Low expression of nicotinic acetylcholine receptor subunit Mdα2 in neonicotinoid‐resistant strains of Musca domestica L.

BACKGROUND: Neonicotinoid action as well as resistance involves interaction with nicotinic acetylcholine receptors (nAChRs). In the housefly, neonicotinoid resistance also involves cytochrome P450, as indicated by bioassay with synergist as well as altered expression. In bioassay, synergism was only partial and indicated possible target‐site resistance. The nAChR α2 subunit is important in neonicotinoid toxicity to insects, and gene expression of the Mdα2 subunit was investigated in field populations and laboratory strains of neonicotinoid‐resistant and insecticide‐susceptible houseflies, Musca domestica L. The genomic sequence covering exon III–VII of Mdα2 was analysed for mutations. RESULTS: Gene expression profiling of Mdα2 revealed notable differences between neonicotinoid‐resistant and insecticide‐susceptible houseflies. On average, the neonicotinoid‐resistant field population 766b and the imidacloprid selected strain 791imi had 60% lower copy numbers of Mdα2 compared with the susceptible reference strain. Sequencing of exon III–VII of the Mdα2, encoding acetylcholine binding‐site regions and three out of four transmembrane domains, did not reveal any mutations explaining the increased neonicotinoid tolerance in the strains examined. CONCLUSION: Previous discoveries and the results of this study suggest that the neonicotinoid resistance mechanism in Danish houseflies involves both cytochrome P450 monooxygenase‐mediated detoxification and reduced expression of the nAChR subunit α2. Copyright © 2010 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     

Cuticular lipids of two resistant and a susceptible strain of houseflies

A study was made of the composition of the cuticular lipids of two resistant strains of houseflies (Rutgers and Fc), both of which show a reduced rate of absorption of insecticides as a partial mechanism of resistance and a susceptible strain (CSMA). Total lipids, monoglycerides, diglycerides and sterol esters (except in the Fc strain), sterols, fatty acids and phospholipid phosphorus were higher in resistant strains than in the susceptible strain. Phosphatidyl-ethanolamine and phosphatidyl-choline were major constituents of the phospholipid fractions and were appreciably higher in the resistant strains. Cuticular wax contents did not differ among strains. Incorporation of lipid precursors, [U-¹⁴]acetate and [³²P]orthophosphate, was greater in the cuticle of one or both resistant strains, depending on the lipid component examined.     

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     

The Pyrethrins and Related Compounds. Part XLI. Structure–Activity Relationships in Non-ester Pyrethroids against Resistant Strains of Housefly (Musca domestica L.)

A series of pyrethroids, related to NRDC 200 and etofenprox (MTI500) in which the central region is represented by a non-ester link, have been tested against one susceptible and two resistant strains (kdr and super-kdr) of houseflies (Musca domestica L.). A range of structural variations in the central region have been examined. Resistance factors mostly fell within narrow ranges for both resistant strains i.e. 10–50-fold resistance against kdr and 50–150-fold against super-kdr; thus no correlation of resistance with structural features was detectable for this region. Other changes examined were the substituent on the phenyl ring in the ‘acid’ component and the bridging group in the ‘alcohol’ component where small variations in response were observed. Examination of the effect of varying the ‘alcohol’ side chain was limited by lack of active analogues.     

Genetic and biochemical studies of resistance to permethrin in a pyrethroid-resistant strain of the housefly (Musca domestica L.)

One or more weak factors of resistance on autosome 2, and barely detectable resistance on autosome 3, confer moderate resistance to several pyrethroids (5–13-fold) in the field-collected Ipswich strain of houseflies. In these flies, which unlike other pyrethroid-resistant strains lack kdr or super-kdr, pyrethroid resistance probably developed in response to prolonged treatment of buildings for animals with pyrethrins synergised with piperonyl butoxide. Substrains, isolated genetically from Ipswich flies and with resistance only on autosome 2, degraded permethrin more rapidly than susceptible flies and produced larger amounts of very polar metabolites. In this, they differed from flies with kdr or super-kdr which resembled susceptible flies in their metabolism of permethrin. NIA 16388 (propyl prop-2-ynyl phenylphosphonate) was a better synergist and reduced the metabolism of permethrin more than piperonyl butoxide in both the susceptible and resistant insects. The slight increase in synergism and minimal decrease in metabolism when piperonyl butoxide was applied with NIA 16388 indicated that the latter also inhibited detoxication that was sensitive to piperonyl butoxide.     

