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     

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     

Evaluation of semiochemical toxicity to Aedes aegypti,Ae. albopictus and Anopheles quadrimaculatus (Diptera: Culicidae)

BACKGROUND: Mosquitoes are the most important vectors of human pathogens. Wide‐scale use of pesticides has led to the development of resistance to most common insecticide groups. The need to develop novel products that have a low impact on human health and the environment is well established. The toxicity of selected semiochemicals with molecular structures indicative of insecticidal activity was determined against adult Aedes aegypti (L.) and Anopheles quadrimaculatus (Say). The two most active insecticides against Ae. aegypti were also evaluated against Ae. albopictus (Skuse). RESULTS: Fifteen semiochemicals classified as terpenoid alcohols, ketones or carboxylic esters showed toxicity to both mosquito species. Geranyl acetone (LC₅₀ = 38.51 µg cm^?2) followed by citronellol (LC₅₀ = 48.55 µg cm^?2) were the most toxic compounds to Ae. aegypti, while geraniol and lavonax, with LC₅₀ values of 31.88 and 43.40 µg cm^?2, showed the highest toxicity to An. quadrimaculatus. Both geranyl acetone and citronellol were highly toxic to Ae. albopioctus. No semiochemical showed fumigation activity against either species. All semiochemicals persisted for less than 24 h when tested on filter paper. CONCLUSION: Quantification of LC₅₀ values of several semiochemicals against Ae. Aegypti, An. quadrimaculatus and Ae. albopioctus showed that semiochemicals not only modify insect behaviors but also hold potential as potent insecticides for mosquito control programs. Copyright © 2010 Society of Chemical Industry     

Laboratory trials of fatty acids as repellents or antifeedants against houseflies,horn flies and stable flies (Diptera: Muscidae)

BACKGROUND: Straight‐chain, saturated fatty acids (particularly C8, C9 and C10) have some known behavioral effects on insects such as mosquitoes, and were tested in combination for potential repellency/antifeedant activity in bioassays against three significant muscoid flies of medical/veterinary importance: houseflies, horn flies and stable flies. RESULTS: Mixtures of C8, C9 and C10 (1:1:1; 15% total actives in formulation) were highly repellent to houseflies and horn flies at or below 1 mg formulation cm^?2. Repellency time varied from < 1 day for houseflies to usually at least 3 days for horn flies. Individual longer‐chain‐length fatty acids were tested, and C11 repelled houseflies for up to 5–8 days, while C12 lasted 2 days. Minimum statistically significant repellency levels of the C8, C9 and C10 mixture (3 h after application) against horn flies were 0.06–0.12 mg cm^?2. A liquid formulation of the 15% C8, C9 and C10 mixture in a silicone oil carrier (at 2.8 mg AI cm^?2) was highly repellent against hungry stable flies in a blood‐feeding membrane bioassay for at least 8 h. CONCLUSION: The low toxicity and reasonable activity and persistence of these carboxylic acids make them good candidates for development as protective materials against pest flies in livestock settings. Copyright © 2009 Society of Chemical Industry     

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     

Involvement of esterases in diazinon resistance and biphasic effects of piperonyl butoxide on diazinon toxicity to Haematobia irritans irritans (Diptera: Muscidae)

Resistance to insecticides remains a major problem for the successful control of the horn fly, Haematobia irritans irritans (L.), one of the most important pests of cattle in many countries including the United States. The organophosphate (OP) insecticide diazinon has been used to control pyrethroid-resistant populations of the horn fly. There are only a few reported cases of horn fly resistance to diazinon in the United States and Mexico. Piperonyl butoxide (PBO) has been used successfully as a synergist of pyrethroid insecticides to control horn flies. PBO-synergized diazinon products are also available for horn fly control in the United States, although PBO is known to inhibit the bio-activation of certain OP insecticides including diazinon. A study was conducted to evaluate the effect of PBO on diazinon toxicity to horn flies using a filter paper bioassay technique. These bioassays in both the susceptible and diazinon-resistant horn fly strains revealed a biphasic effect of PBO on diazinon toxicity to horn flies. PBO inhibited diazinon toxicity when the PBO concentration used was high (5%), and no effect was observed when PBO concentration was intermediate (2%). However, at low concentrations (1% and lower), PBO significantly synergized diazinon toxicity. We demonstrated that enhanced esterase activity was associated with survivability of horn flies exposed to diazinon alone. PBO has been shown to inhibit esterase activity in other insect species. However, results of biochemical assays with esterases from this study suggest that PBO did not have significant effect on the overall esterase activity in the horn fly. The observed synergistic effect of PBO at lower concentrations on diazinon toxicity to horn flies could not be explained by reduced esterase activity due to PBO inhibition. It is likely that PBO synergized diazinon toxicity at lower concentrations by facilitating penetration of diazinon through the cuticle and/or inhibiting the oxidative detoxification of diazinon, and reduced diazinon toxicity at high PBO concentration by inhibiting the bio-activation of diazinon.     

