ArticlePDF Available

Abstract and Figures

Monitoring of acaricide resistance is considered as one of the important facets of integrated tick management. In an attempt of development of resistance monitoring indicators, in the present study two reference tick lines of Rhipicephalus (Boophilus) microplus maintained in the Entomology laboratory, Indian Veterinary Research Institute (IVRI), Izatnagar, India, were studied to determine the possible contributing factors involved in development of resistance to deltamethrin. Electrophoretic profiling of esterase enzymes detected high activities of EST-1 in reference resistant tick colony designated as IVRI-IV whereas it was not detectable in reference susceptible IVRI-I line of R. (B.) microplus. Esterases were further characterized as carboxylesterase or acetylcholinesterase based on inhibitor study using PMSF, eserine sulphate, malathion, TPP and copper sulphate. It was concluded that an acetylcholinesterase, EST-1, possibly plays an important role for development of deltamethrin resistance in IVRI-IV colony of R. (B.) microplus.
This content is subject to copyright. Terms and conditions apply.
Esterase mediated resistance in deltamethrin resistant
reference tick colony of Rhipicephalus (Boophilus)
microplus
Snehil Gupta
1
K. G. Ajith Kumar
1
Anil Kumar Sharma
1
Gaurav Nagar
1
Sachin Kumar
1
B. C. Saravanan
1
Gandham Ravikumar
1
Srikant Ghosh
1
Received: 11 September 2015 / Accepted: 28 February 2016 / Published online: 15 March 2016
ÓSpringer International Publishing Switzerland 2016
Abstract Monitoring of acaricide resistance is considered as one of the important facets
of integrated tick management. In an attempt of development of resistance monitoring
indicators, in the present study two reference tick lines of Rhipicephalus (Boophilus)
microplus maintained in the Entomology laboratory, Indian Veterinary Research Institute
(IVRI), Izatnagar, India, were studied to determine the possible contributing factors
involved in development of resistance to deltamethrin. Electrophoretic profiling of esterase
enzymes detected high activities of EST-1 in reference resistant tick colony designated as
IVRI-IV whereas it was not detectable in reference susceptible IVRI-I line of R. (B.)
microplus. Esterases were further characterized as carboxylesterase or acetylcholinesterase
based on inhibitor study using PMSF, eserine sulphate, malathion, TPP and copper sul-
phate. It was concluded that an acetylcholinesterase, EST-1, possibly plays an important
role for development of deltamethrin resistance in IVRI-IV colony of R. (B.) microplus.
Keywords Esterases India IVRI-IV tick colony Resistance Rhipicephalus
(Boophilus) microplus
Introduction
Rhipicephalus (Boophilus) microplus is a widely prevalent species causing heavy damage
to the livestock industry and considered as the most economically important tick species.
Tick infestations in animals lead to reduction of weight gain, anaemia, low milk pro-
duction, transmission of fatal diseases and increase the risk for myiasis. In India, tick
control is mainly based on the use of various classes of chemical acaricides i.e., synthetic
&Srikant Ghosh
sghoshp@yahoo.co.in; sghoshtick@gmail.com
1
Entomology Laboratory, Division of Parasitology, ICAR-Indian Veterinary Research Institute,
Izatnagar, Bareilly, U.P. 243122, India
123
Exp Appl Acarol (2016) 69:239–248
DOI 10.1007/s10493-016-0032-7
pyrethroids, organophosphates, formamidine compounds and macrocyclic lactones (Ghosh
et al. 2007) and the control cost of ticks and tick-borne diseases was estimated as US$
498.7 million annually (Minjauw and McLeod 2003).
In recent past, many tick researchers reported acaricide resistance in Indian tick species
against various commonly used chemical acaricides (Kumar et al. 2011,2014,2015;
Sharma et al. 2012; Shyma et al. 2012; Kumar Rinesh et al. 2013; Singh et al. 2010,2014).
However, there is paucity of knowledge regarding the possible mechanism of development
of pyrethroid resistance in R. (B.) microplus Indian isolates. Kumar Rinesh et al. (2013)
reported sodium channel mutation at S4-5 linker region of domain II and increase in the
general esterase activity in synthetic pyrethroid (SP) resistant field isolates as possible
mechanisms of resistance. Esterases are the hydrolase enzymes which detoxify a wide
range of pesticides including SP (Montella et al. 2012) and both carboxylesterase and
acetylcholinesterase were correlated with resistance development both qualitatively and
quantitatively in ticks and other arthropods (Temeyer et al. 2004,2006,2007,2013).
