German Cockroach Strains Research Articles

Role of social aggregation in the fitness cost of pyrethroid‐resistant German cockroaches

AbstractLiving in aggregations likely gives fitness benefits to resistant German cockroaches. We first investigated the trade‐offs in life‐history traits between six field pyrethroid‐resistant German cockroach strains and a laboratory susceptible strain (SUS‐1). Second, each strain was placed in groups of 2, 10, 20, 30 and 40 individuals with an equal sex ratio in each group to investigate the effect of social aggregation on fitness and overall population parameters. All resistant strains exhibited reduced nymphal survival (50%–83%) and a prolonged development period in both male (49–63 days) and female (47–62 days) nymphs when compared to SUS‐1 individuals (nymphal development period: 39 ± 2 days for male and 40 ± 3 days for female). Such fitness costs were minimized when the resistant cockroaches were in aggregations. Specifically, male (54 ± 3 days) and female nymphal development periods (52 ± 3 days) of German cockroach significantly decreased to 44 ± 1 days in the largest group. The nymphal survival was substantially enhanced (more than 80% survival) in German cockroaches with elevated cytochrome P450 monooxygenases, or both cytochrome P450 monooxygenases and esterases activities in the largest group. The effect of pyrethroid resistance on the life‐history traits of adult German cockroaches was limited; only adult male longevity was significantly prolonged. Group living did not significantly extend adult longevity and increase the number of offspring per ootheca. Contrary to expectations, the number of nymphs produced by a female and the oothecal hatchability were inversely proportional to the number of individuals in aggregations. Moreover, a lower proportion of viable oothecae were produced in German cockroaches when living in aggregations. Because of low fecundity, population parameters of German cockroaches living in aggregations (net reproductive rate and intrinsic rate of increase) were significantly lower than the parameters of those living in pairs.

Synergists action of piperonyl butoxide and S,S,S-tributyl phosphorotrithioate on toxicity of carbamate insecticides against Blattella germanica.

To determine the synergists action of piperonyl butoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF) on toxicity of carbamate insecticides against Blattella germanica in Tehran city. In the current study, German cockroach strains were collected from several hospitals and dormitories in Tehran. At the beginning, different concentrations of bendiocarb and carbaryl (insecticides belong to carbamate group) were determined by surface contact on a susceptible strain. Then, the level of susceptibility and type of resistance mechanisms in the collected strains from contaminated sites to the aforementioned insecticides were studied by using PBO and DEF synergists with different insecticide ratios to synergist (1:0, 1:1, 1:2, 1:3). The DEF synergist along with bendiocarb and carbaryl completely eliminated the resistance in all strains but PBO did not completely eliminate the resistance in the strains of Mofid, Alvand, Valiasr hospitals and Shariati dormitory. Generally, the impact of DEF was observed in the removing resistance more than PBO. In most of these strains, resistance to bendiocarb and carbaryl is completely eliminated by DEF, showing a very high role of estraze enzymes in resistance to bendiocarb and carbaryl. But in most strains PBO does not remove the resistance because other mechanisms, such as reduced cuticle penetration and insensitivity to the acetylcholine esterase enzyme, may be involved.

Unidirectional Cross-Resistance in German Cockroach (Blattodea: Blattellidae) Populations Under Exposure to Insecticidal Baits.

Insect pests, including the German cockroach, Blattella germanica (L.), are prone to the development of physiological resistance when exposed to a number of insecticide sprays, and cross-resistance is frequently observed. Toxic baits are often used as a primary method of controlling German cockroaches, also resulting in heavy selection pressure from insecticidal baits. In response to this pressure, cockroach populations have developed aversion to specific inert ingredients in bait. Here, we examined the effect of exposure to baits containing fipronil, indoxacarb, or hydramethylnon on the development of physiological resistance to the same and other insecticides in a number of German cockroach strains. We found that prolonged exposure to baits containing fipronil or indoxacarb increased physiological resistance to these compounds. However, no increase in physiological resistance against any insecticide was observed in response to exposure to hydramethylnon bait. Additionally, we found that exposure to fipronil bait increased cross-resistance to indoxacarb. On the other hand, exposure to indoxacarb bait did not increase cross-resistance to fipronil. Neither fipronil nor indoxacarb bait exposure increased resistance to hydramethylnon. Interestingly, the development of insecticide resistance in response to bait exposure was strain-dependent and influenced by bait palatability. Our results demonstrate that exposure to toxic baits, particularly those containing fipronil, plays a significant role in the development of insecticide resistance, including cross-resistance, in German cockroaches. Further, although insecticide resistance in response to baits is mediated by exposure through the oral route, the molecular mechanisms at play are likely different for each insecticide.