Lethal and sublethal effects of chlorantraniliprole on three species of Rhagoletis fruit flies (Diptera: Tephritidae)

BACKGROUND: Chlorantraniliprole formulated as a 350 g kg^?1 WG (Altacor 35WG) for management of apple maggot Rhagoletis pomonella (Walsh), blueberry maggot R. mendax Curran and cherry fruit fly R. cingulata (Loew) (Diptera: Tephritidae) was evaluated in laboratory assays and field trials. RESULTS: A tarsal contact toxicity bioassay showed that a surface residue of 500 mg L^?1 of chlorantraniliprole caused significantly higher mortality of male and female flies of all species compared with a control. Male apple maggot and blueberry maggot mortality was significantly higher than that for females, but there was similar mortality of male and female cherry fruit flies. An ingestion toxicity bioassay showed that 500 mg L^?1 of chlorantraniliprole in diet caused significantly higher mortality of male and female flies of all species than the control, but there were no significant differences among the sexes. Delayed egglaying by females that had ingested chlorantraniliprole was found, but there were no significant sublethal effects on either the number of eggs laid or the egg hatch. Field trials with apple maggot and cherry fruit fly showed that protection of fruit by chlorantraniliprole was comparable with that of standard broad‐spectrum insecticides. CONCLUSIONS: The present data indicate that chlorantraniliprole has suppressant activity against Rhagoletis fruit flies, preventing fruit infestation primarily through direct lethal effects. Copyright © 2008 Society of Chemical Industry     

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.     

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.     

Investigations into carbamate insecticide selectivity: I: Evaluation of potential selectophores

In an effort to improve the generally unfavorable mouse/housefly toxicity ratio of most carbamate insecticides, potential selectophores (nitrile, carbamoyl oxime, carboxylic ester, or amide) were incorporated into a series of phenyl N-methylcarbamates. In addition to the insect and mouse toxicity determinations, the anticholinesterase activity of these compounds was determined for purified housefly head acetylcholinesterase and bovine erythrocyte acetylcholinesterase. The presence of these functional groups, in general, did not give enhanced selectivity ratios and, in one case, (o-N-methylcarbamoyloxyiminomethylphenyl N-methylcarbamate), a very unfavorable selectivity ratio of <0.03 was obtained. A mechanism implicating a Beckmann rearrangement is advanced to rationalize the high rodenticidal activity of this molecule. In general, the carbamates showed poor insecticidal activity when applied alone to the housefly, but, when the flies were pretreated with piperonyl butoxide, the compounds were quite toxic. Finally, an explanation is derived which seeks to justify the inability of these potential selectophores to improve the mouse/housefly toxidity ratio.     

Effects of organic‐farming‐compatible insecticides on four aphid natural enemy species

BACKGROUND: The toxicities of pyrethrins + rapeseed oil, pyrethrins + piperonyl butoxide (PBO), potassium salts of fatty acids and linseed oil were assessed in the laboratory on the parasitic wasp Aphidius rhopalosiphi (Destefani‐Perez), the ladybird Adalia bipunctata (L.), the rove beetle Aleochara bilineata (Gyll.) and the carabid beetle Bembidion lampros (Herbst.). The methods selected were residual contact toxicity tests on inert and natural substrates. RESULTS: Both the pyrethrin products led to 100% mortality in the adult parasitic wasps and ladybird larvae on glass plates and plants. The pyrethrins + PBO formulation was toxic for B. lampros on sand and natural soil, but the pyrethrins + rapeseed oil formulation was harmless for this species. Insecticidal soaps were harmless for all these beneficial species. None of the tested products significantly affected the parasitism of the onion fly pupae by A. bilineata. CONCLUSION: The results indicated the potentially high toxicity of natural pyrethrins for beneficial arthropods. Although this toxicity needs to be confirmed in field conditions, the toxicity levels obtained in the laboratory were similar to or higher than those of several synthetic insecticides known to be toxic in the field. Insecticidal soaps could be considered as an alternative for aphid control in organic farming in terms of selectivity. Copyright © 2010 Society of Chemical Industry     

