Synthetic Pyrethroid Resistance Research Articles

Genetic Diversity of Rhipicephalus (Boophilus) microplus for a Global Scenario: A Comprehensive Review.

Rhipicephalus microplus poses a substantial threat to livestock health and agricultural economies worldwide. Its remarkable adaptability to diverse environments and hosts is a testament to its extensive genetic diversity. This review delves into the genetic diversity of R. microplus, employing three pivotal genetic markers: the cytochrome c oxidase I (COX1) gene, ribosomal genes, and microsatellites. The COX1 gene, a crucial tool for genetic characterization and phylogenetic clustering, provides insights into the adaptability of ticks. Ribosomal genes, such as internal transcribed spacer regions (ITS-1 and2) as well as 18S and 28S, are routinely utilized for species differentiation. However, their use is limited due to indels (insertions and deletions). Microsatellites and minisatellites, known for their high polymorphism, have been successfully employed to study populations and genetic diversity across various tick species. Despite their effectiveness, challenges such as null alleles and marker variations warrant careful consideration. Bm86, a well-studied vaccine candidate, exhibits substantial genetic diversity. This diversity directly influences vaccine efficacy, posing challenges for developing a universally effective Bm86-based vaccine. Moreover, the review emphasizes the prevalence of genes associated with synthetic pyrethroid resistance. Identifying single nucleotide polymorphisms in the acaricide-resistant genes of R. microplus has facilitated the development of molecular markers for detecting and monitoring resistance against synthetic pyrethroids. However, mutations in sodium channels, the target site for synthetic pyrethroid, correlate well with the resistance status of R. microplus, which is not the case with other acaricide target genes. This study underscores the importance of understanding genetic diversity in developing effective tick management strategies. The choice of genetic marker should be tailored based on the level of taxonomic resolution and the group of ticks under investigation. A holistic approach combining multiple markers and integrating additional molecular and morphological data may offer a more comprehensive understanding of tick diversity and relationships. This research has far-reaching implications in formulating breeding programs and the development of vaccine against ticks and tick-borne diseases (TTBDs) as well as strategies for the management of resistant ticks.

A mutation associated with resistance to synthetic pyrethroids is widespread in US populations of the tropical lineage of Rhipicephalus sanguineus s.l

The brown dog tick, Rhipicephalus sanguineus sensu lato (s.l.), is an important vector for Rickettsia rickettsii, causative agent of Rocky Mountain spotted fever. Current public health prevention and control efforts to protect people involve preventing tick infestations on domestic animals and in and around houses. Primary prevention tools rely on acaricides, often synthetic pyrethroids (SPs); resistance to this chemical class is widespread in ticks and other arthropods. Rhipicephalus sanguineus s.l. is a complex that likely contains multiple unique species and although the distribution of this complex is global, there are differences in morphology, ecology, and perhaps vector competence among these major lineages. Two major lineages within Rh. sanguineus s.l., commonly referred to as temperate and tropical, have been documented from multiple locations in North America, but are thought to occupy different ecological niches. To evaluate potential acaricide resistance and better define the distributions of the tropical and temperate lineages throughout the US and in northern Mexico, we employed a highly multiplexed amplicon sequencing approach to characterize sequence diversity at: 1) three loci within the voltage-gated sodium channel (VGSC) gene, which contains numerous genetic mutations associated with resistance to SPs; 2) a region of the gamma-aminobutyric acid-gated chloride channel gene (GABA-Cl) containing several mutations associated with dieldrin/fipronil resistance in other species; and 3) three mitochondrial genes (COI, 12S, and 16S). We utilized a geographically diverse set of Rh sanguineus s.l. collected from domestic pets in the US in 2013 and a smaller set of ticks collected from canines in Baja California, Mexico in 2021. We determined that a single nucleotide polymorphism (T2134C) in domain III segment 6 of the VGSC, which has previously been associated with SP resistance in Rh. sanguineus s.l., was widespread and abundant in tropical lineage ticks (>50 %) but absent from the temperate lineage, suggesting that resistance to SPs may be common in the tropical lineage. We found evidence of multiple copies of GABA-Cl in ticks from both lineages, with some copies containing mutations associated with fipronil resistance in other species, but the effects of these patterns on fipronil resistance in Rh. sanguineus s.l. are currently unknown. The tropical lineage was abundant and geographically widespread, accounting for 79 % of analyzed ticks and present at 13/14 collection sites. The temperate and tropical lineages co-occurred in four US states, and as far north as New York. None of the ticks we examined were positive for Rickettsia rickettsii or Rickettsia massiliae.

