Super-kdr Mutation Research Articles

High nucleotide diversity in the para-like voltage-sensitive sodium channel gene sequence in the western flower thrips (Thysanoptera: Thripidae).

In a search for a pyrethroid resistance diagnostic marker, a partial sequence of the para-like sodium channel gene was obtained from 78 diploid females of the arrhenotokous insect pest species Frankliniella occidentalis (Pergande), the western flower thrips. Although all the insects analyzed came from a single laboratory population, nine different haplotypes were obtained. Two haplotypes did have the well-known L to F kdr mutation, but only one of these could be statistically linked to pyrethroid resistance in our population. This haplotype did not have the superkdr mutation, but did have a unique mutation a few amino acids downstream, at a position already linked to resistance in Plutella. Although this para-like locus seemed to have a role in pyrethroid resistance in our population, other resistance mechanisms were also probably involved. The fact that our laboratory population, open to migration, contained ahigh genetic diversity forthis selected gene shows that "pest tourism" is a major factor for resistance dynamics in this greenhouse pest. This, with the possible occurrence of an original resistance mutation, might preclude the use of very specific approaches for resistance monitoring in the field in this species.

Metabolic detoxication and the kdr mutation in pyrethroid resistant house flies, Musca domestica (L.)

Two house fly strains, ALHF and SeALHF, were collected from Alabama after control failures with pyrethroids. While pyrethroid resistance in ALHF partially conferred by P450 monooxygenase- and hydrolase-mediated metabolism has been reported, no studies have been conducted on resistance of SeALHF. The current study was carried out to investigate mechanisms of pyrethroid resistance in SeALHF and the possible role of target site insensitivity, due to kdr mutation, in pyrethroid resistance of ALHF. Resistance to permethrin in SeALHF was dramatically and partially suppressed by PBO and DEF, respectively, suggesting that P450 monooxygenase-mediated metabolism plays a major role in permethrin resistance in this strain, while hydrolytic metabolism has a minor contribution to resistance. Incomplete suppression of permethrin resistance by PBO and DEF suggests that one or more additional minor mechanisms are involved in overall resistance of SeALHF. Injection did not decrease levels of resistance to permethrin in SeALHF, implying that a decreased rate of cuticular penetration ( pen) does not play a role in permethrin resistance in this strain. A 392 bp para-type sodium channel gene fragment, where kdr (L1014F) and super-kdr (M918T) mutations reside, was generated by RT-PCR from ALHF and SeALHF. The M918T mutation was not detected in ALHF or SeALHF, suggesting that the super-kdr mutation is not important in permethrin resistance of these two house fly strains even though ALHF possesses a much higher level of resistance than SeALHF. The L1014F mutation was present in ALHF, but not in SeALHF, suggesting that the kdr mutation is an important factor in pyrethroid resistance in ALHF. A leucine to histidine (L1014H) substitution at the position corresponding to kdr mutation was detected in SeALHF. The importance of this mutation in resistance is discussed.

Association of novel mutations in a sodium channel gene with fluvalinate resistance in the mite, Varroa destructor

SUMMARYVarroa (Varroa destructor) has recently become resistant to Apistan, a pyrethroid pesticide with tau-fluvalinate as its active ingredient. In many insect pests, resistance to pyrethroid insecticides is due to reduced target-site (sodium channel) sensitivity to pyrethroids in the nervous system, a phenomenon called knockdown resistance (kdr). A number of studies showed that kdr and kdr-type resistance is a result of point mutations in the para family of sodium channel genes. To investigate the molecular mechanism of resistance to fluvalinate in varroa, we have cloned and sequenced a large cDNA fragment corresponding to segment 3 of domain II (IIS3) to segment 6 of domain IV (IVS6) of a para-homologous sodium channel gene (VmNa) from susceptible and resistant mite populations. The deduced amino acid sequence from this cDNA shares 71%, 60%, and 50% identity with the corresponding region of the para-homologous protein of the Southern cattle tick, Boophilus microplus, Drosophila melanogaster Para, and rat brain type II sodium channel α-subunit, respectively. Sequence analysis revealed that four amino acid changes, F758L in IIIS6, L826P in the linker connecting domains III and IV, I982V in IVS5 and M1055I in IVS6, were correlated with fluvalinate resistance in both Florida and Michigan populations. Interestingly, the kdr or super-kdr (which confers much higher level resistance than kdr) mutation previously identified in insects was not detected in the resistant mites. These results support the emerging notion that distinct sodium channel gene mutations are selected in different insects and arachnids in response to pyrethroid selection.