Purification and characterisation of proteins secreted by the entomopathogenic fungus Metarhizium anisopliae with insecticidal activity against adults of the Mediterranean fruit fly,Ceratitis capitata (Diptera: Tephritidae)

BACKGROUND: The control of the Mediterranean fruit fly (medfly) Ceratitis capitata (Wied) is usually performed with protein bait sprays incorporating chemical insecticides that may have adverse effects on humans, non‐target organisms and the environment. In recent years, scientists have sought more environmentally friendly insecticides for medfly control, such as plant‐ and microorganism‐derived compounds. Among these compounds, entomopathogenic fungi are an unexplored source of natural insecticides. RESULTS: The crude soluble protein extract (CSPE) of the entomopathogenic fungus Metarhizium anisopliae (Mestch.) (strain EAMa 01/58‐Su) shows chronic insecticidal activity when administered per os. Mortality in flies exhibits a dose response. The CSPE produces an antifeedant effect in adult flies, a result probably due to a progressive deterioration of the fly midgut after ingestion of the extract. Protease and temperature treatments show that insecticidal activity against C. capitata is due to proteinaceous compounds that are highly thermostable. Four monomeric proteins from this crude extract have been purified by liquid chromatography and gel electroelution. Although all four monomers seem to be involved in the insecticidal activity of the CSPE, the 15 kDa and the 11 kDa proteins appear to be mainly responsible for the observed insecticidal effect. CONCLUSIONS: Four new fungal proteins with insecticidal activity have been purified and identified. These proteins might be combined with insect baits for C. capitata biocontrol. Copyright © 2009 Society of Chemical Industry     

Semiochemicals of the common bed bug,Cimex lectularius L. (Hemiptera: Cimicidae), and their potential for use in monitoring and control

The recent resurgence of the common bed bug, Cimex lectularius L., has driven an increase in research into the biology and behaviour of this pest. Current control is reliant on the application of insecticides, but, owing to the development of insecticide resistance, there is a need for new tools and techniques. Semiochemicals (behaviour‐ and physiology‐modifying chemicals) could be exploited for management of bed bugs. The aim of this review was to evaluate studies undertaken in bed bug chemical ecology to date, with particular reference to how the research could be exploited for monitoring and control. Bed bugs, like many other insects, have a complex olfactory system. Recent studies have characterised the olfactory sensilla, located on the terminal segment of the antennae, to functional classes by electrophysiological screening. Behavioural studies have revealed the presence of an alarm pheromone and potential airborne aggregation semiochemicals, but it is not yet understood if bed bugs use a sex pheromone during mating. Host location cues have been investigated, and carbon dioxide has been found to be highly attractive both in laboratory and in field studies. Recent field trials have tested blends of other potential kairomones, which have been shown to have an additive effect when used in a heated bed bug trap with carbon dioxide. The trap, which combines heat and kairomones, is the only trap currently available with proven efficacy in the field. In order for semiochemicals to be useful for bed bug management, an increased knowledge and understanding of the biology, behaviour and chemical ecology of this insect is essential. Copyright © 2010 Society of Chemical Industry     

A push–pull strategy to suppress stable fly (Diptera: Muscidae) attacks on pasture cattle via a coconut oil fatty acid repellent formulation and traps with m-cresol lures

BACKGROUND

Stable flies [Stomoxys calcitrans (L.)] are economically important pests of cattle and other livestock. As an alternative to conventional insecticides, we tested a push–pull management strategy using a coconut oil fatty acid repellent formulation and an attractant-added stable fly trap.

RESULTS

In our field trials we found that weekly applications of a push–pull strategy can reduce stable fly populations on cattle as well as a standard insecticide (permethrin). We also found that the efficacy periods of the push–pull and permethrin treatments following on-animal application were equivalent. Traps with an attractant lure used as the pull component of the push–pull strategy captured sufficient numbers of stable flies to reduce on-animal numbers by an estimated 17–21%.

