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.     

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.     

The pyrethrins and related compounds part XL— structure-activity relationships of pyrethroidal esters with acyclic side chains in the alcohol component against resistant strains of housefly (Musca domestica)

Activities of a range of pyrethroidal esters, incorporating structural variations in all regions of the acid and alcohol components, have been measured against two fully characterised and homozygous resistant strains of Musca domestica (kdr and super-kdr). The results, limited in this paper to esters of alcohols with acyclic side chains, show the same level of uniform resistance as the kdr strain observed previously, across a range of acid and alcohol variations. Against the super-kdr strain, in contrast to the cyclic side chain set, resistance factors are barely higher than for the kdr strain. Against super-kdr flies, some dependence of resistance factor on the position of the side chain substituent was detected. Polyfluorobenzyl esters confer particularly low resistance factors.     

The pyrethrins and related compounds part XXXIX— structure-activity relationships of pyrethroidal esters with cyclic side chains in the alcohol component against resistant strains of housefly (Musca domestica)

Activities of a range of pyrethroidal esters, incorporating structural variations in all regions of the acid and alcohol components, have been measured against two fully characterised and homozygous resistant strains of Musca domestica L. (kdr and super-kdr). The results, limited in this paper to esters of alcohols with cyclic side chains, indicate uniform resistance to the kdr strain across the whole range of structural variations. Against the super-kdr strain, while variation in the acid component has little effect, the resistance factor is sensitive to the nature of the alcohol component, in particular on whether it contains an α-cyano substituent.     

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.     

DDT and pyrethroids: Receptor binding in relation to knockdown resistance (kdr) in the house fly

The nature of target site or knockdown resistance (kdr) to DDT and pyrethroids was studied by investigating specific binding of [14_C] DDT and [14_C] cis-permethrin to the previously established membrane receptors from the heads of susceptible (sbo) and resistant (kdr) strains of the house fly, Musca domestica L. In vivo studies showed the heads from sbo flies bound two to three times more DDT than those from kdr flies at all doses tested. Reduced binding was also observed in kdr flies in in vitro [14_C] DDT binding assays. Scatchard analysis indicated that kdr flies have the same affinity but fewer receptors per milligram protein in the CNS than sbo flies. Assays with [14_C] cis-permethrin also showed binding was much reduced in kdr flies in comparison with sbo flies. Based on these results, the nature of the target site insensitivity of kdr flies may relate to their having a reduced number of receptors for the insecticides.     

Voltage-dependent Na+ channels in pyrethroid-resistant Culex pipiens L mosquitoes

In some insect species, knockdown resistance (kdr) to pyrethroids and DDT is linked to point mutations in the sequence of the para-type voltage-dependent sodium channel gene. The effects of pyrethroids were assayed on six Culex pipiens strains: two were susceptible to pyrethroids and the four others displayed various levels of resistance, but, in each case, a kdr-type mechanism was strongly suggested. Degenerate primers were designed on the basis of the corresponding sequences of the para orthologous gene reported from several orders of insects. These primers were used to amplify the region of the sodium channel gene which includes the positions where the kdr and super-kdr mutations have been found in Musca domestica. As expected, the amplified fragment was highly homologous to the para sequences. The super-kdr-like mutation (methionine to threonine at position 918 of the M domestica para sequence) was never detected in any strain. In contrast, the same kdr mutation (leucine to phenylalanine at position 1014) was present in some Culex pyrethroid-resistant samples. An alternative substitution of the same leucine to a serine was detected in one strain slightly resistant to pyrethroids but highly resistant to DDT. These data have allowed us to design a PCR-based diagnostic test on genomic DNA to determine the presence or the absence of the kdr allele in single C pipiens collected in several countries. The validity of this test was checked by comparing the frequency of the resistance allele and the toxicological data. © 1999 Society of Chemical Industry     

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.     