Jamroz et al. (2000) electrophoretically proved the significant role of carboxylesterase for
resistance development in the larval homogenates of Mexican tick strains resistant to
coumaphos and permethrin using tri-phenyl phosphate (TPP) and eserine inhibitors. The
possible involvement of general esterases in conferring resistance to pyrethroids in the
Indian field tick population was reported (Vatsya and Yadav 2011; Shyma et al. 2012;
Kumar Rinesh et al. 2013; Singh and Rath 2014). Baffi et al. (2008) reported four bands of
esterases (EST-1 to 4) against both a-naphthyl and b-naphthyl acetate in Brazilian ticks
and classified EST-1 and 2 as acetylcholinesterase and EST-3 and 4 as carboxylesterase.
However, in a limited study using heterozygous field population, 5 bands of esterases
(EST-1 to 5) designated as EST-1, EST-2 and EST-3 as acetylcholinesterases (AchEs),
EST-4 and EST-5 as carboxylesterases (CaEs) and, an extra band of esterase, EST-1A, was
reported without establishing the results in reference homogenous resistant tick line
(Abdullah et al. 2012). The present study was carried out to elucidate the role of specific
esterase, if any, in conferring resistance in reference deltamethrin resistant IVRI-IV colony
and to identify the monitoring indicator for the maintenance of tick colony in the labo-
ratory. The main objective of the study is to establish a country-specific reference resistant
line, an important biological material for tick researchers in the country.
Materials and methods
Reference tick colonies
The colonies of reference acaricide susceptible IVRI-1 (National registration no. NBAII/
IVRI/BM/1/1998, GenBank Accession Nos. GU222462, GU323287, GU323288) and
pyrethroid resistant (RF =194.5) IVRI-IV lines (GenBank Accession No. JQ693154) of
R. (B.) microplus were maintained in Entomology Laboratory, Division of Parasitology,
IVRI, Izatnagar following standardized protocol of Ghosh and Azhahianambi (2007) and
were used for the study.
Animals
Weaned cross-bred (Bos taurus 9B. indicus) male calves were maintained in tick proof
animal houses of the Division of Parasitology, Indian Veterinary Research Institute and fed
240 Exp Appl Acarol (2016) 69:239–248
123
with straw, concentrate mixture, wheat bran, mineral mixture and water ad libitum.
Separate batches of calves were used to maintain acaricide susceptible IVRI-I line, and
resistant IVRI-IV colony. The calves were reared as per the guidelines of the statutory
Indian body, ‘‘Committee for the Purpose of Control and Supervision of Experiments on
Animals (CPCSEA)’’.
SDS-PAGE analysis of larval homogenates
Twelve to fifteen day old larvae of IVRI-I and IVRI-IV colonys stored at -80 °C were
used as biological materials. One hundred larvae were homogenized in 200 ll chilled
0.1 M sodium phosphate buffer, pH 6.5, containing 20 % sucrose and 0.001 M EDTA. The
homogenates were centrifuged at 15,0009g,4°C for 15 min, collected the supernatant and
protein concentration was estimated using Bradford method. The volume containing 40 lg
protein was resuspended in sample buffer (29), boiled for 10 min in a water bath and
volume of the sample was equalized using distilled water. Samples were resolved on 10 %
SDS-PAGE (Laemmli 1970) under denaturing conditions at 100 V for 2–3 h. A pre-
stained marker was used with the sample to determine the molecular weight of the resolved
proteins. The gels were stained with Coomassie Brilliant Blue R-250 and destained for
visualization of the native protein. Molecular weight of various proteins was determined
using Syngene gel documentation and analysis system (Syngene, UK).
