Nicotinoid and pyrethroid insecticide resistance in houseflies (Diptera: Muscidae) collected from Florida dairies

BACKGROUND: The housefly, Musca domestica L., continues to be a major pest of confined livestock operations. Houseflies have developed resistance to most chemical classes, and new chemistries for use in animal agriculture are increasingly slow to emerge. Five adult housefly strains from four Florida dairy farms were evaluated for resistance to four insecticides (beta‐cyfluthrin, permethrin, imidacloprid and nithiazine). RESULTS: Significant levels of tolerance were found in most field strains to all insecticides, and in some cases substantial resistance was apparent (as deduced from comparison with prior published results). At the LC₉₀ level, greater than 20‐fold resistance was found in two of the fly strains for permethrin and one fly strain for imidacloprid. Beta‐cyfluthrin LC₉₀ resistance ratios exceeded tenfold resistance in three fly strains. The relatively underutilized insecticide nithiazine had the lowest resistance ratios; however, fourfold LC₉₀ resistance was observed in one southern Florida fly strain. Farm insecticide use and its impact on resistance selection in Florida housefly populations are discussed. CONCLUSION: Housefly resistance to pyrethroids is widespread in Florida. Imidacloprid resistance is emerging, and tolerance was observed to both imidacloprid and nithiazine. If these insecticides are to retain efficacy, producer use must be restrained. Copyright © 2009 Society of Chemical Industry     

Mechanisms of resistance to diflubenzuron in the house fly, Musca domestica (L.)

The mechanisms of resistance to the chitin synthesis inhibitor diflubenzuron were investigated in a diflubenzuron-selected strain of the house fly (Musca domestica L.) with > 1000 × resistance, and in an OMS-12-selected strain [O-ethyl O-(2,4-dichlorophenyl)phosphoramidothioate] with 380 × resistance to diflubenzuron. In agreement with the accepted mode of action of diflubenzuron, chitin synthesis was reduced less in larvae of the resistant (R) than of a susceptible (S) strain. Cuticular penetration of diflubenzuron into larvae of the R strains was about half that of the S. Both piperonyl butoxide and sesamex synergized diflubenzuron markedly in the R strains, indicating that mixed-function oxidase enzymes play a major role in resistance. Limited synergism by DEF (S,S,S-tributyl phosphorotrithioate) and diethylmaleate indicated that esterases and glutathione-dependent transferases play a relatively small role in resistance. Larvae of the S and R strains exhibited a similar pattern of in vivo cleavage of ³H- and ¹⁴C-labeled diflubenzuron at N₁C₂ and N₁C₁ bonds. However, there were marked differences in the amounts of major metabolites produced: R larvae metabolized diflubenzuron at considerably higher rates, resulting in 18-fold lower accumulation of unmetabolized diflubenzuron by comparison with S larvae. Polar metabolites were excreted at a 2-fold higher rate by R larvae. The high levels of resistance to diflubenzuron in R-Diflubenzuron and R-OMS-12 larvae are due to the combined effect of reduced cuticular penetration, increased metabolism, and rapid excretion of the chemical.     

The status and development of insecticide resistance in Danish populations of the housefly Musca domestica L

Samples of housefly (Musca domestica) field populations were collected from Danish livestock farms in 1997. The tolerance of the first‐generation offspring was determined for a number of insecticides. Dose‐response values were obtained by topical application for the pyrethroids bioresmethrin and pyrethrum, both synergised with piperonyl butoxide, and the organophosphate dimethoate. The organophosphates azamethiphos and propetamphos and the carbamate methomyl were tested in discriminating dose feeding bioassays. Resistance was low to moderate in most of the populations for most of the compounds tested, but this study also revealed the existence of high resistance to pyrethroid, organophosphate and carbamate insecticides in some populations. The resistance factors at LD₅₀ for bioresmethrin/piperonyl butoxide ranged between 2 and 98, and for pyrethrum/piperonyl butoxide between 2 and 29. Our results indicate that pyrethroid resistance in Denmark is increasing, since four of the 21 farms showed more than 100‐fold resistance at LD₉₅, a level of resistance only observed once before. Resistance factors at LD₅₀ for dimethoate ranged from 9 to 100, and showed two distinct trends: populations with either decreasing or increasing resistance. Resistance to azamethiphos was found to be widespread and high. Although two strains with high methomyl and propetamphos resistance were observed, methomyl and propetamphos resistance is moderate and appears not to be increasing. © 2001 Society of Chemical Industry     