Molecular analysis of resistance in a deltamethrin-resistant strain of Musca domestica from China

We investigated the molecular basis of resistance in a strain of house fly (BJD) from Beijing, China. This strain showed 567-fold resistance to commonly used deltamethrin. Flies were 64-fold resistant to deltamethrin synergized by piperonyl butoxide (PBO). The 5′-flanking sequence of the cytochrome P450 gene CYP6D1 in BJD strain had a 15-bp insert as in the LPR strain. Two mutations (kdr, super-kdr) in the voltage sensitive sodium channel (VSSC) were also detected in the BJD strain. Our results showed that a combination of resistance alleles for CYP6D1 and VSSC existed in deltamethrin resistant house flies in China.     

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.     

Biochemical and molecular diagnosis of insecticide resistance conferred by esterase, MACE, kdr and super-kdr based mechanisms in Italian strains of the peach potato aphid, Myzus persicae (Sulzer)

In this paper we analysed the basis of insecticide resistance in 59 Italian strains of the peach potato aphid Myzus persicae using both molecular and biochemical assays. Our data as a whole clearly indicate that most M. persicae strains (76.3%) have high or extremely high production of an esterase enzyme which sequester and detoxify insecticides with esteric group. Kdr genotypes conferring resistance towards pyrethoids are present in 57.7% of the analysed populations. Moreover, 26.5% of the kdr positive strains possess also the M918T mutation conferring super-kdr phenotype. Strains with modified AChE (MACE) are not so numerous (27.1%), although they can be found almost everywhere in Italy. Considering all the strains analysed, both MACE and kdr phenotypes are associated with high levels of esterase activity. In Central–Southern regions, kdr and MACE resistance mechanisms resulted in linkage disequilibrium. Bioassays performed in order to evaluate the efficacy of a pyrethroid insecticide against a strain possessing a F979S mutation within its para-type sodium channel gene suggests that this amino acid substitution could affect the sodium channel responsivity to pyrethroids.     

Depolarization of motor nerve terminals by pyrethroids in susceptible and kdr-resistant house flies

When applied at concentrations of one nM or higher to a house fly larval neuromuscular preparation, deltamethrin (DM) and fenvalerate (FV) greatly increased miniature excitatory postsynaptic potential (mepsp) rate and blocked neuromuscular transmission. The DM-induced mepsp discharge was abolished by tetrodotoxin (TTX), removal of Ca²⁺ from the saline, or by application of hyperpolarizing stimuli to the nerve, indicating that it was due to depolarization of the presynaptic terminals. Also, in the presence of TTX, K⁺ depolarization increased mepsp rate at the same external K⁺ concentration before and after DM treatment, confirming that DM released transmitter by depolarizing the nerve terminals rather than by altering the voltage dependence of transmitter release. The potassium channel blocker tetraethylammonium (TEA) increased mepsp rate somewhat, while aconitine (20 μM), which keeps sodium channels open, increased mepsp rate consistently. Pretreatment of nerves with a subthreshold dose of TEA greatly increased the mepsp rate-increasing activity of DM and aconitine, while a subthreshold level of aconitine did not synergize DM. These observations suggest that DM, like aconitine, depolarized nerves by modifying the sodium channels. Knockdown resistant (kdr) larvae were resistant to the depolarizing action of DM and aconitine but not to that of TEA, indicating that the kdr gene produced a modified sodium channel which was less sensitive to the action of pyrethroids and aconitine. During sustained transmitter release by DM, evoked release gradually declined, resulting in a condition called early block in which spontaneous release was high and release could be evoked by electrotonic depolarization of the nerve terminals, but not by a nerve action potential. Early block was probably due to conduction block in the nerve terminals. Early block eventually gave way to late block, characterized by the decline of spontaneous release to subnormal levels and complete failure of evoked release. After late block, the calcium ionophore X-537A could not release transmitter, suggesting that late block was due to depletion of available transmitter. DM did not have a direct effect upon extrasynaptic muscle membrane. However, after late block, muscles were left insensitive to the putative transmitters glutamate and aspartate when these were bath or iontophoretically applied. A low rate of mepsps persisted after late block, indicating that the muscles were still sensitive to the natural transmitters.     

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.     