Esterase profiling by native-PAGE
For esterase profiling, the larval homogenates were prepared as mentioned above. The total
protein concentration of each sample was determined according to dye-binding method of
Bradford (1976) using a microtiter plate (Axygen, USA) and 40 lg of total protein well
was determined as an optimum and was loaded in non-denaturing, non reducing poly-
acrylamide gel with sample buffer containing sucrose and bromophenol blue. The stacking
and resolving gels were 4 and 10 % polyacrylamide prepared in 1.5 M Tris HCl buffer, pH
8.8 and 1 M Tris HCl buffer, pH 6.8, respectively. Electrophoresis at 40 mA and 140 V
was done in the presence of pre-chilled Tris glycine buffer, pH 8.3, for 4 h at 4 °C. The
buffer was changed at every 30 min. At the end of run, the gels were carefully removed
and transferred to pre-incubation buffer containing 0.1 M sodium phosphate buffer, pH
6.5. Following pre- incubation for 45 min, the gels were put in gel staining solution (25 ml
0.1 M sodium phosphate buffer containing 23 mg fast blue RR salt; Sigma, USA) and
15 mg a-naphthyl acetate or b-naphthyl acetate (Sigma, USA) dissolved in 150 ll acetone
for 1 h at 37 °C in dark to determine the esterase activity. After staining, the gels were
washed thoroughly with water and the photographs were taken using Syngene gel docu-
mentation system and the bands were named from the anodic end.
Inhibitor study
To identify the role of specific esterase, if any, in conferring resistance in IVRI-IV colony,
inhibitor study was carried out using 0.33 % TPP (Supelco, USA), 1.0 mM copper sulfate
(Hi-Media, India), 0.4 mM malathion (Sigma, USA), 1.0 mM eserine sulfate (Sigma,
USA), and 1.0 mM phenylmethylsulphonyl fluoride (PMSF; Merck, India). The concen-
tration of the inhibitors was decided on the basis of reported activity (Baffi et al. 2008;
Miller et al. 1999). The results were interpreted according to the classification of esterases
Exp Appl Acarol (2016) 69:239–248 241
123
by Oakeshott et al. (1993)inDrosophila. The gels were carefully transferred to pre-
incubation buffer containing 0.1 M sodium phosphate buffer (pH 6.5) and then submerged
in sodium phosphate buffer (pH 6.5) containing inhibitors at the given concentration for
45 min. After 45 min incubation in dark, the staining procedure was followed as men-
tioned earlier. In each experiment, two gels were run, one of which was used as control and
in another inhibitor was added. Results were judged on the basis of comparative band
intensity in gels without or with inhibitor.
Results
SDS-PAGE analysis
Comparing with pre-stained molecular weight marker, protein bands of 104, 89, 67, 55, 51,
41, 37, 25, 20 and 17 kDa were detected in larval homogenates.
Native-PAGE profile
Following electrophoretic separation and staining using a-naphthyl acatate, four esterase
bands namely EST-1 to 4 were observed in deltamethrin resistant IVRI-IV colony while
three esterase bands, namely EST-2 to 4 were observed in susceptible IVRI-I line of R. (B.)
microplus (Fig. 1). No difference in the esterase pattern in both the lines was observed
when stained using b-naphthyl acetate.
Inhibitor study
Inhibitor study revealed the variations in band intensity of esterases in comparison to no
inhibitor control. After treatment of gels with eserine sulphate, a carbamate inhibitor, the
staining intensity of the EST-2 band was significantly reduced in comparison to control.
Similarly, probing of gels with malathion, an organophosphate inhibitor, significantly
reduced the staining intensity of EST-1 and EST-2 bands in comparison to control and thus
confirming those bands as acetylcholinesterase. Probing of gels with TPP, a known
organophosphate and carboxylesterase inhibitor, exhibited more intense bands of EST-1
and 2 than those observed in the case of malathion treated gels, but no significant changes
in EST- 3 and EST-4 bands intensity was observed in comparison to malathion treated and
Fig. 1 Native gel profile of esterase activity in IVRI-I and IV lines using a-naphthyl acetate
242 Exp Appl Acarol (2016) 69:239–248
123
untreated gels. In the case of PMSF, a serine protease inhibitor, treated gels no reaction
was observed with EST-1 band while very low intensity bands of EST-2, 3 and 4 in
comparison to no inhibitor control and treatment with malathion, eserine sulphate and TPP
inhibitors were observed. Treatment of gels in the presence of copper sulphate causes no
effect on the staining intensity of any of the esterase bands when compared to control
(Fig. 2).
Discussion
Establishment of characterized reference tick line is one of the important mandates of tick
research laboratory involved in resistance characterization. Till date, ten SP resistant
reference tick lines are established in different laboratories (Table 1) and are being adopted
by many tick researchers for resistance characterization. Due to non-availability of ref-
erence biological materials in India, the subject has not been touched up to the mark and it
is very essential to have such reference tick lines in the country. To attain the objective, we
targeted to characterize the laboratory selected deltamethrin resistant tick colony, IVRI-IV.
