insecticides

Evidence suggests that there are separate binding domains for type I and II pyrethroid insecticides on the voltage gated sodium channel of the nerve cell axon, but there are no studies that have examined the mixture toxicity of nonester pyrethroids and type I and II pyrethroids. Therefore, we examined the effect of nonester pyrethroid (etofenprox), type I (permethrin), and type II (cypermethrin) pyrethroid insecticides alone and in all combinations to Drosophila melanogaster Meigen. The combination of permethrin + etofenprox and permethrin + cypermethrin demonstrated antagonistic toxicity, while the combination of cypermethrin + etofenprox demonstrated synergistic toxicity. The mixture ofpermethrin + cypermethrin + etofenprox demonstrated additive toxicity. The toxicity of permethrin + cypermethrin was significantly lower than the toxicity of cypermethrin alone, but the combination was not significantly different from permethrin alone. The toxicity of permethrin + cypermethrin + etofenprox was significantly greater than the toxicity of both permethrin and etofenprox alone, but it was significantly lower than cypermethrin alone. The mixture of permethrin and etofenprox was significantly less toxic than permethrin. The explanation for the decreased toxicity observed is most likely because of the competitive binding at the voltage-gated sodium channel, which is supported by physiological and biochemical studies of pyrethroids. Our results demonstrate that the assumption that the mixture toxicity of pyrethroids would be additive is not adequate for modeling the mixture toxicity of pyrethroids to insects.     

Effect of temperature on the toxicities of ten pyrethroids to German cockroach (Dictyoptera: Blattellidae).

Of 10 pyrethroids tested by topical application of male German cockroaches, Blattella germanica (L.), d-phenothrin was the least toxic at 19, 26, or 31 degrees C. lambda-cyhalothrin was most toxic. Pyrethroids with the alpha-cyano moiety (fluvalinate, fenvalerate, cypermethrin, esfenvalerate, tralomethrin, cyfluthrin, and lambda-cyhalothrin) were more toxic than those without this functional group (d-phenothrin, resmethrin, and permethrin). At LC50, toxicity was negatively related to temperature. Temperature-toxicity responses of five of seven alpha-cyano pyrethroids were parallel, possibly indicating qualitatively identical but quantitatively different levels of detoxification enzymes.     

A sodium channel mutation identified in Aedes aegypti selectively reduces cockroach sodium channel sensitivity to type I, but not type II pyrethroids

Voltage-gated sodium channels are the primary target of pyrethroid insecticides. Numerous point mutations in sodium channel genes have been identified in pyrethroid-resistant insect species, and many have been confirmed to reduce or abolish sensitivity of channels expressed in Xenopus oocytes to pyrethroids. Recently, several novel mutations were reported in sodium channel genes of pyrethroid-resistant Aedes mosquito populations. One of the mutations is a phenylalanine (F) to cysteine (C) change in segment 6 of domain III (IIIS6) of the Aedes mosquito sodium channel. Curiously, a previous study showed that alanine substitution of this F did not alter the action of deltamethrin, a type II pyrethroid, on a cockroach sodium channel. In this study, we changed this F to C in a pyrethroid-sensitive cockroach sodium channel and examined mutant channel sensitivity to permethrin as well as five other type I or type II pyrethroids in Xenopus oocytes. Interestingly, the F to C mutation drastically reduced channel sensitivity to three type I pyrethroids, permethrin, NRDC 157 (a deltamethrin analogue lacking the ??-cyano group) and bioresemthrin, but not to three type II pyrethroids, cypermethrin, deltamethrin and cyhalothrin. These results confirm the involvement of the F to C mutation in permethrin resistance, and raise the possibility that rotation of type I and type II pyrethroids might be considered in the control of insect pest populations where this particular mutation is present.     

Insecticide toxicity, synergism and resistance in the German cockroach (Dictyoptera: Blattellidae)

The toxicity of synergism of and resistance to insecticides in four strains of German cockroach, Blattella germanica (L.), were investigated. Toxicity of nine insecticides by topical application to the susceptible strain varied greater than 2,000-fold, with deltamethrin (LD50 = 0.004 micrograms per cockroach) and malathion (LD50 = 8.4 micrograms per cockroach) being the most and least toxic, respectively. Resistance to pyrethrins (9.5-fold) in the Kenly strain was unaffected by the synergists piperonyl butoxide (PBO) or S,S,S-tributylphosphorotrithioate (DEF), suggesting that the metabolism is not involved in this case. Malathion resistance in the Rutgers strain was suppressible with PBO, implicating oxidative metabolism as a resistance mechanism. The Ectiban-R strain was resistant to all the pyrethroids tested, and cypermethrin resistance was not suppressible with PBO or DEF. These findings support results of previous studies that indicated this train has a kdr-like mechanism. Bendiocarb resistance in both the Kenly and Rutgers strains was partially suppressed by either PBO or DEF, suggesting that oxidative and hydrolytic metabolism are involved in the resistance. Trends between the effects of the synergists on the susceptible versus resistant strains are discussed.     