Insecticide Resistance of Several Field-Collected German Cockroach (Dictyoptera: Blattellidae) Strains.

German cockroaches, Blattella germanica (L.), remain one of the most difficult indoor insect species to control because of its ability to develop resistance to insecticides. The toxicity and resistance levels of five technical-grade insecticides (permethrin, chlorpyrifos, propoxur, imidacloprid, and fipronil) were determined for adult males of seven strains of the German cockroach, a laboratory-reared susceptible strain (S) and six field-collected strains (B, D, E, G, H, and I). Using topical application methods, fipronil was the most toxic insecticide to all seven strains. The LD50 values of fipronil in the susceptible strain (S) and the field-collected strains B, D, E, G, H, and I were 1.33, 2.62, 11.53, 5.07, 7.66, 5.15, and 10.15 ng/insect, respectively. The field-collected strains were most resistant to permethrin among the five insecticides, except for strain H. The resistance ratios of strains B, D, E, G, and I to permethrin were 31.8, 37.3, 51.9, 34.9, and 37.5, respectively. With a resistance ratio of 6.4, the field-collected strain H was most resistant to chlorpyrifos. The field-collected strains were not significantly resistant to propoxur. Strains B, H, and I were not significantly resistant to imidacloprid when compared with the susceptible strain. Based on the different resistance ratios for each insecticide, we conclude that there are high rates of insecticide resistance in German cockroaches from Franklin County, NC, and that the field-collected strains most likely had different treatment histories.

Indoxacarb biotransformation in the German cockroach

Insecticides that are used for pest control undergo physical and biological (enzymatic) degradation. Indoxacarb is an oxadiazine class sodium channel blocker insecticide used for German cockroach (Blattella germanica L.) control. At present, no information is available on enzymatic biotransformation or metabolism of indoxacarb in this important urban pest. We studied the biotransformation pathways of indoxacarb in one susceptible and three field strains with varying susceptibility levels using liquid chromatography and high-resolution mass spectrometry. As shown in other insect species we found evidence for hydrolase-based bioactivation of indoxacarb to a toxic decarbomethoxylated metabolite, DCJW. In addition, both indoxacarb and DCJW were further metabolized to hydroxy, oxadiazine ring-opened and hydroxylated ring-opened metabolites. In general, higher indoxacarb disappearance, increased formation of DCJW and the above-mentioned metabolites were observed in the three field strains. In vitro biotransformation studies showed that hydroxylated and oxadiazine ring-opened metabolite formation is NADPH/cytochrome P450-dependent. Bioassays and in vivo metabolism experiments using the enzyme-inhibiting insecticide synergists, piperonyl butoxide (PBO) and S,S,S,-tributyl phosphorotrithioate (DEF), provided insights into potential indoxacarb resistance mechanisms that may proliferate in German cockroach field strains following unchecked selection pressures. The information presented here is an essential step toward developing indoxacarb resistance management programs and also reveals mechanisms of secondary/tertiary indoxacarb toxicity.

Mechanisms Underlying Fipronil Resistance in a Multiresistant Field Strain of the German Cockroach (Blattodea: Blattellidae)

German cockroaches (Blattella germanica L.) have significant impacts on human health, most notably they are implicated as causes of childhood asthma. Gel bait formulations of fipronil, a phenylpyrazole insecticide, have been in use for German cockroach control in the United States since 1998. Previously, dieldrin resistant German cockroach strains were shown to have 7- to 17-fold cross-resistance to fipronil. More recently, a field-collected strain (GNV-R) displayed approximately 36-fold resistance to topically applied fipronil at the LD50 level, which is the highest level of fipronil resistance reported to date in the German cockroach. The aim of the current research was to identify mechanism(s) responsible for high-level fipronil resistance in the GNV-R strain. Synergist bioassays conducted using topical and injection application methods implicated cytochrome P450-mediated detoxification in resistance. Electrophysiological recordings using the suction-electrode technique revealed the nervous system of the GNV-R strain is insensitive to fipronil. In agreement with electrophysiology results, the alanine to serine (A302S) mutation encoded by the gamma-amino butyric acid-gated chloride channel subunit gene resistance to dieldrin, which confers limited cross-resistance to fipronil, was detected in 95% of GNV-R strain individuals. Logistic regression analysis showed that A302S mutation frequency correlates with neurological insensitivity as shown by electrophysiology data. Overall, results of synergism bioassays, electrophysiological recordings, and A302S mutation frequency measurements suggest that fipronil resistance in the GNV-R strain is caused by the combined effects of enhanced metabolism by cytochrome P450s and target-site insensitivity caused by the A302S-encoding mutation in the resistance to dieldrin gene.