Toxicity of spinosad to susceptible and resistant strains of house flies,Musca domestica

The toxicity of spinosad, a new insecticide derived from the bacterium Saccharopolyspora spinosa, was evaluated against susceptible and resistant strains of house fly (Musca domestica L.). Spinosad was highly toxic to house flies based on 72-h LD₅₀ values and the symptoms of poisoning were consistent with a neurotoxic mechanism of action. Spinosad was relatively slow acting, with the maximum toxicity noted at 72 h. Piperonyl butoxide and S,S,S,-tribu-tylphosphorotrithioate synergized the toxicity of spinosad by 3·0- and 1·8-fold, respectively, while diethyl maleate had no significant effect. These results suggest that there is a small degree of monooxygenase-mediated spinosad detoxification in house flies, while hydrolases may be only minimally important and glutathione transferases may have no role. There were no substantial levels of cross-resistance detected, except in the LPR strain where a low 4·3-fold cross-resistance was observed. The cyclodiene-resistant OCR strain was 2·7-fold more sensitive to spinosad than the susceptible strain (CS). These results suggest that cross-resistance may not be a limiting factor for the use of spinosad against house flies. © 1998 Society of Chemical Industry     

Electrophysiological identification of site-insensitive mechanisms in knockdown-resistant strains (kdr,super-kdr) of the housefly larva (Musca domestica)

The effects of pyrethroids were studied upon isolated segmental nerves and neuromuscular junctions in both susceptible (Cooper) and knockdown-resistant (kdr; super-kdr) strains of housefly larvae (Musca domestica L.). Isolated segmental nerves contained neither cell bodies nor synaptic contacts; thus, any effects of pyrethroids were attributed solely to their actions upon voltage-dependent Na⁺ channels. Threshold concentrations of the type II pyrethroid, deltamethrin, required to elevate the spontaneous firing rate of these nerves were determined. Both resistant strains were about ten times less sensitive to deltamethrin than the susceptible strain, but insensitivity of super-kdr nerves was no greater than in the less resistant kdr strain. At neuromuscular junctions, the minimum concentrations of pyrethroids needed to trigger massive increases in the frequency of miniature excitatory postsynaptic potentials (mEPSPs) were determined for deltamethrin and the type I pyrethroid, fenfluthrin. With fenfluthrin there was no detectable difference between the junctions of kdr and super-kdr strains, which were both about ten-fold less sensitive than Cooper junctions. With deltamethrin, kdr junctions were about 30 times less sensitive than those of Cooper; super-kdr junctions were dramatically insensitive to deltamethrin, being some 10000- and 300-fold less sensitive than those of Cooper and kdr respectively. Thus, in the synaptic assay, super-kdr conferred an extension in resistance over kdr only against the type II pyrethroid, it being ineffective against fenfluthrin. We suggest that kdr resistance comprises at least two site-insensitive areas within the nervous system. One involves insensitivity of the Na⁺ channel and has similar efficacy in both kdr and super-kdr strains against type I and II pyrethroids; the other is associated with the presynaptic terminal and is particularly effective in super-kdr resistance against type II pyrethroids. The latter could be associated with Ca²⁺-activated phosphorylation of proteins involved with neurotransmitter release. Such phosphorylation reactions are known to be perturbed by pyrethroids, especially by type II compounds.     

Absence of enhanced detoxication of permethrin in pyrethroid-resistant house flies

The mechanisms of resistance to pyrethroids were studied in a permethrin-selected (147-R) strain of the house fly, Musca domestica L. Approximately 12-fold synergism was obtained with a mixture of (1R)-trans-permethrin:piperonyl butoxide (1:5) so that the resistance decreased from 97-fold to 22-fold. Tests with the esterase inhibitor S,S,S-tributyl phosphorotrithioate produced very little synergism in either the resistant (R) strain (1.6-fold) or the susceptible (S) strain (1.9-fold). An investigation of the microsomal components revealed that compared to the S strain, the R strain demonstrated twice as much cytochrome P-450 and cytochrome b₅ and double the rate of NADPH-cytochrome c reductase activity. In addition, the rate of p-nitroanisole O-demethylation was found to be six times greater in the R strain. An in vivo accumulation study showed that the R strain displayed a decreased rate of penetration of trans-[¹⁴C]permethrin. When treated at equitoxic doses the R strain was found to tolerate 50-fold more internal permethrin than the S strain. An in vitro metabolism study indicated that there was no difference between strains in the overall rate of metabolism of trans-[¹⁴C]permethrin. The evidence obtained supports the conclusion that several resistance factors are involved but that decreased sensitivity of the nervous system to the action of pyrethroids is the principal mechanism of resistance in the 147-R strain.     