Knockdown resistance mutations are common and widely distributed in Xenopsylla cheopis fleas that transmit plague in Madagascar.

Plague, caused by the bacterium Yersinia pestis, remains an important disease in Madagascar, where the oriental rat flea, Xenopsylla cheopis, is a primary vector. To control fleas, synthetic pyrethroids (SPs) have been used for >20 years, resulting in resistance in many X. cheopis populations. The most common mechanisms of SP resistance are target site mutations in the voltage-gated sodium channel (VGSC) gene. We obtained 25 collections of X. cheopis from 22 locations across Madagascar and performed phenotypic tests to determine resistance to deltamethrin, permethrin, and/or dichlorodiphenyltrichloroethane (DDT). Most populations were resistant to all these insecticides. We sequenced a 535 bp segment of the VGSC gene and identified two different mutations encoding distinct substitutions at amino acid position 1014, which is associated with knockdown resistance (kdr) to SPs in insects. Kdr mutation L1014F occurred in all 25 collections; a rarer mutation, L1014H, was found in 12 collections. There was a significant positive relationship between the frequency of kdr alleles and the proportion of individuals surviving exposure to deltamethrin. Phylogenetic comparisons of 12 VGSC alleles in Madagascar suggested resistant alleles arose from susceptible lineages at least three times. Because genotype can reasonably predict resistance phenotype, we developed a TaqMan PCR assay for the rapid detection of kdr resistance alleles. Our study provides new insights into VGSC mutations in Malagasy populations of X. cheopis and is the first to report a positive correlation between VGSC genotypes and SP resistance phenotypes in fleas. Widespread occurrence of these two SP resistance mutations in X. cheopis populations in Madagascar reduces the viability of these insecticides for flea control. However, the TaqMan assay described here facilitates rapid detection of kdr mutations to inform when use of these insecticides is still warranted to reduce transmission of plague.

Myzus persicae (Sulzer, 1776) (Hemiptera: Aphididae) popülasyonlarında neonikotinoid ve sentetik piretroid insektisitlerin tekli ve karışımlarına karşı direnç durumu

The peach potato aphid, Myzus persicae (Sulzer, 1776) (Hemiptera: Aphididae), a vector of many plant virus diseases, causes damage to its wide range of hosts by direct feeding. Chemical control has been the primary method to control this species, and the intensive use of insecticides has led to the development of resistance. In this study, conducted between the years 2017-2019, firstly resistance ratio of five M. persicae populations from Antalya, Türkiye were determined by leaf-dip bioassay method. The field populations showed significant resistance to thiamethoxam (between 201-332 fold) and lambda-cyhalothrin (between 50-103 fold) when compared to susceptible population. To identify whether resistance mediated by mutations in sodium channel and nicotinic acetylcholine receptor, DNA regions that encompass “mutation hot-spot” were sequenced. This revealed no population contained R81T mutation that has been previously linked with neonicotinoid resistance. As to synthetic pyrethroid resistance, the L1014F kdr mutation was fixed in all field populations. This study is the first description of kdr mutation in M. persicae populations from Türkiye. Bioassay results also indicated that the toxicity of thiamethoxam and lambda-cyhalothrin mixture was higher than that of lambda-cyhalothrin alone. Our findings can make significant contributions to M. persicae resistance management.