Insecticide resistance in houseflies from the middle east and North Africa with notes on the use of various bioassay techniques

The susceptibility to pyrethroid, organochlorine, organophosphorus and carbamate insecticides, of 20 strains of houseflies (Musca domestica L.) collected in the Middle East and North Africa, was assessed by topical application. No resistance to pyrethroids was found but most flies were resistant to DDT, gamma-HCH, organophosphorus and carbamate insecticides. Numerical factors of resistance for a susceptible and two different resistant strains, obtained using different bioassay techniques, were compared. High mortality (≥95%) was achieved with ‘resisted’ insecticides in tests with space sprays, but only low, variable mortality resulted from deposit tests. If this occurs under practical field conditions, moderately resistant populations of flies could be controlled by using space sprays containing comparatively high concentrations of active ingredient, but increased levels of deposit would be ineffective.     

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     

Investigating the potential of selected natural compounds to increase the potency of pyrethrum against houseflies Muscadomestica (Diptera: Muscidae)

BACKGROUND: A study was undertaken to determine the efficacy of seven natural compounds compared with piperonyl butoxide (PBO) in synergising pyrethrum, with the intention of formulating an effective natural synergist with pyrethrum for use in the organic crop market. RESULTS: Discriminating dose bioassays showed PBO to be significantly more effective at synergising pyrethrum in houseflies than the seven natural compounds tested, causing 100% mortality in insecticide‐susceptible WHO and resistant 381zb strains of housefly. The most effective natural synergists against WHO houseflies were dillapiole oil, grapefruit oil and parsley seed oil, with 59, 50 and 41% mortality respectively, compared with 18% mortality with unsynergised pyrethrum. Against 381zb houseflies, the most effective natural synergists were parsley seed oil and dillapiole oil. Esterase inhibition by the natural compounds and PBO in vitro showed no correlation with pyrethrum synergism in vivo, whereas the inhibition of oxidases in vitro more closely correlated with pyrethrum synergism in vivo. CONCLUSION: Dillapiole oil and parsley seed oil showed the greatest potential as pyrethrum synergists. PBO remained the most effective synergist, possibly owing to its surfactant properties, enhancing penetration of pyrethrins. The results suggest the involvement of oxidases in pyrethroid resistance in houseflies, with the efficacy of synergists showing a high correlation with inhibition of oxidases. Copyright © 2011 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.     

Interaction between the factor delaying penetration of insecticides and the desethylation mechanism of resistance in organophosphorus-resistant houseflies

The desethylation (gene a) and penetration delaying (pen) factors of resistance to organophosphorus insecticides isolated by genetical methods from the diazinon-selected SKA strain of houseflies (Musca domestica L.) were inbred into a strain whose resistance was compared with that of flies with single factors of resistance and the SKA strain. Pen alone decreases kill marginally, and gene a alone raises LD₅₀ to 1·4–48 times that of susceptible flies, depending on the insecticide. The two factors together increase resistance greatly to many organophosphorus insecticides (up to 5–10 times or more to diazinon, malathion-ethyl and chlorthion-ethyl than of flies with gene a only) indicating that the two factors interact. Interaction is greater against the thionates than the corresponding phosphates, probably because pen delays entry of thionates more than of the corresponding phosphates. The role in resistance of each factor when hetero-and homo-zygous, and the reasons for interaction, are discussed.     

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.     

Metabolism of methyl bromide by susceptible and resistant strains of the granary weevil, Sitophilus granarius (L.)

Methyl bromide was metabolized by susceptible and resistant strains of adult granary weevil, Sitophilus granarius (L.), mainly by conjugation with glutathione. S-Methyl glutathione and S-methyl cysteine were produced by both strains and S-methyl glutathione sulfoxide was identified as a metabolite in the resistant strain. In the untreated insects, no significant difference was observed in glutathione S-transferase activity but the resistant contained approximately twice as much glutathione per insect as the susceptible strain. When the insects were treated with methyl bromide, the glutathione content of both strains was lowered; proportionally, however, the decrease was considerably higher in the susceptible than in the resistant strain. These results indicate that conjugation of methyl bromide with glutathione is a major detoxication pathway and tolerance to this fumigant is related, in part at least, to the level of glutathione in the granary weevil.     

