Cypermethrin and Ivermectin Resistance in Field Populations of Rhipicephalus Sanguineus Sensu Lato (Latrielle, 1806) Collected From Dogs in South India

The abuse of acaricides for control of Rhipicephalus sanguineus sensu lato ticks led to a strong selection pressure for acaricide resistance. Data on acaricide resistance in R. sanguineus s.l. populations from India is meager though veterinarians frequently report treatment failures. The present study was aimed to characterize the level of resistance in R. sanguineus s.l. against most commonly used drugs, cypermethrin and ivermectin in Andhra Pradesh, south India. Fourteen adult female tick populations were collected from naturally infested dogs at veterinary clinics, residence and stray dog in nine state municipal corporations/municipalities. Information on the history of dog treatment with acaricides was obtained by interviewing dog owners. The larval packet test (LPT) and larval immersion test (LIT) were used on the larvae of ticks to test for resistance to cypermethrin and ivermectin, respectively. Mortality data of each tick population was analyzed by probit analysis. Corresponding to the most susceptible population, thirteen (92.6%) and six (42.9%) tick populations were regarded as resistant to cypermethrin and ivermectin, respectively. The phenotypic level of cypermethrin (resistance ratios at LC50% varied from 1.55 to 13.29) and ivermectin (resistance ratios at LC50% ranged from 1.16 to 4.79) resistance varied distinctly between the populations. Additionally, all the populations were tested using PCR to demonstrate the frequency of the point mutation in sodium channel gene that corresponds with resistance of R. sanguineus s.l. to cypermethrin. A nucleotide substitution (T2134C) on domain III segment VI of the sodium channel gene was noticed in phenotypically resistant tick populations on DNA sequencing. Ivermectin resistance in the brown dog ticks is reported for the first time from India.

A field study on the relationship between tick burden and deltamethrin resistance in Rhipicephalus microplus isolates of Indian household cattle

A total of 2809 ixodid ticks were collected by screening 4560 household cattle from six different agro-climatic zones of Tamil Nadu, a southern state of India. Among the ixodid ticks, Rhipicephalus microplus tick isolates were subjected to Adult Immersion Test (AIT) and Larval Packet Test (LPT) using deltamethrin. The mean % of ixodid tick infested cattle population was 63.2%±10.9. Positive correlation was found between the tick burden on household cattle and Resistance Factor (R=0.66). Further, 44% variance in the tick burden on cattle is explained by LPT based resistance factor suggesting deltamethrin resistance level is one of the contributing factors to the tick load on cattle. Analysis of DNA sequence of sodium channel gene domain II-S4-5 linker region of all deltamethrin resistant R. microplus tick isolates, confirmed the absence of point mutation at the 190th nucleotide which suggests the possible mutation in other regions of sodium channel gene of R. microplus tick isolates of south India and/or alternate resistance mechanisms.

Permethrin Resistance in Aedes aegypti Affects Aspects of Vectorial Capacity

Aedes aegypti, as one of the vectors transmitting several arboviruses, is the main target in mosquito control programs. Permethrin is used to control mosquitoes and Aedes aegypti get exposed due to its overuse and are now resistant. The increasing percentage of permethrin resistant Aedes aegypti has become an important issue around the world and the potential influence on vectorial capacity needs to be studied. Here we selected a permethrin resistant (p-s) Aedes aegypti population from a wild Florida population and confirmed the resistance ratio to its parental population. We used allele-specific PCR genotyping of the V1016I and F1534C sites in the sodium channel gene to map mutations responsible for the resistance. Two important factors, survival rate and vector competence, that impact vectorial capacity were checked. Results indicated the p-s population had 20 times more resistance to permethrin based on LD50 compared to the parental population. In the genotyping study, the p-s population had more homozygous mutations in both mutant sites of the sodium channel gene. The p-s adults survived longer and had a higher dissemination rate for dengue virus than the parental population. These results suggest that highly permethrin resistant Aedes aegypti populations might affect the vectorial capacity, moreover, resistance increased the survival time and vector competence, which should be of concern in areas where permethrin is applied.