Novel point mutations in the German cockroach para sodium channel gene are associated with knockdown resistance (kdr) to pyrethroid insecticides

Knockdown resistance (kdr) to pyrethroid insecticides has been attributed to point mutations in the para sodium channel gene in more than a half dozen insect pest species. In this study, we identified two novel para mutations in five highly resistant kdr-type German cockroach strains. The two mutations, from glutamic acid (E434) to lysine (K434) and from cysteine (C764) to arginine (R764), respectively, are located in the first intracellular linker connecting domains I and II. E434K is located near the beginning of the linker (closest to domain I), whereas C764R is found toward the end of the linker (closest to domain II). Two additional mutations from aspartic acid (D58) to glycine (G58), and from proline (P1880) to leucine (L1888), respectively, were found in one of the resistant strains. The four mutations coexist with the previously identified leucine to phenylalanine (L993F) kdr mutation in IIS6, and are present only in the highly resistant individuals of a given strain. These findings suggest that these mutations might be responsible for high levels of knockdown resistance toward pyrethroid insecticides in the German cockroach.     

Insecticide Susceptibilities of Anopheles sinensis (Diptera: Culicidae) Larvae from Paju-shi, Korea

A study was conducted to determine the susceptibility of Anopheles sinensis larvae, malaria vector, from Paju‐shi (Gyonggi‐do) to 24 pesticides including 11 pyrethroids, 11 organophosphates, 1 pyrazole analogue and another pesticide. The mosquito larvae showed the most highly susceptible to chlorfenapyr with LC₅₀ of 0.0063 ppm followed by spinosad, temephos, fenthion and beta‐cyfluthrin with 0.03, 0.0366, 0.0367 and 0.0998 ppm, respectively, and fluvalinate (>128 ppm) was the least susceptible. Also, 11 pesticides of them were compared with Goyang strain (Gyonggi‐do) of data 1992. The development of resistance was noticed to cypermethrin and permethrin as much of 73.59 and 6.23 folds at LC₅₀, respectively, and also fluvalinate was showed high resistance development. In generally, the pyrethroid insecticides were showed higher development of resistance than organophosphorus insecticides.     

Extended selections for pyrethroid resistance in the German cockroach (Dictyoptera: Blattellidae).

Selection experiments with a pyrethrins-susceptible and a pyrethrins-resistant strain of German cockroaches, Blattella germanica (L.), were conducted for 17 generations with either permethrin or fenvalerate as the selecting agent. Large nymphs were left on treated glass surfaces for extended periods of time each generation. Mortality was assessed at 24 h. The level of resistance was determined periodically by time-mortality testing. The VPI-susceptible strain served as the basis for comparison. The pyrethrins-susceptible strain developed resistance to pyrethrins early in the selection process; this strain ultimately became resistant to allethrin, phenothrin, permethrin, fenvalerate, cyfluthrin, and cypermethrin. Fenvalerate caused faster development of resistance than did permethrin. The pyrethrins-resistant strain, selected with fenvalerate, quickly became resistant to allethrin, permethrin, phenothrin, and fenvalerate. Ultimately, it developed resistance to all nine pyrethroids tested.     

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.     

Efficacy of abamectin fed to German cockroaches (Dictyoptera: Blattellidae) resistant to pyrethroids

Ten field-collected strains of the German cockroach, Blattella germanica (L.), with varying levels of pyrethroid resistance were tested for possible cross-resistance to abamectin administered as a bait. The time-mortality method was used for comparison of response with a known susceptible strain in 10-d feeding experiments. Essentially no resistance to abamectin was found. Extensive feeding inhibition occurred after a few days, but the amount of abamectin consumed varied substantially from strain to strain. Abamectin may have considerable potential as a cockroach bait insecticide.     