Interstrain comparison of glutathione-dependent reactions in susceptible and resistant houseflies

Glutathione S-alkyl- and S-aryltransferase activities and the glutathione-dependent reactions involved in the metabolism of diazinon, parathion, DDT and γ-BHC were determined in two susceptible and three resistant housefly strains. The relative rate of formation of desethyl diazinon and desethyl parathion and the degradation of γ-BHC paralleled the activities of the alkyl and aryltransferases in the various strains of houseflies suggesting that a single enzyme might be involved. DDT-dehydrochlorinase showed different relative rates among the strains indicating that the dechlorination was catalyzed by a different enzyme. The enzyme responsible for the conjugation of the pyrimidinyl moiety of diazinon appears to be different from the one which catalyzes the conjugation of the p-nitrophenyl moiety of parathion. The dearylation reactions were not mediated by the glutathione S-aryltransferase in the various housefly strains.     

Neonicotinoid resistance in rice brown planthopper, Nilaparvata lugens

BACKGROUND: Rice brown planthopper, Nilaparvata lugens Stål, is a primary insect pest of cultivated rice, and effective control is essential for economical crop production. Resistance to neonicotinoid insecticides, in particular imidacloprid, has been reported as an increasing constraint in recent years. In order to investigate the extent of resistance, 24 samples of N. lugens were collected from China, India, Indonesia, Malaysia, Thailand and Vietnam during 2005 and 2006. Their responses to two diagnostic doses of imidacloprid (corresponding approximately to the LC₉₅ and 5 × LC₉₅ of a susceptible strain) were examined. RESULTS: Ten of the 12 samples collected during 2005 were found to be susceptible to imidacloprid, but two late‐season samples from India showed reduced mortality at both diagnostic doses. All 13 strains collected in 2006 showed reduced mortality at both doses when compared with the susceptible strain. Dose–response lines showed resistance in one of the most resistant field strains to be approximately 100‐fold compared with the susceptible standard. CONCLUSION: The data demonstrate the development and spread of neonicotinoid resistance in N. lugens in Asia and support reports of reduced field efficacy of imidacloprid. Copyright © 2008 Society of Chemical Industry     

Factors affecting the sequential acquisition by Danish houseflies (musca domestica L.) of resistance to organophosphorus insecticides

The prolonged use of dimethoate, introduced into Denmark to control houseflies (Musca domestica L.) that had become resistant to parathion and diazinon, resulted ultimately in dimethoate resistance. Selection with dimethoate led to the disappearance of the hydrolytic phosphatase, a major mechanism of resistance to parathion and diazinon, and its replacement by the acetylcholinesterase AChER with somewhat decreased sensitivity to inhibition by organophosphorus (OP) insecticides. The hydrolytic phosphatase probably disappeared because low substrate turn-over made it ineffective against dimethoxon (O, O-dimethyl S-methylcarbamoylmethyl phosphorothioate, also known as omethoate). which accumulates at higher concentrations than paraoxon (diethyl4-nitrophenyl phosphate) in the haemolymph. Dimethoate selected AChER preferentially because it improved the chances of houseflies surviving against the relatively poor AChE inhibitor dimethoxon, whereas its relatively small insensitivity to OP insecticides, unimportant against good inhibitors such as paraoxon, prevented its selection by parathion.     

Characterization of the structure-activity relationship of kdr and two variants of super-kdr to pyrethroids in the housefly (Musca domestica L.)

Structure-activity relationships (SARs) for 10 pyrethroids against susceptible, kdr and super-kdr strains of houseflies (Musca domestica L.) were investigated by Principal Components Analysis. In the three strains with kdr_Latina' all only slightly to moderately (2.6 to 26-fold) resistant to pyrethroids, no correlation between the structure and Levels of resistance could be discerned. In flies with super-kdr, SARs were influenced by the nature of the alcoholic portion of the ester. Resistance was strongest to esters of a-cyano-3-phenoxybenzyl alcohol (74 to 430-fold) and to permethrin (48 to 55-fold). It was weak (6.2 to 11-fold) to cyclopentenone derivatives, being barely stronger than for flies with kdr (2-6 to 6.3-fold). Two variants of super-kdr (3D and A2) were distinguished on the basis of their differential response to esters of 5-benzyl-3-furylmethanol. It is presumed that kdr_Latina, super-kdr_A2 and super-kdr_3D form an allelic series in which kdr_Latina represents ground level insensitivity, and the two super-kdrs the progressive extension of strong resistance to more types of ester. The strong differences in resistance to different pyrethroid esters by super-kdr flies provides scope for improving management of resistance to pyrethroid insecticides and for modifying the SAR of pyrethroids to favour weak resistance.     