Optimasi Analisis Melting Curve untuk Skrining Cepat dan Sensitif Mutasi V1016G pada Aedes aegypti Resisten Sintetik Piretroid dengan Reaksi Rantai Polimerase Spesifik Alel

Detection of V1016G mutation is important for identifying the mechanism of synthetic pyrethroid resistance in Aedes aegypti population. The previous method has described an allele specific polymerase chain reaction (AS-PCR) using conventional PCR to detect the mutation. Although the method has great differentiating power and reproducibility, faster and more sensitive genotyping method is essential to accurately detect the mutation. This study evaluate the used of SYBR® Green real-time PCR and melting curve analysis (MCA) to identify the V1016G mutation. The collection of homozygous 1016G, heterozygous, and wild type (1016 V) mosquitoes DNA genome was extracted using genomic DNA mini kit. The SsoAdvanced™ Universal SYBR® Green Supermix was used to identify alleles by real-time PCR followed melting curve analysis of the amplicons. Melting curve analysis produced reproducible results for the loci tested. The melting temperature was reached at 78.5 oC for homozygous 1016G mosquito and at 86 oC for wild type mosquito. Meanwhile, the heterozigous mosquito revealed two peaks of melting temperature at both 78.5 oC and 86 oC. These easily interpretable and distinguishable melting curve results were consistent with AS-PCR results obtained for the same alleles. The described MCA application for screening V1016G mutation is fast and widely accessible also could be implemented under field conditions

Insecticide resistance status of important malaria vectors in a tribal district of Rajasthan

Investigations have been carried-out in a malaria prone district of Rajasthan to determine insecticide resistance status of important malaria vector species viz. Anopheles culicifacies, An. annularis and An. stephensi, against insecticides being used under National Malaria Control Programme in two Block Public Health Centres of Banswara district of Rajasthan. An. culicifaies, except one canal irrigated village, where it was found susceptible to DDT, was found either resistant or intermediate resistant. Against Malathion An. culicifacies was found susceptible in one rain-fed irrigation village, whereas in all other villages, it was found either resistant or intermediate resistant. Against synthetic pyrethroids An. culicifacies was found susceptible to both Alpha Cypermethrin and Cyfluthrin, except one rain-fed irrigation village, where against Cyfluthrin, the species exhibited intermediate resistance. This is a first report of synthetic pyrethroid resistance from Rajasthan. An. annularis, was found resistant to DDT in both canal irrigated and rain-fed irrigation villages, however, against Malathion it exhibited resistance in canal irrigated village and intermediate resistance in rain-fed irrigation village, however, against Alpha Cypermethrin and Cyfluthrin the species was found totally susceptible in both the areas. An, stephensi was tested only against Alpha Cypermethrin in a canal irrigated village and found totally susceptible. In the study area An. culicifacies have exhibited triple resistance, showing resistance to DDT, Malathion and intermediate resistance to cyfluthrin, however, An. annularis exhibited double resistance to DDT and Malathion.

Efficacy of two commercial synthetic pyrethroids (cypermethrin and deltamethrin) on Amblyomma variegatum and Rhipicephalus microplus strains of the south-western region of Burkina Faso

Since 2011, period of the livestock invasion by the cattle tick Rhipicephalus microplus in Burkina Faso (BF), tick-control problems were exacerbated. Based on farmer’s reports, most commonly used commercial acaricides were found to be ineffective in Western South part of the country. To investigate the occurrence and extent of such acaricidal ineffectiveness, we performed the standardized larval packet test (LPT) with commercial deltamethrin (vectocid) and cypermethrin (cypertop), on two cattle tick species, the native Amblyomma variegatum and the invasive R. microplus. The resistance ratios (RR) were computed with susceptible Hounde strain of Rhipicephalus geigyi as reference. The R. microplus population showed resistance to the two acaricides tested with the highest lethal concentration (LC) values, and different resistance ratios higher than 4 (deltamethrin: RR50 = 28.18 and RR90 = 32.41; cypermethrin: RR50 = 8.79 and RR90 = 23.15). In the contrary, A. variegatum population was found to be highly susceptible to acaricides tested with low lethal concentrations and resistance ratio values (deltamethrin: RR50 = 0.5 and RR90 = 0.48; cypermethrin: RR50 = 0.68 and RR90 = 0.79). These data demonstrate high synthetic pyrethroid resistance in R. microplus strain, leading to conclude that the acaricide ineffectiveness in tick populations control remains a concern in BF.

Deltamethrin resistant alleles predominate in Rhipicephalus sanguineus sensu lato in South India.