Susceptibility of Field Populations of Blattella germanica (Blattaria: Blattellidae) to Spinosad

The German cockroach is an important household insect pest worldwide and acts as a mechanical vector and reservoir for pathogenic agents. The aim of this study was to examine the basic laboratory toxicity of Blattella germanica to spinosad. The M, T, A22, AZAR4, BOOSTAN7 and ABAN21 strains were collected from field populations of six infested kitchen student dormitories and the SAMAN strain was collected from a residential area after insecticide spraying control failure in Tehran, Iran. Technical grade spinosad was delivered in 0.5 microL acetone to the first abdominal sternum of briefly CO2-anesthetize adult male cockroaches by topical application bioassay. Treated males monitored for mortality. Mortality data from the replicates was assessed by probit analysis. The average LD50 of susceptible strain was 494.3, 148.8 and 55.1 ng per insect after 24, 48 and 72 h, respectively. The LD50 of spinosad decreased with time in the field population strains. All German cockroach strains showed a similar susceptibility or lower tolerance (1.6-folds) for spinosad compared with the susceptible laboratory strain and the steep slopes of dose-response curves indicated that the field population of these German cockroach strains was homogenous in response to spinosad. These results indicated that the spinosad was relatively slow-acting in topical application bioassay, with LD50 values decreasing until 72 h and becoming stable thereafter. The effectiveness of spinosad against susceptible and the field population German cockroach strains in laboratory condition showed that spinosad probably could be useful for the control of the German cockroach.

Iranian Non-Responding Contact Method German Cockroach Permethrin Resistance Strains Resulting from Field Pressure Pyrethroid Spraying

The present study investigated the probable responsible reasons for non-response the B. germanica to permethrin only in World Health Organization glass jar method after insecticide spraying control failure with pyrethroid insecticide groups in Islamic Republic of Iran. Eleven German cockroach strains were collected from field populations of nine infested kitchen student dormitories and two infested hospitals after insecticide spraying control failure with pyrethroid insecticide groups in Iran. The current study, in World Health Organization glass jar and knock down methods conducted on newly emerged adult males. All feral strains, with various levels of resistance to pyrethroids, in World Health Organization glass jar method at 400 min (6 h) time exposures, was not observed mortality, while the susceptible strain was observed 100% mortality in 25 min time exposures. Susceptible strain at LT50 after assessing on mortality data from the replicates by probit analysis in World Health Organization glass jar method was 15.3 min. In this study, all feral strains in World Health Organization glass jar method after 400 min (6 h) time exposures, was not observed mortality, that showed these strains very high-level resistance to permethrin. In the knock down method, the resistance ratios were 3.6 to 26.1-folds compare with the susceptible reference strain. In a comparison, among this study and previous studies resistance ratios of 8.6 to 17.7-folds for permethrin in topical application, indicated that German cockroach have had under pressure spraying. German cockroach have had in vicinity to pyrethroid insecticides especially permethrin in these locations in long period for non-responding to permethrin insecticide only in World Health Organization glass jar method, and the field evident confirm this subject.

Duration of Fipronil WHO Glass Jar Method Toxicity Against Susceptible and Feral German Cockroach Strains

Duration of Fipronil WHO Glass Jar Method Toxicity Against Susceptible and Feral German Cockroach Strains

Overexpression of the cytochrome c oxidase subunit I gene associated with a pyrethroid resistant strain of German cockroaches, Blattella germanica (L.)