Sodium channel gene expression in mosquitoes, Aeries albopictus （S.）

A mosquito strain of Aedes albopictus, HAmAal^G0, from Huntsville, Alabama, USA, showed a normal susceptibility and low tolerance to permethrin and resmethrin （pyrethroid insecticides） compared to a susceptible Ikaken strain, even though these pyrethroid insecticides have been used in the field for a long period of time in Alabama. Recently, we treated HAmAal^G0 in the laboratory with permethrin for five generations and detected no significant change in the level of resistance to permethrin in the selected mosquitoes, HAmAal^G0, compared with the parental strain HAmAal^G0. We then examined the allelic expression at the L-to-F kdr site of the sodium channel gene in the Aedes mosquitoes to address our hypothesis that the L-to-F kdr mutation was not present in HAmAal^G0 and HAmAal^G5 mosquitoes. We found that every tested individual in Ikaken, HAmAal^G5, and HAmAal^G5 populations expressed a codon of CTA at the L-to-F kdr site encoding Leu, strongly corresponding to their susceptibility to insecticides.     

Measuring cypermethrin resistance in the German cockroach (Orthoptera: Blattellidae).

Cypermethrin resistance in the German cockroach, Blattella germanica (L.), was assessed by tests of surface contact and topical application. Topical application provided the most sensitive measure of resistance in a field strain. The resistance ratio (RR) measured by topical application was 122.6 for cypermethrin at LD50. As measured by surface contact, the resistance ratio at KT50 was 2.9. Differences between the walking movement of cockroaches of susceptible (VPI) and field (RHA) strains on deposits of cypermethrin influenced KT50 values. A bioassay with unrestricted movement resulted in RHA strain cockroaches accumulating a larger dose on the tarsal pads and subsequent reduction of the resistance ratio at KT50. Less walking by the VPI strain resulted in less insecticide accumulation on their tarsal pads. On a bioassay in which movement was restricted, the amount of insecticide accumulating on the tarsi was equalized, resulting in an increased resistance ratio at KT50. Differences in susceptibility were more accurately measured when the two strains were topically treated (either on the thorax or the tarsal pads) with known doses of insecticide.     

Organophosphorus insecticides synergize pyrethroids in the resistant strain of cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) from West Africa

Helicoverpa armigera (Hübner) populations from West Africa recently developed resistance to pyrethroid insecticides through enhanced metabolism by mixed-function oxidases. The combination index method was used to study the synergism of pyrethroids by organophosphorus insecticides. Several mixtures of insecticides currently registered to control cotton pest complex in West Africa were tested, including: cypermethrin/ethion, cypermethrin/profenofos, deltamethrin/ triazophos, deltamethrin/chlorpyriphos, cyfluthrin/chlorpyriphos, and betacyfluthrin/chlorpyriphos. In the resistant strain, the organophosphorus insecticides significantly increased the toxicity of pyrethroids suppressing the resistance effect, either by additive or synergistic effects. Significant synergism was shown for the following mixtures: cypermethrin/ethion, deltamethrin/triazophos, and deltamethrin/chlorpyriphos. The use of synergism from these insecticide mixtures should prove to be an additional tool in the overall resistance management strategy because the pyrethroid resistance in H. armigera from West Africa is not yet stable, decreasing between cotton seasons and increasing with treatments. In absence of selection, the susceptibility of H. armigera to insecticides should be restored.     

Quantification of pyrethroid insecticides from treated bednets using a mosquito recombinant glutathione S-transferase

Recombinant glutathione S-transferase (agGST1-6) from the malaria vector mosquito Anopheles gambiae Giles (Diptera: Culicidae) was expressed in Escherichia coli using a pET3a vector system. The expressed enzyme was biochemically active with reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Activity of agGST1-6 with GSH and CDNB was inhibited to different degrees by both alpha-cyano and non-alpha-cyano pyrethroid insecticides. This inhibition was used to develop an assay for quantification of pyrethroids. Standard curves of insecticide concentration against percentage of enzyme inhibition or volume of iodine solution were established by spectrophotometry and iodine volumetric titration, respectively, for permethrin and deltamethrin. These assays allowed estimation of pyrethroid concentrations both spectrophotometrically and visually. For the residue assay of each insecticide, a cut-off point of 50% of the initial pyrethroid impregnation concentration was used, which should differentiate between biologically active and inactive treated bednets. The cross-reactivity of the primary permethrin photodegradants (3-phenoxyalcohol and 3-phenoxybenzoic acid) with the recombinant agGST1-6 was assayed in the same system. No agGST1-6 inhibition by the insecticide metabolites was observed, suggesting that the system is unaffected by primary permethrin metabolites and will accurately measure insecticide parent compound concentrations. The estimated pyrethroid insecticide concentrations, given spectrophotometrically and by iodine titration assay, were comparable to those obtained by direct HPLC quantification of residues extracted from bednets. Hence, it should be relatively easy to adapt this method to produce a test kit for residue quantification in the field.