Brown dog tick, Rhipicephalus sanguineus sensu lato, an important ectoparasite transmitting several pathogens, is the most common tick species infesting dogs. Control of ticks being central to the control of fatal tick-borne diseases, this study attempted to assess the susceptibility/resistance of brown dog ticks to synthetic pyrethroids, the commonly used acaricides against ticks. Larval packet assay revealed 60% of isolates tested to be resistant and tolerant to deltamethrin as per the resistance factor that ranged from 1 to 53.7. Sequence analysis of the PCR amplified product of domain II S4-5 linker of sodium channel gene in R. sanguineus revealed novel polymorphisms, viz., C190A, G215T and T270C. In domain III S6 region of the gene, a T2134C mutation was observed. Genotyping with allele-specific PCR targeting domain II S4-5 linker region using single larvae revealed that most R. sanguineus larvae in the study population were homozygous resistant (RR) genotypes, followed by heterozygous (RS) and homozygous susceptible (SS) genotypes. A higher proportion of RS genotypes was also observed in domain III S6 region. This first report of genotyping of Indian R. sanguineus to analyse synthetic pyrethroid resistance highlights the need to devise alternate control strategies to reduce the brown dog tick population.

High-resolution melt (HRM) analysis for detection of SNPs associated with pyrethroid resistance in the southern cattle fever tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

The southern cattle fever tick, Rhipicephalus (Boophilus) microplus, is the most economically important ectoparasite of cattle worldwide. A limitation for sustainable control and eradication is the emergence of acaricide resistance among tick populations. Molecular diagnostic tools offer the opportunity to detect resistance rapidly, which can be complemented with confirmatory bioassays with larvae and adult ticks that are more resource and time consuming to generate. Synthetic pyrethroid resistance is one of the most prevalent and well-studied forms of resistance in arthropods, being linked with target site alterations in the sodium ion channel gene. Here, we report research on a novel molecular method to detect mutations in the para-sodium channel gene of R. microplus associated with acaricide resistance that is based on quantitative PCR high-resolution melt (HRM) analysis. Genomic DNA fragments of domains II and III of the para-sodium channel gene were amplified by real-time PCR in the presence of EVA®Green dye to test resistant and susceptible reference ticks from the U.S., Brazil, and Mexico. Larval packet tests with discriminating doses and a modified lethal time analysis were performed to confirm resistance to permethrin, cypermethrin, deltamethrin, and flumethrin in laboratory strains. Tick specimens collected from cattle that were inspected at the United States Port-of-Entry at the Texas-Mexico border were also genotyped. Previously described mutations associated with pyrethroid resistance (T170C, C190A, G184C, and T2134A) were successfully detected by qPCR-HRM in different genotypes and confirmed by sequencing. A novel non-synonymous SNP located at domain III (C2136A) and the G215T mutation in domain II, previously described only in Asian R. microplus and R. australis, were also detected with the HRM and confirmed by sequencing. This technique could be adapted for high-throughput screening, detection, and discovery of allele-specific mutations in cattle tick outbreak populations to inform eradication strategies in the USA. This knowledge could also be applied to integrated control programs in other parts of the world where R. microplus is endemic and where similar SNPs have been identified associated with pyrethroid resistance. This study highlights the existence of several mutations in the para-sodium channel gene in different combinations in field populations of R. microplus from Mexico.

Synthetic pyrethroid resistance in Rhipicephalus (Boophilus) microplus ticks from north-western Himalayas, India.

In the present study, adult immersion test (AIT) was used for evaluation of resistance against synthetic pyrethroids (deltamethrin and cypermethrin) in Rhipicephalus (Boophilus) microplus ticks collected from nine districts of three agro-climatic zones of north-western Himalayan region of India. Resistance factors (RFs) were calculated between 0.94 to 50.71 for deltamethrin and 0.32 to 13.18 for cypermethrin. Resistance to deltamethrin was detected at level I in two, level II in four, level III and level IV in one isolate each while one isolate was susceptible. Against cypermethrin, resistance at levels I and II was detected in three isolates each while three isolates were found susceptible. The low altitude sub-tropical zone revealed higher density of resistant ticks where intensive animal husbandry practices were followed and the synthetic pyrethroid usage was common. Data generated on pyrethroid resistance status of ticks in north-western Himalayan region will provide new insights in acaricidal resistance particularly from remote areas of this region and will help in formulating suitable control measures.