A cytochrome c oxidase subunit I gene ( COXI) was identified and isolated as a differentially expressed gene between insecticide susceptible ACY and resistant Apyr-R German cockroach strains using PCR-selected subtractive hybridization and cDNA array techniques. The cDNA sequence of COXI has an open reading frame of 1533 nucleotides encoding a putative protein of 511 amino acid residues. Northern blot analysis indicated that levels of COXI expression were similar in three life stages (eggs, nymphs, and adults) of the susceptible ACY strain. The expression of COXI in the resistant Apyr-R strain was developmentally regulated, with low expression in eggs, an increase (~1.4-fold) in nymphs, and rose to a maximum (~3-fold) in both adult females and males. Comparison of COXI expression between ACY and Apyr-R strains indicated that there was no difference in the eggs of the two strains, but expression was higher (~1.5-fold) in nymphs and much higher (~3- to 4-fold) in adult males and females of the Apyr-R strain. The levels of COXI mRNA showed about 1.4- and 1.7-fold increase in the abdomen tissues compared with the head+thorax tissues of ACY and Apyr-R strains, respectively. Although expression patterns of COXI in head+thorax and abdomen tissues were similar (i.e. lower in the head+thorax tissues and higher in the abdomen tissues) in both the ACY and Apyr-R strains, the expression of COXI was about 2.5-fold higher in the head+thorax and ~3-fold higher in the abdomen tissues of the Apyr-R strain compared with the corresponding ACY samples. The overexpression of COXI in resistant German cockroaches merits the investigation of the importance of the gene in insecticide resistant German cockroaches.

Overexpression of CYP4G19 associated with a pyrethroid-resistant strain of the German cockroach, Blattella germanica (L.)

A new cytochrome P450 gene, CYP4G19, was identified and isolated as a differentially expressed gene between insecticide susceptible ACY and resistant Apyr-R German cockroach strains using PCR-selected subtractive hybridization and cDNA array techniques. The cDNA sequence of CYP4G19 has an open ready frame of 1638 nucleotides encoding a putative protein of 546 amino acid residues. Sequence analysis shows that CYP4G19 putative protein contains (1) a highly hydrophobic N terminus, (2) a P450 protein signature motif, FXXGXRXCXG, known to be an important ligand for heme binding, and (3) an important characteristic motif, EVDTFMFEGHDTT, for the family 4. Northern blot analysis indicated that levels of CYP4G19 expression were low in eggs, nymphs, and adults of the susceptible ACY strain with a similar expression pattern. The expression of CYP4G19 in the resistant Apyr-R strain was developmentally regulated, with very low expression in eggs, increasing in nymphs, and reaching a maximum in both female and male adults. Comparison of CYP4G19 expression between ACY and Apyr-R strains indicated that there was no difference in their eggs, but expression was higher (∼1.7-fold) in the nymphs and much higher (∼5-fold) in the male and female adults of the Apyr-R strain. Levels of CYP4G19 mRNA were readily detectable in head+thorax tissues and increased (∼5-fold) in the abdomens of the ACY strain. In the Apyr-R strain, however, levels of CYP4G19 mRNA were relatively low in head+thorax tissues and were about 7-fold increase in the abdomen. Although expression patterns of CYP4G19 in head+thorax and abdomen tissues were similar (i.e. lower in head+thorax tissues and higher in abdomen tissues) in both the ACY and Apyr-R strains, the overexpression was more evident in the Apyr-R strain in both head+thorax and abdomen tissues than in the ACY strain.

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.

Differences in the Rapid Knockdown and Lethal Effects of Aerosol Formulations against German Cockroach (Blattaria, Blattellidae) Strains

ABSTRACT The knockdown and lethal efficacies of five aerosol formulations including Combat Speed® (AIs: 0.1 % imiprothrin and 0.3% cyphenothrin), Raid Power® (AIs: 1.0% pyrethrin and 0.2% permethrin), Home Keeper®, (AIs: 0.2% tetramethrin and 0.3% permethrin), Super Killer® (AIs: 0.32% tetramethrin and 0.08% bioresmethrin), and Perma Kill‐K® (AIs: 0.3% dichlorvos and 0.1% tetramethrin) against five strains of the German cockroach, Blattella germanica (L.) were assessed. The results show that the mean value of KT50 (5.4 sec.) of Combat Speed® was 4.5 and 3.1‐folds lower than those of Perma Kill‐K® and Home Keeper®, respectively. The mean value of KT90 (9.0 sec; slope = 10.02) of Combat Speed® was 3.8 to 5.8‐folds lower than Perma Kill‐K®, Supper Killer® and Home Keeper®. As lethal effects, the mean value of LT50 (17.3 sec.) of Combat Speed® was over 26 folds lower than Supper Killer® and Perma Kill‐K®. The mean value of LT90 (32.9 sec.) of Combat Speed® was 37.4 and 15.1‐folds lower than those of Supper Killer® and Perma Kill‐K®, respectively. In general, Combat Speed® and Raid Power® were considered the insecticide aerosols with faster knockdown and higher lethal effects than Supper Killer®, Perma Kill‐K®, and Home Keeper® against five strains of German cockroaches in Korea. Also, the knockdown and lethal effects of Supper Killer®, Perma Kill‐K®, and Home Keeper® were highly variable depends on the strains.