Resistance to bifenthrin and resistance mechanisms of different strains of the two-spotted spider mite (Tetranychus urticae) from Turkey

Nine different strains of the two-spotted spider miteTetranychus urticae Koch (Acari: Tetranychidae) were collected on cotton from Adana, Antalya, Izmir, Manisa and Urfa in Turkey. Their responses to bifenthrin were investigated using conventional bioassay and biochemical assays. LC₅₀ and LC₉₀ values of bifenthrin were determined for all strains by using a residual bioassay with a petri dish-spray tower. Resistance ratios were determined by comparing the samples with a standard susceptible strain, GSS. The resistance ratios of the strains ranged from <1 to 669-fold (at LC₅₀). Of the investigated field strains, only three (two from Adana and one from Urfa) were resistant to bifenthrin. There was a correlation between esterase enzyme activity and bifenthrin resistance according to polyacrylamide gel electrophoresis and microtiter plate assays in the three resistant strains. http://www.phytoparasitica.org posting May 17, 2005.     

Potentiation of Super-kdr resistance to deltamethrin and other pyrethroids by an intensifier (factor 161) on autosome 2 in the housefly (Musca domestica L.)

An intensifier (factor 161) identified on the second autosome in a pyrethroid-resistant strain of houseflies (Musca domestica L.) was isolated and introduced into a strain with super-kdr. Unlike E_0.39, which on its own also confers very weak (< × 3) resistance to pyrethroids, factor 161 very strongly intensified super-kdr resistance to pyrethroids. Together, factor 161 and super-kdr conferred immunity to deltamethrin in female houseflies (LD₅₀ > 20 μg fly^?1) but produced much less intensification of resistance to WL 48281, the (1R)cis (αS) isomer of cypermethrin, which differs from deltamethrin only in having chlorine instead of bromine substituents in the acid side-chain. Intensification was strongly decreased by piperonyl butoxide and propyl prop-2-ynylphenylphosphonate (NIA) but was unaffected by S,S,S-tributyl phosphorotrithioate (DEF). This synergism suggests involvement of oxidative rather than esteratic metabolism in the intensification of super-kdr by factor 161.     

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.     

High-throughput approach to detection of knockdown resistance (kdr) mutation in mosquitoes, Culex quinquefasciatus, based on real-time PCR using single-labelled hybridisation probe/melting curve analysis

BACKGROUND: Knockdown resistance (kdr) mutation (L1014F) is a well‐defined mechanism of resistance to pyrethroids and DDT in many insect species. Sensitive detection of the mutations associated with resistance is a prerequisite for resistance management strategies. The authors have developed a new real‐time molecular diagnostic assay based on SimpleProbe^®/melting curve analysis for large‐scale kdr genotyping in the wild population of Culex quinquefasciatus Say, the principal vector of bancroftian filariasis. Melting curve analysis is based on the thermal stability difference between matched and mismatched DNA duplexes. The application of SimpleProbe^® chemistry in insects described here is novel in entomology research. RESULTS: The mosquitoes homozygous for knockdown‐resistant and knockdown‐susceptible allele showed melting peaks at 60.45 °C ( ± 0.25) and 64.09 °C ( ± 0.24) respectively. The heterozygous mosquitoes yielded both peaks at approximately 60.5 °C ( ± 0.2) and 64.20 °C ( ± 0.23). Among the 92 samples genotyped, 16 were found to be homozygous resistant, 44 homozygous susceptible and 32 heterozygous. Comparative assessments were made of all the reported methods for kdr genotyping. CONCLUSION: The present method is cheaper, faster, more reliable and versatile than other alternatives proposed in detecting correct kdr genotypes in mosquitoes. This is the first report using a single‐labelled hybridisation probe to detect point mutations in insect populations. Copyright © 2010 Society of Chemical Industry     

Molecular studies of knockdown resistance to pyrethroids: cloning of domain II sodium channel gene sequences from insects