Molecular mechanism of synthetic pyrethroid and organophosphate resistance in field isolates of Rhipicephalus microplus tick collected from a northern state of India.

The frequently used chemical control method to manage Rhipicephalus microplus is limited by the emergence of resistance populations. Understanding of resistance mechanisms is essential to develop strategy for sustainable management. The present study was focused on working out the molecular mechanisms of resistance against synthetic pyrethroids (SPs) and organophosphates (OPs) in field isolates of R. microplus collected from six districts of Uttar Pradesh, India. Adult immersion test with discriminating concentrations (AIT-DC) was used to determine resistance status of isolates to SPs (deltamethrin, cypermethrin) and OPs (diazinon, coumaphos). All the six isolates were found resistant to SPs with resistance factor (RF) of 2.9-58.6 and to one of the OP compounds, diazinon having RF of 3.5-13.7 but susceptible to coumaphos (RF < 1.4). Three R. microplus genes, viz. para-sodium channel domain II S4-5 linker, carboxylesterase (372bp) and acetylcholinesterase 2 (1692bp) were sequenced and compared with respective sequences of reference susceptible IVRI-I, reference OP resistant population (IVRI-III), IVRI-IV and multi-acaricide resistant population (IVRI-V) of R. microplus. A C190A mutation in the domain II S4-5 linker region of sodium channel gene leading to L64I amino acid substitution was detected in all six isolates. The G1120A mutation in the carboxylesterase gene could not be detected in any isolate. Five nucleotide substitutions viz., G138A, G889A, T1090A, C1234T and G1403A were identified in the acetylcholinesterase 2 gene leading to four amino acid substitutions. The findings of the study corroborate the role of mutation in sodium channel and acetylcholinesterase 2 genes in SP and OP resistance in this part of India.

Efficiency of a walk-through fly trap for Haematobia irritans control in milking cows in Uruguay.

Haematobia irritans (horn fly) is a bloodsucking insect that affects grazing cattle. Since this fly's introduction into Uruguay in 1992, pest management practices used to control the insect have been exclusively based on the use of insecticides, which has caused synthetic pyrethroid resistance. The use of insecticides is a major constraint to livestock production due to food safety and environmental concerns. This study evaluated the effectiveness of a walk-through fly trap for horn fly control. The field trail was conducted in 18 trail evaluations dates from 2015 to 2016 in Holstein-Frisian lactating cows on two dairy farms in southern Uruguay. The traps were placed at the exit of the milking parlor. Two digital cameras were used to record video at the entrances and exits of the traps. On each of the 18 trail evaluation, between 30 and 158 cows were randomly selected for fly counting according to video records. On Farm 1, a total of 718 cows were assessed. The median number of flies per cow at the entrance of the traps was 22 (ranging from 1 to 199), while the median number at the exit was three flies per cow (ranging from 0 to 22). The median efficiency of the trap was 88%. Farm 2 had 345 observations, and the median fly count at the entrance of the traps was of 22 flies per cow (ranging from 1 to 129) and four flies at the exit (ranging from 0 to 35) with a median efficiency of 82%. It was observed that the effectiveness of the fly traps varied depending on the number of flies at entry, the season of the year and the farm site. In conclusion, fly traps could be used for the control of H. irritans in milking cows without the use of insecticides.

Biochemical Characterization of Insecticide Resistance and Exposure in Aedes Aegypti Population From Wonosobo, Central Java, Indonesia

Resistance to insecticides mainly occurs due to changes in insect metabolic enzyme. Increased metabolism is often caused by qualitative or quantitative changes of esterase and glutathione S-transferase. Susceptibility test and biochemical assay to detect organophosphate and synthetic pyrethroid resistance were conducted on Aedes aegypti from Wonosobo (new highland Dengue endemic area). The test were performed on F1 generation of Ae.aegypti field caught mosquitoes which aimed to determine the resistance mechanisms regarding two detoxifying enzymes i.e. esterase and glutathione S-transferase. Susceptibility test showed 23.4 and 46.7% mortalities after exposure to 0.8% malathion and 0.05% cypermethrin. The biochemical assay result suggested that esterase, monooxygenase and glutathione s-transferase activity tend to increase in Ae.aegypti in Wonosobo. Interview and questionaires conclude that synthethic pyrethroid was the only insecticide type used in vector control program by Wonosobo Health Office and was the most frequent insecticide type to be used in household by Wonosobo society to control Ae.aegypti population. Knowledge of localized resistance and underlying mechanisms helps in making rational decisions in selection of appropriate and effective insecticides in the event of a dengue outbreak.