Beta-cypermethrin resistance associated with high carboxylesterase activities in a strain of house fly, Musca domestica (Diptera: Muscidae)

A housefly strain, originally collected in 1998 from a dump in Beijing, was selected with beta-cypermethrin to generate a resistant strain (CRR) in order to characterize the resistance and identify the possible mechanisms involved in the pyrethroid resistance. The resistance was increased from 2.56- to 4419.07-fold in the CRR strain after 25 consecutive generations of selection compared to a laboratory susceptible strain (CSS). The CRR strain also developed different levels of cross-resistance to various insecticides within and outside the pyrethroid group such as abamectin. Synergists, piperonyl butoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF), increased beta-cypermethrin toxicity 21.88- and 364.29-fold in the CRR strain as compared to 15.33- and 2.35-fold in the CSS strain, respectively. Results of biochemical assays revealed that carboxylesterase activities and maximal velocities to five naphthyl-substituted substrates in the CRR strain were significantly higher than that in the CSS strain, however, there was no significant difference in glutathione S-transferase activity and the level of total cytochrome P450 between the CRR and CSS strains. Therefore, our studies suggested that carboxylesterase play an important role in beta-cypermethrin resistance in the CRR strain.     

Spinosad resistance in female Musca domestica L. from a field-derived population

BACKGROUND: Bait-formulated spinosad is currently being introduced for housefly (Musca domestica L.) control around the world. Spinosad resistance was evaluated in a multiresistant field population and strains derived from this by selection with insecticides. Constitutive and spinosad-induced expression levels of three cytochrome P450 genes, CYP6A1, CYP6D1 and CYP6D3, previously reported to be involved in insecticide resistance, were examined. RESULTS: In 2004 a baseline for spinosad toxicity of Danish houseflies where all field populations were considered to be susceptible was established. In the present study, females of a multiresistant field population 791a were, however, 27-fold spinosad resistant at LC₅₀, whereas 791a male houseflies were susceptible. Strain 791a was selected with spinosad, thiamethoxam, fipronil and imidacloprid, resulting in four strains with individual characteristics. Selection of 791a with spinosad did not alter spinosad resistance in either males or females, but counterselected against resistance to the insecticides thiamethoxam and imidacloprid targeting nicotinic acetylcholine receptors. A synergist study with piperonyl butoxide, as well as gene expression studies of CYP6A1, CYP6D1 and CYP6D3, indicated a partial involvement of cytochrome P450 genes in spinosad resistance. CONCLUSION: This study reports female-linked spinosad resistance in Danish houseflies. Negative cross-resistance was observed between spinosad and neonicotinoids in one multiresistant housefly strain. Spinosad resistance involved alterations of cytochrome P450 gene expression. Copyright © 2011 Society of Chemical Industry     

Evaluation of semiochemical toxicity to houseflies and stable flies (Diptera: Muscidae)

BACKGROUND: The housefly, Musca domestica L., and stable fly, Stomoxys calcitrans (L.) are cosmopolitan pests of both farm and home environments. Houseflies have been shown to be resistant to a variety of insecticides, and new chemistries are slow to emerge on the market. Toxicities of selected semiochemicals with molecular structures indicative of insecticidal activity were determined against adults from an insecticide‐susceptible laboratory strain of houseflies. The three most active semiochemicals were also evaluated against recently colonized housefly and stable fly strains. RESULTS: Nineteen semiochemicals classified as aliphatic alcohols, terpenoids, ketones and carboxylic esters showed toxicity to houseflies and stable flies. Rosalva (LC₅₀ = 25.98 µg cm^?2) followed by geranyl acetone and citronellol (LC₅₀ = 49.97 and 50.02 µg cm^?2) were identified as the most toxic compounds to houseflies. Permethrin was up to 144‐fold more toxic than rosalva on the susceptible strain. However, it was only 35‐fold more toxic to the insecticide‐tolerant field strain. The compounds generated high toxicity to stable flies, with LC₅₀ values ranging from 16.30 to 40.41 µg cm^?2. CONCLUSION: Quantification of LC₅₀ values of rosalva, citronellol and geranyl acetone against susceptible housefly and field‐collected housefly and stable fly strains showed that semiochemicals could serve as potent insecticides for fly control programs. Copyright © 2010 Society of Chemical Industry     

