Association between overexpression of cytochrome P450 genes and deltamethrin resistance in Rhipicephalus microplus.

Cytochrome P450 monooxygenases mediated metabolic detoxification has been recognized as one of the mechanisms involved in resistance to pyrethroids, which is a class of pesticides that includes acaricides such as deltamethrin. Several cytochrome P450 (CYP) genes were identified in arthropod pests which are upregulated in response to exposure to pesticides used as acaricides. However, to date, limited information is available with respect to CYP genes and their response to acaricide exposure in ticks. We cloned and sequenced four CYP genes, the CYP41, CYP3006G8, CYP319A1 and CYP4W1 from reference susceptible IVRI-I strain of Rhipicephalus microplus. The expression pattern of the genes was investigated using qPCR in reference susceptible IVRI-I, pyrethroid-resistant IVRI-IV and multi-acaricide resistant IVRI-V strains. The effect of a single exposure of deltamethrin, at a concentration of 2600 µg/mL and 299.7 µg/mL on IVRI-IV and IVRI-V strains, respectively, on the expression of the four CYP genes was evaluated. In IVRI-IV strain, the CYP41 gene was highly overexpressed (FC 8.72) while CYP3006G8 was underexpressed with FC of 0.06. All the four genes were overexpressed in IVRI-V strain. After exposure to deltamethrin, the CYP3006G8 transcript levels were significantly upregulated at all time intervals in both resistant strains with the highest FC of 11.62 at 12 h in IVRI-IV and 13.38 at 3 h in IVRI-V. Our results suggest that the constitutive overexpression of CYP41 and deltamethrin induced upregulation of CYP3006G8 contribute to the development of pyrethroid resistance, specifically deltamethrin, in these two reference strains.

Voltage-gated sodium channel gene mutations and pyrethroid resistance in Rhipicephalus microplus.

Pyrethroid pesticides are extensively used to manage animal and human disease vectors including the southern cattle tick Rhipicephalus microplus (Canestrini). The indiscriminate and incorrect use of pyrethroids has led to the almost ubiquitous development of resistance to this pesticide class for this tick species. Voltage-gated sodium channels (Na-channels) are the primary target-site of pyrethroids and several studies on the involvement of mutations in the coding gene among pyrethroid-resistant R. microplus populations from different parts of the world have shown that there are various single nucleotide polymorphisms (SNPs) that are associated with resistance to pyrethroids. Identification of the exact location of the mutations in the protein coding regions of the targeted gene facilitates the design of various molecular tools for genotyping the resistant populations and thus promotes the rapid detection of resistance. This review aims to provide an update on the identification of pyrethroid resistance-associated Na-channel mutations from R. microplus.The database of diverse mutations from different regions of the world helped us to develop the molecular markers for resistance monitoring in a rapid and efficient manner. Their role and the development of different forms of molecular tools for genotyping ticks for mutations in the Na-channel gene are also discussed. In this review, the word mutation is used interchangeably with SNP.

Menace of acaricide resistance in cattle tick, Rhipicephalus microplus in India: Status and possible mitigation strategies.

Livestock in general and dairy sector in particular plays a vital role in the Indian economy and in the socio-economic development of millions of people. Rhipicephalus microplus, a one-host and the most prevalent cattle tick in various agro climatic zones of India. This tick species is responsible for severe economic losses to livestock owners both through the direct effects of blood sucking, injection of toxins and indirectly by transmitting pathogens. Due to inadvertent and indiscriminate use of chemicals to kill ticks, field tick population has developed resistance to almost all chemicals that are used to manage them. The intention of this review is to provide the current status of resistance in ticks, the possible mechanisms of resistance operating in the tick population, factors contributing to the development of resistance and the managemental strategies.

Acaricide resistance in Rhipicephalus microplus collected from selected districts of Madhya Pradesh, Uttar Pradesh and Punjab states of India.

The isolates of Rhipicephalus microplus collected from Madhya Pradesh (MP), Punjab (PJB) and Uttar Pradesh (UP) states of India were characterized using laboratory standardized adult immersion test (AIT) against macrocyclic lactone (ivermectin), synthetic pyrethroids (cypermethrin and deltamethrin), organophosphates (coumaphos and diazinon) and phenylpyrazole compounds (fipronil). Out of the six isolates tested, five isolates except MTH were resistant to deltamethrin and cypermethrin at level II with RF ranging from 16.4 to 24.02 and 7.05 to 13.2, respectively. In case of organophosphates, coumaphos was less effective showing resistance level II (RF 8.52-11.2) in all the six populations compared with diazinon to which three isolates (MHW, RWA and AGS) were resistant at level II. Except MTH, other five isolates were categorized at level I with RF ranging from 1.53 to 3.02 against ivermectin. The phenylpyrazole compound however was found effective, and none of the isolates could survive at a discriminating concentration. The possible strategy for the management of multi-acaricide-resistant ticks in the surveyed districts was discussed in the present study.