Negative cross-resistance to bentazone and pyridate in atrazine-resistant Amaranthus cruentus and Amaranthus hybridus biotypes

Plants of Amaranthus cruentus and Amaranthus hybridus resistant to atrazine and cyanazine were found in maize fields in north-eastern Spain. Both resistant foiotypes survived doses of 5 kg ha^?1 of atrazine and 2–4 kg ha^?1 of cyanazine but were controlled by lower doses of bentazone and pyridate than were susceptible biotypes. Such a negative cross-resistance was not found for chloroacetamides and MCPA. Chlorophyll fluorescence studies revealed that atrazine, bentazone, cyanazine and pyridate (10 mg litre^?1) caused inhibition of photosynlhetic electron transport in susceptible leaves, while in resistant plants, atrazine and cyanazine had no effect. Conversely, bentazone and pyridate inhibited photosynthesis to a greater extent in resistant than in susceptible biotypes. Isolated chloroplast membranes from resistant biotypes showed resistance factors of 366 and 501 to atrazine and 39 and 60 to cyanazine for A. hybridus and A. cruentus, respectively. Bentazone and pyridate were found to be more effective in chloropiasts of the resistant biotypes than those of the susceptible plants. It is suggested that enhanced susceptibility to bentazone and pyridate in triazine-resistant A. cruentus and A. hybridus biotypes may be associated with the alteration of the D-I polypeptide subunit of photosystem II, as found in triazine-resistant plants.     

A simple bioassay for detecting and characterising insecticide resistance

A simple and rapid paralysis assay was developed to detect and characterise knockdown resistance in larvae of the house fly and pink bollworm. Pyrethroidtreated larvae unable to perform a stereotypic curling movement when probed with a warm needle, 10 min (house fly) or 60 min (pink bollworm) after treatment, were considered to be paralysed. Responses in a susceptible strain of house fly were compared with those of the pyrethroid-resistant strains kdr and super-kdr. In this assay, the kdr strain displayed over 28 fold resistance to deltamethrin, and super-kdr larvae were unaffected by doses up to 100μg. These results are in good qualitative agreement with previous studies. The assay detected no significant fenvalerate resistance in pink bollworm larvae collected from the field; this was consistent with mortality bioassays performed on wild adult males. The limitations and potential utility of the paralysis bioassay in resistance screening are discussed.     

Selection for abamectin resistance in colorado potato beetle (coleoptera: Chrysomelidae)

There was no difference in dose response to abamectin between a laboratory strain of Colorado potato beetle (Leptinotarsa decemlineata (Say))from North Carolina and a multiple resistant strain from Massachusetts (LD₅₀= 1.95 and 1.98 ng larva^?1, respectively). Two abamectin-resistant strains were generated by separate means. The mutagen, ethyl methanesulfonate, was used to generate an abamectin-resistant strain (LD₅₀= 29.4 ng larva^?1) from the susceptible laboratory strain. The other resistant strain (LD₅₀ = 45.3 ng larva^?1) was generated through an intense selection with abamectin of a field strain contained in cages set up in existing potato fields. Resistance ratios calculated from LD₅₀values for the two abamectin-resistant strains were 15-fold and 23-fold, respectively. Resistance ratios calculated from LD₉₇values (21-fold and 38-fold, respectively) were higher than those calculated at LD₅₀. Also, there were larvae in both resistant strains which were capable of surviving doses up to 100 ng larva^?1, while the susceptible strain had no survivors at 10 ng larva^?1. Although the logit mortality regression analysis produced statistically different lines for the two abamectin-resistant strains, the slopes of each appear to be the same. Both resistance factors were determined to be autosomal and incompletely recessive (0.26 to 0.4, respectively) via reciprocal F₁crosses to the susceptible laboratory strain.