Status of Insecticide Resistance in Spodoptera litura in Andhra Pradesh,India

Twenty-two strains of the tobacco caterpillar, Spodoptera litura (F.) (Lepidoptera: Noctuidae), collected from groundnut crops of eight locations in Andhra Pradesh, India, between 1991 and 1996 were assayed in the F1 generation for resistance to commonly used insecticides. Resistance levels ranged as follows: cypermethrin, 0·2- to 197-fold; fenvalerate, 8- to 121-fold; endosulfan, 1-to 13-fold; quinalphos, 1- to 29-fold; monocrotophos, 2- to 362-fold and methomyl, 0·7- to 19-fold. In nearly all strains pre-treatment with the metabolic inhibitor, piperonyl butoxide, resulted in complete suppression of cypermethrin resistance (2- to 121-fold synergism), indicating that enhanced detoxification by microsomal P450-dependent monooxygenases was probably the major mechanism of pyrethroid resistance. Pre-treatment with the synergist DEF, an inhibitor of esterases and the glutathione S-transferase system, resulted in a 2- to 3-fold synergism with monocrotophos indicating that esterases and possibly glutathione S-transferases were at least to some extent contributing to organophosphate resistance. © 1997 SCI.     

GABA receptors of insects susceptible and resistant to cyclodiene insecticides

Toxicity tests revealed up to 40-fold resistance to a number of cyclodiene insecticides in a laboratory-reared, cyclodiene-resistant (CYW) housefly strain (Musca domestica L.). Using [³⁵S] TBPS as a probe for convulsant sites in insects, saturable specific binding was detected in thorax and abdomen membranes prepared from housefly strains susceptible (CSMA) and resistant (CYW) to cyclodienes. Scatchard analysis of[³⁵S] TBPS binding data to CSMA and CYW membranes failed to provide evidence for significant differences between the two strains in either the affinity (K_d) or density (B_max) of saturable binding sites. For several polychlorocycloalkane insecticides, the ligand displacement profile of [³⁵S] TBPS binding was almost identical for the CSMA and CYW houseflies. Therefore, using [³⁵S] TBPS as a probe for convulsant sites, a 40-fold resistance to cyclodienes in the CYW housefly strain cannot be accounted for only in terms of alterations in TBPS binding sites.     

The current status of insecticide resistance in Musca domestica in England and Wales and the implications for housefly control in intensive animal units

Following the 1984–85 housefly (Musca domestica L.) resistance survey, a similar survey was carried out between January 1990 and April 1992 in order to monitor changes in UK housefly resistance. Samples of fly populations were collected from 35 farms throughout England and Wales. Dose-response data were obtained by topical application and feeding test methods. For both methods the knockdown (KD) after 48 h was used for all the dose-response comparisons. The ranges of resistance factors for the topical application method were for methomyl, 1·6–20·0 at KD₅₀ and 4·5–34·4 at KD₉₅; for azamethiphos, 2·5–58·5 at KD₅₀ and 5·0–2604 at KD₉₅; for pyrethrins+piperonyl butoxide, 1·2–9·6 at KD₅₀ and 1·6–14·7 at KD₉₅; and for permethrin, 2·2–118·8 at KD₅₀ and 4·3–200·0 at KD₉₅. The ranges of the resistance factors for the feeding tests were for methomyl, 1·2–56·1 at KD₅₀ and 3·1–80·0 at KD₉₅, and for azamethiphos, 3·9–125·0 at KD₅₀ and 4·4–380·0 at KD₉₅. The means of resistance factors for all of the insecticides showed increases over those obtained in a previous survey carried out in 1984–85. The significance of the results for housefly control in intensive animal units in the UK is discussed.     

Genetic and biochemical mechanisms limiting fipronil toxicity in the LPR strain of house fly,Musca domestica

Fipronil is a new insecticide which exerts its toxic action by interacting with the insect GABA-gated chloride channel. Previous studies have shown that cyclodiene-resistant insects have low to moderate levels of cross-resistance to fipronil, while other resistant strains are usually susceptible. In contrast, we recently found a strain (LPR) of house fly (Musca domestica L) with 15-fold cross-resistance to fipronil that was not associated with cyclodiene resistance. Fipronil cross-resistance in LPR was inherited as an intermediately dominant, autosomal, multigenic trait. [¹⁴C]Fipronil was observed to penetrate into LPR flies more slowly than into susceptible flies. S,S,S-tributylphosphorotrithioate and diethyl maleate pretreatment did not reduce the level of fipronil cross-resistance, while piperonyl butoxide resulted in a slight decrease. These results indicate that decreased penetration and monooxygenase-mediated detoxification may be mechanisms contributing to fipronil cross-resistance in the LPR strain. © 1999 Society of Chemical Industry     