Deltamethrin resistance in south Indian isolates of Rhipicephalus (Boophilus) microplus

The present communication deals with the detection and characterization of deltamethrin resistance in tick populations using biological (larval packet test), biochemical (esterase enzyme assay) and molecular assays. Ticks were collected from cattle farms of Korutla, Telangana (KOR), Mehboob Nagar, Telangana (MBN), Nagpur, Maharashtra (NAG), Parbani, Maharashtra (PBN), Madhavaram, Tamil Nadu (MAD), Cuddalore, Tamil Nadu (CUD), Sakhleshpur, Karnataka (SAK) and Buvenduvella, Karnataka (BUV). Out of eight field isolates, seven were identified as Rhipicephalus (Boophilus) microplus while one isolate (CUD) was identified as R. (B.) annulatus. The LC50 values and resistance factors (RF) of field isolates were assessed by larval packet test (LPT). RF values of two isolates viz., Korutla and Parbhani (KOR, PAR) were close to that of reference susceptible isolate. R. (B.) microplus isolate from Nagpur (NAG) and Sakleshpur (SAK) revealed slightly higher RF values (6.42 and 4.51). They revealed slightly elevated esterase enzyme activity too. Other isolates did not reveal higher values for RF or esterase activity. Previously identified mutations conferring synthetic pyrethroid resistance in R. (B.) microplus populations were analysed by sequencing the mutation flanking regions of the carboxyl esterase and the sodium channel genes (domain III S6 and domain II S4–5 linker region). However, these point mutations were not detected in the field isolates. The results of the present study revealed that low levels of synthetic pyrethroid resistance had developed in field populations of ticks of southern India.

Esterase and glutathione S-transferase levels associated with synthetic pyrethroid resistance in Hyalomma anatolicum and Rhipicephalus microplus ticks from Punjab, India

Larval packet test was used for assessment of resistance status against cypermethrin and deltamethrin in Hyalomma anatolicum and Rhipicephalus microplus from various districts of Punjab (India). Among the various field isolates of H. anatolicum susceptible status was recorded against cypermethrin in all isolates, whereas against deltamethrin resistance status (level I-III) was recorded. In R. microplus lower resistance levels (I-II) were recorded against cypermethrin in comparison to deltamethrin (level I-IV). Quantitative analysis of general esterase activity revealed a range of 4.21 ± 0.46 to 6.05 ± 0.55 and 2.23 ± 0.23 to 2.66 ± 0.24 µmol/min/mg protein for α- and β-esterase activity, respectively, in different field isolates of H. anatolicum and the increase in comparison to susceptible was not significant (P > 0.05). In contrast to H. anatolicum, the α- and β-esterase activity in all field isolates (except Jalandhar) of R. microplus was higher (range of 3.89 ± 0.26 to 10.85 ± 0.47 and 1.75 ± 0.08 to 5.87 ± 0.29 µmol/min/mg protein, respectively) (P < 0.001). The glutathione-S-transferase (GST) activity in field isolates of H. anatolicum and R. microplus was in the range of 0.01 ± 0.001 to 0.03 ± 0.001 and 0.02 ± 0.0003 to 0.03 ± 0.001 mM/mg/min. The enzyme ratios (α-and β-esterase and GST) and RR95 against deltamethrin of H. anatolicum isolates were correlated (P < 0.05), whereas in R. microplus only α-and β-esterase and RR50 against deltamethrin were correlated (P < 0.05).