Molecular markers and their application in the monitoring of acaricide resistance in Rhipicephalus microplus.

Monitoring acaricide resistance and understanding the underlying mechanisms are critically important in developing strategies for resistance management and tick control. Identification of single nucleotide polymorphisms in the acaricide-resistant associated gene of Rhipicephalus microplus has enabled the development of molecular markers for detection and monitoring of resistance against different types of acaricide. There are many molecular markers developed for resistance monitoring, including mutations on target genes such as sodium channel, acetylcholinesterase, carboxylesterase, ß-adrenergic octopamine receptor, octopamine-tyramine etc. Molecular genotyping through molecular markers can detect the presence of resistance-associated genes in a tick population before it reaches high frequency. This review aims to provide an update on the various molecular markers discovered to date from different regions of the world.

Characterization and establishment of a reference deltamethrin and cypermethrin resistant tick line (IVRI-IV) of Rhipicephalus (Boophilus) microplus.

The problem of ticks and tick borne diseases is a global threat and growing reports of resistance to commonly used insecticides further aggravated the condition and demands for country specific resistance monitoring tools and possible solutions of the problem. Establishment of standard reference is prerequisite for development of monitoring tools. For studying possible role of different mechanisms involved in development of resistance in Rhipicephalus (Boophilus) microplus population and to develop newer drug to manage the problem of resistance, a deltamethrin exposed and selected tick colony, referred to as IVRI-IV, was characterized using reference susceptible IVRI-I tick line as control. The RF values of IVRI-IV ticks against deltamethrin, cypermethrin and diazinon were determined as 194.0, 26.6, 2.86, respectively, against adults. The esterase enzyme ratios of 2.60 and 5.83 was observed using α-naphthyl and ß-naphthyl acetate while glutathione S-transferase (GST) ratio was 3.77. Comparative analysis of IVRI-I and IVRI-IV carboxylesterase gene sequences revealed 13 synonymous and 5 non synonymous mutations, reported for the first time. The C190A mutation in the domain II S4-5 linker region of sodium channel gene leading to leucine to isoleucine (L64I) amino acid substitution was also detected in the IVRI-IV population. In the present study, monitorable indicators for the maintenance of the reference IVRI-IV colony, the first established deltamethrin and cypermethrin resistant tick line of India, were identified.

Survey of acaricides resistance status of Rhipiciphalus (Boophilus) microplus collected from selected places of Bihar, an eastern state of India.

Monitoring acaricide resistance in field ticks and use of suitable managemental practices are essential for controlling tick populations infesting animals. In the present study, the acaricide resistance status in Rhipicephalus (Boophilus) microplus ticks infesting cattle and buffaloes of five districts located in the eastern Indian state, Bihar were characterized using three data sets (AIT, Biochemical assays and gene sequences). Adult immersion test (AIT) was adopted using seven field isolates and their resistance factor (RF) was determined. Six isolates (DNP, MUZ, BEG, VSH, DRB and SUL) were found resistant to both deltamethrin and diazinon and except VSH all were resistant to cypermethrin. One isolate (PTN) was susceptible with a RF below 1.5. To understand the possible mode of resistance development, targeted enzymes and gene sequences of the para sodium channel and achetylcholinesterase 2 (AChE2) were analyzed. The esterase, monooxygenase and glutathione-S-transferase (GST) activity of reference susceptible IVRI-I line was determined as 2.47 ± 0.007 nmol/min/mg protein, 0.089 ± 0.0016 nmol/mg of protein and 0.0439 ± 0.0003 nmol/mg/min respectively, which increased significantly in the resistant field isolates. However, except esterases, the fold increase of monooxygenase (1.14-2.27 times) and GST (0.82-1.53 times) activities were not very high. A cytosine (C) to adenine (A) nucleotide substitution (CTC to ATC) at position 190 in domain II S4-5 linker region was detected only in one isolate (SUL) having RF of 34.9 and in the reference deltamethrin resistant line (IVRI-IV). However, the T2134A mutation was not detected in domain IIIS6 transmembrane segment of resistant isolates and also in reference IVRI-IV line despite of varying degree of resistance. The flumethrin specific G215T and the recently identified T170C mutations were also absent in domain II sequences under study. Four novel amino acid substitutions in AChE2 gene of field isolates and in organophosphate (OP) resistant reference IVRI-III line were identified which can possibly have a role in resistance development.