Synergism of teflubenzuron and chlorfluazuron in an acylurea-resistant field strain of Plutella xylostella L. (lepidoptera: Yponomeutidae)

Topical application of the synergists piperonyl butoxide (PB) and S,S,S-tributyl phosphorotrithioate (DEF) to second-instar larvae of a standard laboratory strain (FS) and an unselected Malaysian field strain (CH) of the diamondback moth Plutella xylostella had no significant effect on the toxicity of the acylurea insecticides, chlorfluazuron and teflubenzuron, in a subsequent leafdip bioassay. In contrast, pre-treatment with PB or DEF in acylurea-selected subpopulations of the CH strain with varying levels of cross-resistance to chlorfluazuron and teflubenzuron significantly increased (up to 34-fold and 28-fold, respectively) the toxicity of both compounds, suggesting that microsomal monooxygenases and esterases may be involved in resistance. The addition of a mineral oil, ‘Sunspray 6E’, to topically-applied chlorfluazuron consistently reduced its LD₅₀ value, and the effect of the oil appeared to be greatest on the most resistant population of P. xylostella. However, the effects of the oil were not significant (P > 0·05) and further studies are necessary to determine whether a penetration factor is present in the CH strain.     

Insecticide resistance in house flies from caged‐layer poultry facilities

The frequency of resistance of eight strains of house flies, Musca domestica L., collected from caged‐layer poultry facilities across New York state, to nine insecticides (dimethoate, tetrachlorvinphos, permethrin, cyfluthrin, pyrethrins, methomyl, fipronil, spinosad and cyromazine) was measured relative to a laboratory susceptible strain. Percentage survival was evaluated at five diagnostic concentrations: susceptible strain LC₉₉, 3 × LC₉₉, 10 × LC₉₉, 30 × LC₉₉ and 100 × LC₉₉. The highest levels of resistance were noted for tetrachlorvinphos, permethrin and cyfluthrin. There was substantial variation in the levels of resistance to the different insecticides from one facility to another, independent of their geographical location. There was very little cross‐resistance detected in these populations to either fipronil or spinosad. Overall, there was a good correlation between insecticide use histories and the levels of resistance. The apparent isolation of fly populations within poultry facilities suggests that there are good opportunities for the implementation of successful resistance management strategies at these facilities. Differences between these results and those of a resistance survey on New York dairy farms in 1987 are discussed. © 2000 Society of Chemical Industry     

Development of resistance to pyrethroids in insects resistant to other insecticides

Resistance to pyrethroids in insects is rare, but its recent rapid development in the field suggests that this resistance may be facilitated by previous exposure to or by resistance to insecticides of unrelated groups. To test this houseflies of strain 49_r2b, originally resistant to dimethoate in the field, were selected eight times during ten generations with either pyrethrum extract or bioresmethrin with or without piperonyl butoxide or with dimethoate. Selecting with any of the pyrethroids led to resistance to these insecticides and in particular to pyrethrum/piperonyl butoxide. Selecting with pyrethrum/piperonyl butoxide resulted in strongest resistance to the pyrethroids tested, whereas selecting with bioresmethrin/piperonyl butoxide resulted in least resistance. These results show that dimethoate-resistant flies selected with pyrethroids can readily develop resistance to these insecticides, but development of resistance can be minimised by using bioresmethrin/piperonyl butoxide. The implications of these findings on the sequential use of insecticides are discussed.     

Synergism of insecticides provides evidence of metabolic mechanisms of resistance in the obliquebanded leafroller Choristoneura rosaceana (Lepidoptera: Tortricidae)