The molecular interactions of pyrethroid insecticides with insect and mammalian sodium channels.

Recent progress in the cloning of alpha (para) and beta (TipE) Na channel sub-units from Drosophila melanogaster (fruit fly) and Musca domestica (housefly) have facilitated functional expression studies of insect Na channels in Xenopus laevis oocytes, assayed by voltage clamp techniques. The effects of Type I and Type III pyrethroids on the biophysical properties of these channels are critically reviewed. Pyrethroid resistance mutations (termed kdr and super-kdr) that reduce the sensitivity of the insect Na channel to pyrethroids have been identified in a range of insect species. Some of these mutations (e.g. L1014F, M918T and T929I) have been incorporated into the para Na channel of Drosophila, either individually or in combination, to investigate their effects on the sensitivity of this channel to pyrethroids. The kdr mutation (L1014F) shifts the voltage dependence of both activation and steady-state inactivation by approximately 5 mV towards more positive potentials and facilitates Na channel inactivation. Incorporation of the super-kdr mutation (M918T) into the Drosophila Na channel also increases channel inactivation and causes a > 100-fold reduction in deltamethrin sensitivity. These effects are shared by T929I, an alternative mutation that confers super-kdr-like resistance. Parallel studies have been undertaken using the rat IIA Na channel to investigate the molecular basis for the low sensitivity of mammalian brain Na channels to pyrethroids. Rat IIA channels containing the mutation L1014F exhibit a shift in their mid-point potential for Na activation, but their overall sensitivity to permethrin remains similar to that of the wild-type rat channel (i.e. both are 1000-fold less sensitive than the wild-type insect channel). Mammalian neuronal Na channels have an isoleucine rather than a methionine at the position (874) corresponding to the super-kdr (M918) residue of the insect channel. Replacement of the isoleucine of the wild-type rat IIA Na channel with a methionine (I874M) increases deltamethrin sensitivity 100-fold. In this way, studies of wild-type and mutant Na channels of insects and mammals are providing a molecular understanding of kdr and super-kdr resistance in insects, and of the low pyrethroid sensitivity of most mammalian Na channels. They are also giving valuable insights into the binding sites for pyrethroids on these channels.

Molecular and biochemical survey of acaricide resistance mechanisms in larvae from Mexican strains of the southern cattle tick, Boophilus microplus

We examined the larvae of several organophosphate and pyrethroid-resistant Mexican strains of Boophilus microplus using biochemical and molecular tests to investigate the mechanisms conferring acaricide resistance. The electrophoretic profiles of esterase activity in protein extracts from coumaphos and permethrin-resistant strains compared to the susceptible strain revealed distinct differences, which inhibitor studies attributed to carboxylesterases. Esterase hydrolysis assays showed significant enhancement of both total and permethrin hydrolysis in one pyrethroid-resistant strain, with no enhancement in two other strains with very high resistance to pyrethroids. Sequence analysis of sodium channel mRNA fragments in all pyrethroid-resistant strains determined that they did not possess the classic kdr and super-kdr mutations known to confer pyrethroid resistance in several insect species. Using reverse transcriptase–polymerase chain reaction (RT–PCR) with degenerate primers designed from conserved regions of insect esterase amino acid sequences, a B. microplus larval cDNA fragment was isolated whose deduced amino acid sequence was significantly similar to esterases from a wide range of species. In Northern blot RNA analysis the cDNA hybridized to a 2.1 kb mRNA that was abundant in all resistant strains except one, in which a very low abundance could provide a marker for the mechanism conferring resistance in this strain.