Variability of resistance mechanisms in pyrethroid resistant German cockroaches (Dictyoptera: Blattellidae)

Mechanisms of pyrethroid resistance were compared in German cockroach strains, Apyr-R, Bpyr-R, and Cpyr-R, collected from Opelika, Alabama, after control failures with pyrethroid insecticides. Resistance to permethrin in Bpyr-R (54-fold, compared with a susceptible strain, ACY) and Cpyr-R (46-fold) was slightly suppressed by piperonyl butoxide (PBO) and S,S,S,-tributylphosphorotrithioate (DEF). Given that DEF is not a completely specific inhibitor of hydrolases and that it can inhibit microsomal oxidases at high concentration, these data should be interpreted with caution. These results suggest that P450 monooxygenases and, perhaps, hydrolases play minor roles in permethrin resistance in both strains. However, resistance to deltamethrin in both Bpyr-R (50-fold) and Cpyr-R (47-fold) was not decreased by PBO or DEF. These results were different from that in Apyr-R where levels of resistance to permethrin and deltamethrin were dramatically decreased by PBO and DEF. It is likely that these German cockroach strains from the same geographic origin exhibit diverse mechanisms of pyrethroid resistance. Injection did not decrease levels of resistance to either permethrin or deltamethrin in Bpyr-R and Cpyr-R, which is similar as the result in Apyr-R, suggesting that a decreased rate of cuticular penetration does not play an important role in pyrethroid resistance in these strains. A 408 bp para-homologous sodium channel gene fragment where the para ( kdr) mutation resides was generated by RT-PCR from German cockroach strains. We found that the para mutation was present in Apyr-R, but not in ACY, Bpyr-R, and Cpyr-R, suggesting that the para mutation is one of the important factors in pyrethroid resistance in Apyr-R but not an important contributing factor to pyrethroid resistance in Bpyr-R and Cpyr-R. Interestingly, a new mutation, T to C at nt 2996 resulting in a phenylalanine 999 to serine 999 amino acid change, was detected in both Bpyr-R and Cpyr-R. The importance of this mutation in resistance is 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.

Mechanisms Responsible for Cypermethrin Resistance in a Strain of German Cockroach, Blattella germanica

Cypermethrin resistance level and mechanisms responsible for the resistance were investigated in a strain (Aves) of German cockroach, Blattella germanica (L.), collected recently from a Gainesville, Florida residence. Topical bioassay data revealed that the Aves strain was highly resistant to cypermethrin, exhibiting a resistance ratio of 93-fold, which was reduced to 29-fold when cockroaches were pretreated with piperonyl butoxide and reduced to 18-fold when pretreated with S,S,S-tributyl phosphorotrithioate. The synergist data implicated enhanced oxidative and hydrolytic metabolism as resistance mechanisms in the Aves German cockroach strain. This conclusion was further supported by significantly higher oxidative (2.4- to 4.2-fold) and hydrolytic (1.6- to 3.6-fold) detoxification enzyme activities toward surrogate substrates and significantly higher in vitro [14C]cypermethrin metabolism. Microsomal NADPH-dependent (1.8-fold) and NADPH-independent (2.2-fold) [14C]cypermethrin metabolism were significantly greater in the Aves strain than in the Orlando insecticide-susceptible strain. In vivo penetration studies with [14C]cypermethrin indicated that decreased cypermethrin penetration may also be a contributing resistance mechanism in the Aves strain. Finally, the Leu993Phe mutation, shown previously to be associated with knockdown resistance (kdr), was present in the Aves strain.