The interactions between six insecticides (indoxacarb, cypermethrin, chlorpyrifos, azinphosmethyl, tebufenozide and chlorfenapyr) and three potential synergists, (piperonyl butoxide (PBO), S,S,S-tributyl phosphorotrithioate (DEF) and diethyl maleate (DEM)) were studied by dietary exposure in a multi-resistant and a susceptible strain of the obliquebanded leafroller, Choristoneura rosaceana (Harris). The synergists did not produce appreciable synergism with most of the insecticides in the susceptible strain. Except for tebufenozide, PBO synergized all the insecticides to varying degrees in the resistant strain. A very high level of synergism by PBO was found with indoxacarb, which reduced the resistance level from 705- to 20-fold when PBO was administered alone and to around 10-fold when used in combination with DEF. DEF also synergized indoxacarb, cypermethrin, chlorpyrifos, azinphosmethyl and tebufenozide in the resistant strain. DEM produced synergism of indoxacarb, chlorpyrifos, azinphos-methyl and chlorfenapyr in the resistant strain. DEM was highly synergistic to cypermethrin, and to some extent to tebufenozide in both the susceptible and resistant strains equally, implying that detoxification by glutathione S-transferases was not a mechanism of resistance for these insecticides. The high level of synergism seen with DEM in the case of cypermethrin may be due to an increase in oxidative stress resulting from the removal of the antioxidant, glutathione. These studies indicate that enhanced detoxification, often mediated by cytochrome P-450 monooxygenases, but with probable esterase and glutathione S-transferase contributions in some cases, is the major mechanism imparting resistance to different insecticides in C. rosaceana.     

Stability and Biological Activity of Cyfluthrin Isomers

A GC-MS method capable of completely separating the four pairs of diastereoisomers of cyfluthrin is presented and the method used to show that isomerisation of the cyfluthrin enantiomers occurs in methanol. This methanol-induced isomerisation could also be demonstrated by bioassays using water fleas. The biological activities of the various cyfluthrin isomers contained in the commercial products cyfluthrin and beta-cyfluthrin were assayed using several strains of lepidopteran larvae including Plutella xylostella, Heliothis virescens and Spodoptera frugiperda. With the susceptible strains, the efficiencies of the isomers mixtures of cyfluthrin and beta-cyfluthrin were shown to obey the rules of additivity. However, in tests with a resistant strain of P. xylostella originating from Thailand, the ‘inactive’ isomer III acted synergistically with the active isomer IV. Resistance factors in strains of H. virescens and P. xylostella were found to be higher with cis than with trans isomers. This probably contributes to the superior action of cyfluthrin and beta-cyfluthrin against various pests of agricultural importance since the commercial products contain a high content of trans isomers (‘high trans pyrethroids’).     

A new resistance to pyrethroids in tribolium castaneum (herbst)

The characteristics of a new high-level, field-derived resistance to pyrethroids in Tribolium castaneum (Herbst) were investigated using impregnated-paper and treated-grain assays. Piperonyl butoxide almost completely suppressed the resistance, suggesting that the major resistance mechanism was microsomal oxidation. Resistance extended to all pyrethroids tested and to carbaryl but not to organophosphorus insecticides or to methoprene. Resistance was strongest against α—CN phenoxybenzyl cyclopropanecarboxylate pyrethroids and was correlated with structural modifications of the pyrethroid molecule, results also consistent with oxidative resistance. This resistance will ultimately result in failures to control T. castaneum if pyrethroids, such as deltamethrin, cypermethrin or cyfluthrin, are used in the field, even if they are synergised with piperonyl butoxide. The resistance does not jeopardise organophosphorus materials (e.g. fenitrothion, chlorpyrifos-methyl, pirimiphos-methyl, methacrifos) or methoprene.     

Resistance to insecticides in winter- and summer-forms of pear psylla,Psylla pyricola

Summer-form pear psylla, Psylla pyricola Foerster, from sprayed pear were resistant to endosulfan (2·4-fold), methiocarb (2·5-fold), ethylan (5·8-fold), azinphos-methyl (7·7-fold), and fenvalerate (40·1-fold). Esterase (3·8-fold), glutathione transferase (1·8-fold), and cytochrome P-450 monooxygenase (1·6-fold) detoxification enzyme activity was higher in resistant than in susceptible summer forms. Synergism by piperonyl butoxide and S,S,S-tributylphosphorotrithioate (DEF) was added evidence for cytochrome P-450 monooxygenases and esterases as resistance mechanisms. Reduced penetration may also have contributed to resistance, as indicated by a 1·6-fold slower penetration of azinphos-methyl in resistant than susceptible summer-forms. Similar differences in insecticide toxicity and esterase and glutathione transferase activities were observed between winter-forms of resistant and susceptible pear psylla. Winter-forms of P. pyricola were up to three times more tolerant to insecticides than summer-forms. Higher cytochrome P-450 monooxygenase activity (1·7-fold) and slower azinphosmethyl penetration (2·1-fold) in winter-forms may have contributed to their greater insecticide tolerance; however, sequestration may also have been involved.