Survey of pyrethroids resistance in Indian isolates of Rhipicephalus (Boophilus) microplus: identification of C190A mutation in the domain II of the para-sodium channel gene.

Monitoring acaricide resistance and understanding the underlying mechanisms are critically important in developing strategies for resistance management and tick control. Eighteen isolates of Rhipicephalus (Boophilus) microplus collected from four agro-climatic regions of India were characterized and the resistant data were correlated with bioassay results, esterase enzyme activities and with the presence/absence of point mutation in the para-sodium channel gene. The adult immersion test was standardized to assess the level of resistance and resistant factors (RF) in the range of 1.2-95.7 were detected. Out of eighteen isolates, three were categorized as susceptible (RF<1.4), five isolates at level I (RF=1.5-<5), eight at level II (RF=5.1-<25), and one isolate each at level III (RF=26-<40) and level IV (RF=>41). The esterase enzyme ratio and survival% of tick isolates was observed significantly (p<0.001) correlated with correlation coefficient (r) in α- and ß-esterase activity. The correlation of determination (R(2)) for α- and ß-esterase activity indicated that 73.3% and 55.3% data points of field isolates were very close to the correlation lines. For detection of point mutation, three sites (mutation in domain IIS6, T2134A mutation in domain IIIS6 and C190A mutation in domain IIS4-5 linker) of sodium channel gene were amplified and sequenced. Comparative sequence analysis identified a cytosine (C) to adenine (A) nucleotide substitution (CTC to ATC) at position 190 in domain II S4-5 linker region of para-sodium channel gene in six isolates and in reference deltamethrin resistant IVRI-IV line. The occurrence of mutation in the tick isolates having high resistance factor suggested that target site insensitivity and enhanced esterase activity is the possible mechanism of resistance to deltamethrin in the Indian isolates of R. (B.) microplus. These results also concluded that the mutation site in Indian tick isolates is similar to Australian and Brazilian tick isolates while it is different in tick isolates from Mexico and North America. This is the first report of occurrence of mutation in para-sodium channel gene of deltamethrin resistant Indian isolates of R. (B.) microplus.

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 (LC(95)) of deltamethrin (29.6 ppm in AIT and 35.5 ppm in LPT) and cypermethrin (349.1 ppm in AIT and 350.7 ppm in LPT). The AIT with a discriminating dose (2 × LC(95)) 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.

Diazinon resistant status in Rhipicephalus (Boophilus) microplus collected from different agro-climatic regions of India.

The resistance status of Rhipicephalus (Boophilus) microplus to "Diazinon" was evaluated in 20 locations situated at various agro-climatic regions of India. Adult immersion test (AIT) was optimized using laboratory reared acaricide susceptible IVRI-I strain of R. (B.) microplus and minimum effective concentration of Diazinon was determined as 635.2 ppm. The discriminating dose (DD) was worked out as 1270.4 ppm and was tested on female ticks collected from organized and unorganized farms located at different agro-climatic regions of India. 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 average resistance factor (RF) of 6.1 (level II) was recorded in the ticks collected from the northern sub-temperate trans-gangetic plains while high average RF values of 26.65 (level III) was recorded in the ticks collected from tropical middle-gangetic plains. The tropical middle gangetic plain has a very high density of animal populations where farmers use Diazinon for tick control, for agricultural practices and for mosquito control. Due to the continuous use of OP compounds the environmental load of Diazinon has become high in the area. This is the first experimental data generated on Diazinon resistant status in ticks of India.

In vitro and in vivo efficacy of Acorus calamus extract against Rhipicephalus (Boophilus) microplus.

To develop a environment friendly control measure against cattle tick, Rhipicephalus (Boophilus) microplus, medicinally important plants were identified and extracts were prepared. Twelve 95% ethanolic, thirteen 50% hydroethanolic and nine hot water extracts were prepared and tested against laboratory reared homogenous colony of R. (B.) microplus. Amongst the 34 extracts, 26 extracts showed no mortality within 72 h of application while 12.0 ± 4.9% to 35.0 ± 9.6% mortality of treated ticks was recorded in other extracts. Of the effective extracts, the extract prepared from rhizome of Acorus calamus proved highly efficacious and 100% final mortality within 14 DPT was recorded. The LC85 value of the extract was determined as 11.26. In vivo experiments confirmed the efficacy of the extract up to 42%, and repeat application was required after 7 DPT. The extract was found safe and no reaction was observed when animals were treated with 50% of the concentration, which was five times of the concentration used for in vivo studies. The possibility of using the extract for the control of cattle tick is discussed.