First detection of F1534C knockdown resistance mutation in Aedes aegypti (Diptera: Culicidae) from Cameroon

Background Aedes borne viral diseases, notably dengue, are increasingly reported in Cameroon with Aedes aegypti being a major vector. Data on insecticide resistance of this vector and underlying mechanisms needed for outbreak preparedness remain scarce in Cameroon. Here, we present the nationwide distribution of insecticide resistance in Ae. aegypti and investigate the potential resistance mechanisms involved. Methods Immature stages of Ae. aegypti were collected between March and July 2017 in 13 locations across Cameroon and reared until G1/G2/G3 generation. Larval, adult bioassays, and piperonyl butoxide (PBO) synergist assays were carried out according to World Health Organization guidelines. F1534C mutation was genotyped using allele specific polymerase chain reaction in field collected adults (Go) and the polymorphism of the sodium channel gene was assessed. The χ2 test was used to compare the mortality rate between bioassays with insecticides only and bioassays after preexposure to PBO synergist. Results Larval bioassay revealed that all the three populations tested with temephos were susceptible. Adult bioassays showed a good level of susceptibility toward both pyrethroids tested, 0.25% permethrin and 0.05% deltamethrin, with six out of 10 populations susceptible. However, two populations (Douala and Edéa) were resistant (deltamethrin [73.2–92.5% mortality], permethrin [2.6–76.3% mortality]). The resistance to 4% dichlorodiphenyltrichloroethane was observed in four out of 10 populations tested (16.8–87.1% mortality). Resistance was also reported to carbamates including 0.1% propoxur (60.8–87.1% mortality) and to 0.1% bendiocarb (82.9% mortality). All populations tested were fully susceptible to 1% fenitrothion. A partial recovery of susceptibility was observed in the pyrethroid resistant population of Douala after pre-exposed to PBO suggesting the implication of cytochrome P450 monoxygenases permethrin resistance. Genotyping and sequencing detected the F1534C kdr mutation in the two pyrethroid resistant locations of Edéa and Douala, with allelic frequency of 3.3% and 33.3% respectively. However, the high genetic diversity of the sodium channel gene supports the recent introduction of this mutation in Cameroon. Conclusions This study revealed the contrasting resistance profiles to insecticides of Ae. aegypti populations in Cameroon suggesting that, instead of a unique nationwide control approach, a regionally adapted strategy will be needed to control this vector. The localised distribution of the F1534C kdr mutation supports this region-specific control strategy.

Physiological evidence that three known mutations in the para-sodium channel gene confer cypermethrin knockdown resistance in Rhipicephalus microplus