CONCLUSIONS

This is the first proof-of-concept field trial demonstrating the effectiveness of a push–pull strategy using a coconut oil fatty acid-based repellent formulation and traps with an attractant lure to manage stable flies on pasture cattle. Also notable is that the push–pull strategy had an efficacy period equivalent to that of a standard, conventional insecticide under field conditions. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Status of pesticide resistance in arthropod pests in Israel

A complex of events and factors, pertinent to a specific insect and insecticide, governs the development of resistance to insecticides. In Israel, resistance to conventional and novel insecticides occurred in insect pests such asBemisia tabaci andSpodoptera littoralis (that damage agricultural crops),Tribolium castaneum and other flour beetles (that contaminate stored products), andPediculus humanus spp., house flies and mosquitoes (that threaten public health). In the mid-1980s an insecticide resistance management (IRM) strategy was established for all cotton grown in Israel and is being adjusted on a yearly basis as needed. At present, insect pest management and IRM strategies are being developed and implemented area-wide for three regions in Israel: Bet She’an Valley, western Galilee, and western Negev. There are several research groups now working in Israel on various aspects of resistance including occurrence, mechanisms, and management practices. This paper offers a tentative review of the status of insecticide and acaricide resistance in pests in Israel.     

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     

Further evaluation of permethrin for biting-fly control

Laboratory tests in a fly chamber and field trials in the UK and Australia have shown that permethrin, (3-phenoxybenzyl (1R,1S)-cis,trans-3-(2,2-dichlorovinyl)-2, 2-dime-thylcyclopropanecarboxylate) containing 25: 75 cis: trans isomers, applied as water emulsions to cattle and horses will control biting flies. Satisfactory protection against the stable fly, Stomoxys calcitrans (L.) and excellent control against the horn fly, Haematobia (Lyperosia) irritans (L.) was achieved on cattle. Good control was obtained on horses against the stable fly and the sand fly Culicoides spp. These early results are encouraging and toxicity and residue data suggest that permethrin would be a safe insecticide to use on livestock for the control of biting flies of veterinary importance.     

An electrophysiological investigation of the susceptible (cooper) and resistant (kdr; super-kdr) strains of the adult housefly,Musca domestica L

The electrical activity of abdominal nerves of the housefly, Musca domestica L., was used as a bioassay to study nerve sensitivity to DDT and deltamethrin in susceptible (Cooper) and resistant (kdr, super-kdr) strains. By this technique the resistant strains were less sensitive (approximately 10 000-fold) than Cooper, but the bioassay could not distinguish between super-kdr and kdr in their responses to either compound and so could not account for the greater resistance shown by flies with super-kdr above kdr flies when these insecticides are applied topically. Although factors other than nerve insensitivity may be involved, the compounds were applied to the preparation in aqueous saline solutions at, or close to, their solubility limits and this could have masked differences in responses of nerves from the resistant strains.     

Susceptibility to thiamethoxam of Musca domestica from Danish livestock farms

BACKGROUND: Neonicotinoid baits are currently replacing anticholinesterase baits for control of adult houseflies (Musca domestica L.). Introduction of new insecticides includes evaluation of their cross-resistance potential, which was assessed for thiamethoxam in field populations from Denmark. RESULTS: In feeding bioassay with a susceptible strain, thiamethoxam LC(50) at 72 h was 1.7 microg thiamethoxam g(-1) sugar, making it 19-fold, 11-fold and threefold more toxic to houseflies than azamethiphos, methomyl and spinosad respectively. The field populations were 6-76-fold resistant to thiamethoxam. There was no correlation between the toxicities of thiamethoxam and spinosad, dimethoate, methomyl, bioresmethrin or azamethiphos. The toxicity in feeding bioassay at 72 h of imidacloprid in a susceptible strain was 32 microg imidacloprid g(-1) sugar at LC(50), making it 19-fold less toxic to houseflies than thiamethoxam. There was a strong significant correlation between the toxicities of thiamethoxam and imidacloprid in field populations.CONCLUSION: Neonicotinoid-resistant houseflies were present at a detectable and noticeable level before thiamethoxam and imidacloprid were introduced for housefly control in Denmark. The toxicity of thiamethoxam is explained by other parameters than the toxicities of spinosad, dimethoate, methomyl, bioresmethrin or azamethiphos. The cross-resistance between thiamethoxam and imidacloprid indicates a coincidence of mechanism of the toxicity and resistance in the field populations.     

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.