Knockdown resistance (kdr) is a target-site resistance mechanism that confers nerve insensitivity to DDT and pyrethroid insecticides. In the housefly, Musca domestica, molecular cloning of the para-type sodium channel gene has revealed two amino acid mutations that are associated with kdr and super-kdr resistance phenotypes. Both mutations are located in the domain II region of the channel; Leu1014 to Phe in the hydrophobic segment IIS6 and Met918 to Thr in the IIS4-IIS5 linker. To investigate whether these mutations also occur in other insects, we have designed degenerate primers based on conserved sequences in the domain II region of the sodium channel and used these to PCR amplify this region from insecticide-susceptible strains of eight diverse insect species representing four different insect Orders: Helicoverpa armigera, Plutella xylostella, Spodoptera littoralis (Lepidoptera), Blattella germanica (Dictyoptera), Tribolium castaneum (Coleoptera), Myzus persicae, Aphis gossypii and Phorodon humuli (Hemiptera). The primers amplified closely related para-type sodium channel sequences from each insect with a minimum of 85% amino acid identity between species. All of the sequences contained ‘susceptible’ Leu and Met residues at the positions associated with kdr and super-kdr resistance in the housefly. Recent results detailing the presence of a kdr-type Leu to Phe mutation in pyrethroid-resistant strains of two important agricultural pests, P. xylostella and M. persicae, are discussed. ©1997 SCI     

Incidence and spread of knockdown resistance (kdr) in German Colorado potato beetle (Leptinotarsa decemlineata Say) populations

The Colorado beetle, Leptinotarsa decemlineata (Say), is one of the most important pests in many potato‐producing regions. Colorado beetle infestations are normally kept under economic damage thresholds by applying insecticides such as pyrethroids. Pyrethroids are known to act on voltage‐gated sodium channels and have been used for several decades to control L. decemlineata. Their continuous and widespread use has resulted in the development of resistance, which is often linked to a L1014F target‐site mutation in the voltage‐gated sodium channel, known as knockdown resistance (kdr). Since pyrethroids are used in many potato‐growing regions in Germany, more than 140 L. decemlineata samples were collected and analysed for the presence of the kdr allele by pyrosequencing diagnostics. Results showed that kdr is present in many German L. decemlineata populations, but even its homozygous presence does not substantially compromise the efficacy of recommended label rates of pyrethroids, as demonstrated by bioassays and crossing experiments. The implications of these findings for resistance management are briefly discussed.     

Frequency and intensity of pyrethroid resistance through the CDC bottle bioassay and their association with the frequency of kdr mutations in Aedes aegypti (Diptera: Culicidae) from Mexico

BACKGROUND

The control of Aedes aegypti (L.), the main urban vector that causes arboviral diseases such as dengue, Chikungunya and Zika, has proved to be a challenge because of a rapid increase in insecticide resistance. Therefore, adequate monitoring of insecticide resistance is an essential element in the control of Ae. aegypti and the diseases it transmits. We estimated the frequency and intensity (Resistance Frequency Rapid Diagnostic Test [F‐RDT] and Resistance Intensity Rapid Diagnostic Test [I‐RDT]) of pyrethroid resistance in populations of Ae. aegypti from Mexico using the bottle bioassay and results were related to the frequencies of knockdown resistance (kdr) mutations V1016I and F1534C.

RESULTS

All populations under study were resistant to the pyrethroids: bifenthrin (99%), d‐(cis–trans)‐phenothrin (6.3% cis, 91.7% trans) and permethrin (99.5%) according to F‐RDT, and showed moderate to high‐intensity resistance at 10× the diagnostic dose (DD) in I‐RDT. Frequencies of the kdr mutation V1016I in Ae. aegypti populations were correlated with moderate permethrin resistance at 10× DD, whereas F1534C mutation frequencies were correlated with high bifenthrin resistance at 5× DD. Both I1016 and C1535 were highly correlated with high‐intensity phenothrin resistance at 1× to 10× DD.

CONCLUSIONS

This study showed that high frequencies of kdr mutations V1016I and F1534C are reflected in the results of F‐RDT and I‐RDT tests. Bioassays in conjunction with the characterization of genetic resistance mechanisms are indispensable in the strategic and rational management of resistance in mosquitoes. © 2018 Society of Chemical Industry