Activation of Drosophila sodium channels promotes modification by deltamethrin. Reductions in affinity caused by knock-down resistance mutations.

kdr and super-kdr are mutations in houseflies and other insects that confer 30- and 500-fold resistance to the pyrethroid deltamethrin. They correspond to single (L1014F) and double (L1014F+M918T) mutations in segment IIS6 and linker II(S4-S5) of Na channels. We expressed Drosophila para Na channels with and without these mutations and characterized their modification by deltamethrin. All wild-type channels can be modified by <10 nM deltamethrin, but high affinity binding requires channel opening: (a) modification is promoted more by trains of brief depolarizations than by a single long depolarization, (b) the voltage dependence of modification parallels that of channel opening, and (c) modification is promoted by toxin II from Anemonia sulcata, which slows inactivation. The mutations reduce channel opening by enhancing closed-state inactivation. In addition, these mutations reduce the affinity for open channels by 20- and 100-fold, respectively. Deltamethrin inhibits channel closing and the mutations reduce the time that channels remain open once drug has bound. The super-kdr mutations effectively reduce the number of deltamethrin binding sites per channel from two to one. Thus, the mutations reduce both the potency and efficacy of insecticide action.

Voltage-dependent Na+channels in pyrethroid-resistantCulex pipiensL 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

Role of the kdr and super-kdr sodium channel mutations in pyrethroid resistance: correlation of allelic frequency to resistance level in wild and laboratory populations of horn flies ( Haematobia irritans)

The kdr and super-kdr point mutations found in the insect sodium channel gene are postulated to confer knockdown resistance (kdr) to pyrethroids. Using an allele-specific PCR assay to detect these mutations in individual horn flies, Haematobia irritans (L.), we determined the allelic frequency of the kdr and super-kdr mutations in several wild and laboratory populations. Wild populations with very similar allelic frequencies had resistance levels that ranged widely from 3- to 18-fold relative to a susceptible population. Conversely, the kdr allele frequency in a lab population with 17-fold resistance was nearly double that found in a heavily pressured wild population with 18-fold resistance. We conclude that, although the kdr mutation confers significant levels of pyrethroid resistance, a substantial component of resistance in insecticidally pressured populations is conferred by mechanisms that are PBO-suppressible. High super-kdr allele frequencies were detected in two resistant lab populations, but in wild populations with equivalent resistance the super-kdr allele frequency was very low. Interestingly, in over 1200 individuals assayed, the super-kdr mutation was never detected in the absence of the kdr mutation.

How Much Safety Is Too Much Danger?

The tomato leafminer, Tuta absoluta, is a major pest of tomato crops worldwide. This study surveyed the resistance of T. absoluta populations from four regions in Brazil to pyrethroid insecticides, the frequencies of L1014F, T929I and M918T Na channel mutations, and the role of detoxification metabolism in the resistance. Resistance ratios varied from 1- to 11-times among populations and insecticides, but control failure likelihood assays showed that all pyrethroids assessed exhibited no efficacy at all (and thus, 98–100% control failure likelihood) against all T. absoluta populations. The activity of glutathione S-transferase and cytochrome P450-mediated N-demethylation in biochemical assays was significantly correlated with the level of resistance to deltamethrin and permethrin suggesting that these enzymes may play a role in resistance. TaqMan assays were used to screen for the presence of knockdown resistance (kdr) mutations and revealed that the L1014F kdr mutation was fixed in all populations and associated with two super-kdr mutations, M918T and particularly T929I, at high frequency. Altogether, results suggest that control failures are because of mutations in the domain II of the sodium channel, as a prevailing mechanism of resistance to pyrethroids in populations of T. absoluta in Brazil. But, enhanced cytochrome P450-dependent monooxygenases and GST activities also play an important role in the resistance of some populations, which reinforce that pyrethroids must not be used overall to control T. absoluta.