Acaricide and ivermectin resistance in a field population of Rhipicephalus microplus (Acari: Ixodidae) collected from red deer (Cervus elaphus) in the Mexican tropics

In the Neotropics the control of tick infestations in red deer (Cervus elaphus) is achieved primarily through the use of acaricides and macrocyclic lactones. In Mexico, resistance to one or multiple classes of acaricides has been reported in Rhipicephalus microplus infesting cattle, but information on acaricide susceptibility in R. microplus infesting red deer is lacking. In this study we report the level of resistance to different classes of acaricides and ivermectin in R. microplus collected from red deer in the Mexican tropics. Engorged R. microplus females were collected from a red deer farm in Yucatan, Mexico. The larval packet test was used to detect resistance to the organophosphates (OPs) chlorpyrifos and coumaphos, synthetic pyrethroids (SPs) cypermethrin and permethrin, and the phenylpyrazol, fipronil. Resistance to the formamidine amitraz (Am), and ivermectin was ascertained using the larval immersion test. Data were subjected to probit analysis to determine lethal concentrations and resistance ratios to kill 50% (RR50) and 99% (RR99) of the tick population under evaluation in relation to susceptible reference strains. Additionally, allele specific polymerase chain reaction was used to detect the sodium channel F1550I mutation associated with SP resistance in R. microplus. The R. microplus population from red deer in Yucatan showed very high resistance to the two SPs evaluated (RRs>72.2 for cypermethrin; RR for permethrin resistance was so high a dose–response curve was not possible). All individual larvae tested to detect the sodium channel F1550I mutation associated with SP resistance in R. microplus were homozygous. The same tick population showed different levels of resistance to OPs (chlorpyrifos: RR50=1.55, RR99=0.63; coumaphos: RR50=6.8, RR99=5.9), fipronil (RR50=1.8, RR99=0.9), and amitraz (RR50=2.3, RR99=4.4). Resistance to ivermectin was regarded as moderate (RR50=7.1, RR99=5.0). This is the first report of R. microplus ticks collected from red deer in Mexico with different levels of resistance to four acaricide groups and ivermectin.

Autosomal inheritance of alphamethrin, a synthetic pyrethroid, resistance in Anopheles stephensi – Liston, a malaria mosquito

Anopheles stephensi–Liston (Culicidae: Diptera) is an important urban malarial vector in the Indian sub-continent, accounting for about 15% of the total annual malaria incidence. Chemical control represents a key strategy in the management of this insect vector. However, owing to erratic and continuous application of insecticides, resistance has become a common phenomenon among them and their control has become an uphill task. The genetics of alphamethrin, a synthetic pyrethroid resistance was studied to determine its mode of inheritance. The late third instar larvae were selectively inbred for 27 and ten generations to synthesize homozygous resistant (R) and susceptible (S) stocks, respectively, to the diagnostic dose of 0.12 mg l−1. The log-dosage probit mortality relationships and degree of dominance (D) were calculated. Resistance was observed in both sexes, the dosage-mortality (d-m) line of F 1 was towards the resistant parent and the ‘D’ value was found to be 0.8 indicating alphamethrin resistant (amr) gene to be autosomal and incompletely dominant. The d-m lines of F 2/backcross exhibited a clear plateau of mortality across a range of doses indicating monogenic resistance. The null hypothesis for monogenic resistance was tested from mortality data of backcross progeny compared with theoretical expectations using the χ2 test and was found to be non-significant. Understanding genetics of insecticide resistance is significant in prediction and management of resistant insects. The amr genes can be used as genetic marker in A. stephensi, which can be used in several applications in conducting basic and applied genetic research.

In vitro diagnosis of the first case of amitraz resistance in Rhipicephalus microplus in Santo Tomé (Corrientes), Argentina