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.

Toxicological and Biochemical Studies with Field Populations of the German Cockroach,Blattella germanica

Topical bioassays with cypermethrin, λ-cyhalothrin, permethrin, propoxur, and chlorpyrifos were conducted on 12 German cockroach strains recently collected from the field. Resistance levels ranged from 3- to 159-fold for cypermethrin, 2- to 88-fold for permethrin, 4- to 55-fold for λ-cyhalothrin, 5- to 33-fold for propoxur, and 3- to 19-fold for chlorpyrifos. The synergists piperonyl butoxide (PBO) andS,S,S-tributylphoshorotrithioate (DEF) affected cypermethrin resistance to varying degrees depending on the strain. Piperonyl butoxide pretreatment decreased cypermethrin resistance in only 5 strains, but caused an increase in resistance level in 7 strains. Conversely, DEF pretreatment reduced the resistance level in 10 of the strains and increased the resistance level in only 2 strains. Correlation analysis of resistance ratios for each strain and insecticide indicated a direct relationship between resistance level of one insecticide and another, especially among the pyrethroids. All field strains exhibited significantly higher microsomal oxidase, glutathioneS-transferase, and esterase activities toward surrogate substrates as compared with the insecticide-susceptible strain. However, levels of cytochrome P450 content, aldrin epoxidase activity, methoxyresorufinO-demethylase activity, and glutathioneS-transferase activity were not correlated with pyrethroid resistance, suggesting that these activities are poor indicators of pyrethroid-resistance magnitude. Interestingly, significant correlations were found between general esterase activity and cypermethrin (P= 0.002), permethrin (P= 0.007), cyhalothrin (P= 0.002), and propoxur (P= 0.001) resistance levels. The data support the conclusion of esterase involvement in cypermethrin resistance determined by synergist (DEF) bioassay. However, the significance of this relationship, in the context of resistance detection, requires further examination.

The Knockdown Resistance (kdr) Mutation in Pyrethroid-Resistant German Cockroaches

A point mutation in thepara-homologous sodium channel gene has been shown to be associated with knockdown resistance (kdr) in several insect species including the German cockroach. In this study, we analyzed the genomic organization of the region where thekdrmutation resides and then performed polymerase chain reaction (PCR) and sequencing using genomic DNA as the template to detectkdrmutation in 24 pyrethroid-resistant German cockroach strains, most of which have been collected recently from the field. Thekdrmutation, G to C at nt 2979 resulting in a leucine to phenylalanine amino acid substitution, was detected in 20 strains including 2 strains from overseas (China and Germany). Our results clearly indicate that thekdrmutation is widespread in German cockroach populations. However, the super-kdrmutation detected in super-kdrhouse flies was not found in any of the 4 strains that showed higher levels of knockdown resistance. Little correlation was observed between the presence of thekdrmutation and the level of knockdown resistance, suggesting the existence of multiple resistance mechanisms in many of these strains.

Cytochrome p450 purification and immunological detection in an insecticide resistant strain of german cockroach ( Blattella germanica, l.)

A German cockroach strain, Munsyana (MA) had 80-fold resistance to the pyrethroid insecticide cypermethrin, 4.5-fold greater total cytochrome P450 content and 2.5-fold greater cytochrome P450-mediated N-demethylation of 4-chloro- N-methylaniline compared to the susceptible Johnson Wax (JWax) strain. Immobilized artificial membrane high performance liquid chromatography (IAM-HPLC) of microsomal proteins from the MA strain enriched cytochrome P450 greater than 70-fold. Following purification, a single protein band of M r=49 000 (P450 MA), was detected by silver-staining SDS PAGE gels. Antiserum to the purified protein from the MA strain (anti-P450 MA) was produced in mice. Anti-P450 MA inhibited cytochrome P450-mediated N-demethylation by 4-fold in both MA and JWax strains. In Western blots of microsomal proteins, anti-P450 MA differentiated single MA and JWax individuals by recognizing an M r 49 000 protein band in only the MA strain. In JWax cockroaches, the M r 49 000 band was only detectable in Western analysis following induction with pentamethylbenzene (PMB). PMB induction also increases N-demethylation 2.6 and 8.0-fold in the MA and JWax strains, respectively. These results are consistent with the hypothesis that insecticide resistance in the MA strain is due to over-expression of a cytochrome P450.