Background: Acaricide resistance is a central problem for the control of the cattle tick Rhipicephalus microplus. Genetic changes that confer resistance get fixed in the population, however, the physiological effects and phenotypes of these mutations are not always well understood or characterized. Single Nucleotide Polymorphisms (SNPs) in the para-sodium channel gene that confer cypermethrin knockdown resistance (kdr) were identified in several arthropods, and homologous mutations have been reported in R. microplus populations. To our knowledge, R. microplus populations that have these homologous SNPs have been associated and correlated with pyrethroid resistance but there is no direct physiological evidence that their presence does confer kdr in this organism. Methods: Resistance profiles from resistant and susceptible strains used in this work were obtained using the larval packet discriminating dose assay. The relevant genomic regions of the para-sodium channel of these strains were amplified using standard PCR; SNPs were detected by sequencing the corresponding amplicons. Ovary response to cypermethrin exposure/treatment was evaluated using videometrical analysis.Results: In this work we present historical evidence that the pyrethroid resistance trait is stable in a resistant reference strain after many years without selection, thus suggesting that the primary resistance mechanism is caused by mutations fixed in the population. In our experimental system, the mechanism that allows the maintenance of the contraction of the ovary after treatment with pyrethroids, is likely to be mediated by a change in the structure of the presynaptic para-sodium channel. We found that the resistant strain has the G184C, the C190A and the T2134A mutations in the para-sodium channel gene. SNPs G184C and T2134A have been reported to be genetically linked in resistant populations and are always found together. These mutations were confirmed to be absent in the susceptible strain used as control. Finally, using videometric analysis, we demonstrate that cypermethrin blocks ovary contraction in cypermethrin-susceptible ticks. We also show that ovaries from populations that carry the kdr associated SNPs still contract at cypermethrin concentrations that completely block ovary contraction in the susceptible strain. The configuration of the experimental system excludes a xenobiotic detoxification mechanism. Conclusions: This is the first report that presents physiological evidence that the presence of the G184C, the C190A, and the T2134A mutations in the para-sodium channel correlates with the ability of maintaining muscle contractility in R. microplus when exposed to cypermethrin. These results confirm that these SNPs may confer cypermethrin resistance in this organism by avoiding presynaptic blockage that in turn causes flaccid muscle paralysis that is characteristic of this acaricide. This work also demonstrates that the videometric assay that we previously validated can be used to detect more rapidly than other assays that involve larval mortality kdr-like cypermethrin resistant tick strains, because adult preengorged females can be directly assayed after they are collected on the field without waiting until eggs are laid and the larvae eclose.

Physiological evidence that three known mutations in the para-sodium channel gene confer cypermethrin knockdown resistance in Rhipicephalus microplus

Background: Acaricide resistance is a central problem for the control of the cattle tick Rhipicephalus microplus . Genetic changes that confer resistance get fixed in the population, however, the physiological effects and phenotypes of these mutations are not always well understood or characterized. SNPs in the para-sodium channel gene that confer cypermethrin knockdown resistance ( kdr ) were identified in several arthropods, and homologous mutations have been reported in R. microplus populations. To our knowledge, R. microplus populations that have these homologous SNPs have been associated and correlated with pyrethroid resistance but there is no direct physiological evidence that their presence does confer kdr in this organism. Methods: Resistance profiles from resistant and susceptible strains used in this work were obtained using the larval packet discriminating dose assay. The relevant genomic regions of the para-sodium channel of these strains were amplified using standard PCR; SNPs were detected by sequencing the corresponding amplicons. Ovary response to cypermethrin exposure/treatment was evaluated using videometrical analysis. Results: In this work we present historical evidence that the pyrethroid resistance trait is stable in a resistant reference strain after many years without selection, thus suggesting that the primary resistance mechanism is caused by mutations fixed in the population. In our experimental system, the mechanism that allows the maintenance of the contraction of the ovary after treatment with pyrethroids, is likely to be mediated by a change in the structure of the presynaptic para-sodium channel. We found that the resistant strain has the G184C, the C190A and the T2134A mutations in the para-sodium channel gene. SNPs G184C and T2134A have been reported to be genetically linked in resistant populations and are always found together. These mutations were confirmed to be absent in the susceptible strain used as control. Finally, using videometric analysis, we demonstrate that cypermethrin blocks ovary contraction in cypermethrin-susceptible ticks. We also show that ovaries from populations that carry the kdr associated SNPs still contract at cypermethrin concentrations that completely block ovary contraction in the susceptible strain. The configuration of the experimental system excludes a xenobiotic detoxification mechanism. Conclusions: This is the first report that presents physiological evidence that the presence of the G184C, the C190A, and the T2134A mutations in the para-sodium channel correlates with the ability of maintaining muscle contractility in R. microplus when exposed to cypermethrin. These results confirm that these SNPs may confer cypermethrin resistance in this organism by avoiding presynaptic blockage that in turn causes flaccid muscle paralysis that is characteristic of this acaricide. This work also demonstrates that the videometric assay that we previously validated can be used to detect more rapidly than other assays that involve larval mortality kdr -like cypermethrin resistant tick strains, because adult preengorged females can be directly assayed after they are collected on the field without waiting until eggs are laid and the larvae eclose.