In Argentina, the cattle tick Rhipicephalus microplus has already developed resistance to organophospates and synthetic pyrethroids. However, no cases of amitraz resistance have ever been recorded in this country despite its heavy use. A recent failure of amitraz to control ticks in a farm located in Santo Tomé, province of Corrientes, resulted in the collection of samples for acaricide resistance diagnosis. The modified Drummond adult immersion test (AIT) and the larval tarsal test (LTT) were performed separately in Argentina and Switzerland to evaluate efficacy of amitraz and other acaricides. The AIT showed that oviposition in the Santo Tomé field isolate was not inhibited when it was challenged to 250 and 500ppm amitraz, and 50ppm deltamethrin. However, oviposition was reduced by 90.6% when this field isolate was challenged to a combination of 400ppm ethion and 100ppm cypermethrin. To confirm the results obtained with the AIT, 2 additional tick samples were collected and shipped to Switzerland for resistance diagnosis of amitraz, cypermethrin and flumethrin, using the LTT. With this bioassay, the resistance ratios of the 2 field isolates were 32.5 and 57.0 for amitraz and between 5.9 and 27.2 for the synthetic pyrethroids. Both in vitro bioassays confirmed amitraz and synthetic pyrethroid resistance in the Santo Tomé samples. These results account for the first evidence of amitraz resistance in R. microplus in Argentina.

Deltamethrin and cypermethrin resistance status of Rhipicephalus (Boophilus) microplus collected from six agro-climatic regions of India

A cross sectional study was conducted to assess the prevalence of synthetic pyrethroids (SP) resistance in Rhipicephalus (Boophilus) microplus in India. Twenty-seven areas located in six agro-climatic regions were selected for the collection of engorged ticks using two stage stratified sampling procedure.Adult immersion test (AIT) and larval packet test (LPT) were optimized using laboratory reared susceptible line of R.(B.) microplus (IVRI-I) for determination of 95% lethal concentration (LC95) of deltamethrin (29.6ppm in AIT and 35.5ppm in LPT) and cypermethrin (349.1ppm in AIT and 350.7ppm in LPT). The AIT with a discriminating dose (2× LC95) was used to detect deltamethrin and cypermethrin resistance in the field isolates of R.(B.) microplus.On the basis of the data generated on three variables viz., mortality, egg masses and reproductive index, the resistance level was categorized as I, II, III and IV. The overall prevalence of SP-resistant R.(B.) microplus among the sampled farms was 66.6% (18/27). Out of these 18 areas, resistance to deltamethrin at level I was detected in 08 areas (resistance factor=2.0–4.9), at level II in 09 areas (RF=5.2–11.8), at level III in 01 area (RF=34.9) and at level IV in 01 area (RF=95.7). The resistance to cypermethrin was detected in 16 areas and level of resistance was detected at level I in 10 areas (RF=2.06–4.64) and at level II in 06 areas (RF=5.13–9.88). The middle-gangetic and trans-gangetic plains revealed higher density of resistant ticks where intensive cross bred cattle population are reared and the SP compounds are commonly used. The data generated on acaricide resistant status in ticks will help in formulating tick control strategy for the country.

First report of a Rhipicephalus microplus tick population multi-resistant to acaricides and ivermectin in the Mexican tropics

We document the presence of a Rhipicephalus microplus tick population resistant to acaricides (organophosphates (OP), synthetic pyrethroids (SP), amitraz) and macrocyclic lactones (ML) (ivermectin). Engorged females of R. microplus were collected from a cattle farm in Veracruz, Mexico, to evaluate acaricide and ivermectin resistance. The modified larval packet test (LPT) was used to detect OP (chlorpiriphos and diazinon) and SP (flumethrin, deltamethrin and cypermethrin) resistance and the larval immersion test (LIT) to detect resistance to amitraz and ivermectin. Both, LPT and LIT were performed twice at different times with different collected samples. Mortality data with ivermectin were subjected to probit analysis to obtain lethal concentrations and resistance ratios (RR) using an ivermectin-susceptible strain (Deutch) as a reference. The R. microplus population showed resistance to all acaricides tested, with different mortalities at the discriminate dose: chlorpiriphos (1%), diazinon (24.2%), flumethrin (92.8%), deltamethrin (94.2%), cypermethrin (98.0%) and amitraz (1.5%). The studied tick population also showed resistance to ivermectin with a resistance ratio at 99% of 9.58 and 6.52 in the first and second evaluation, respectively. We report for the first time a R. microplus population in Mexico with different levels of resistance to OP, SP, amidines (Am) and ivermectin. The uncontrolled use of these products in the study area may promote the complete failure of tick control within a short period of time.