Mainly three enzymes viz., esterase, monoxygenase and glutathione-s-transferase are
involved in metabolic detoxification and often correlated with development of acaricide
resistance. Quantitative estimation of the three enzymes activity by tick specific laboratory
standardized protocol is under validation (Data not shown). Alternatively, the SDS-PAGE
profile of the extracts of IVRI-IV colony was compared with the results obtained earlier.
For example; the predominant 67 kDa band observed in the present study was corre-
sponding to a-naphthyl esterase enzyme (Rosario-Cruz et al. 2009) and is reported in larval
homogenates. He et al. (1999) purified GST from R.(B.) microplus having a molecular
mass of 25.8 kDa which was also prominent in the present study. Crampton et al. (1999)
reported a monoxygenase, CYP41 from R.(B.) microplus having molecular weight of
59 kDa which was close to the protein band of 55 kDa observed in the present study.
Although the presence of same molecular mass bands corresponding to functional enzymes
reported earlier gave some indication on the possible role of theses enzymes in conferring
resistance, the results need to be confirmed by enzyme substrate reaction assay.
Amongst the Mexican tick strains, the larval homogenates of OP resistant, Tuxpan and
in permethrin resistant Coatzacoalcos, Corrales and San Felipe strains, 21 out of 22 bands
Fig. 2 Esterase pattern in IVRI-IV line probing with inhibitors. (WI without inhibitor, Eeserine sulphate,
CS copper sulphate, PM phenylmethyl sulphonylfluoride, TP triphenyl phosphate, Mmalathion)
Exp Appl Acarol (2016) 69:239–248 243
123
exhibited activity against a-naphthyl acetate and 18 bands against b-naphthyl acetate in
native PAGE. Among the bands detected in Tuxpan and Coatzacoalcos strains, the car-
boxylesterase, EST-9 and EST-10, respectively, were reported to be responsible for the
Table 1 List of characterized synthetic pyrethroid resistant reference Rhipicephalus (Boophilus) microplus
tick lines established and available for tick research
S.
no
Tick strain Country/laboratory Characters References
1 Corrales Mexican, maintained at Cattle fever
Tick Research Laboratory
(CFTRL) in Mission, Texas, USA
Target site
insensitivity
(Domain III S6)
Miller et al. (1999), He
et al. (1999)
2 San Felipe Permethrin
(RR1840);
Coumaphos
(RR 1.4)
Domain III S6
sodium channel
mutation
Foil et al. (2004), He
et al. (1999), Guerrero
et al. (2001)
3 Coatzacoalcos Permethrin
(RR 250);
Coumaphos
(RR 3.6)
Overproduction of
a specific
esterase,
designated
CzEst9
Foil et al. (2004),
Jamroz et al. (2000)
4 Aldama Flumethrin and
deltamethrin.
Amaury (2013), Tapia-
Perez et al. (2003)
5 Rio Bravo Permethtrin
Target site
insensitivity
(Domain III
mutation)
Lovis et al. (2012)
6 Parkhurst Australian, maintained at the
Queensland Department of
Primary Industries and Fisheries
(DPI&F), Animal Research
Institute
Flumethrin,
Deltamethrin and
Cypermethrin
Nolan et al. (1989),
Jonsson et al. (2000),
Rodrı
´guez-Vivas et al.
(2006)
7 Marmor Deltamethrin and
cypermethrin,
susceptible to
flumethrin
Nolan et al. (1989),
Rodrı
´guez-Vivas et al.
(2006)
8 Lamington Flumethrin,
susceptible to
deltamethrin and
cypermethrin
Nolan et al. (1989),
Nolan (1990), Jonsson
et al. (2000)
9 Santa Luiza Brazilian, maintained at CFTRL,
Mission, Texas, USA
Permethrin
(RR 93)
Domain II
mutation
Li et al. (2008),
Guerrero et al. (2012)
10 Ultimo CSIRO, Australia and Novartis
Animal Health Research Center
(CRA), St-Aubin, Switzerland
OP, all SPs and
amitraz
Kunz and Kemp (1994),
Jonsson and Hope
(2007); Lovis et al.
(2013)
244 Exp Appl Acarol (2016) 69:239–248
123
development of resistance (Jamroz et al. 2000). On the contrary, four esterase bands were
found in cypermethrin and deltamethrin resistant adult ticks and three esterase bands in the
susceptible adult ticks (Baffi et al. 2005,2008). Similar profiles in resistant larval
homogenate (IVRI-IV) were observed in the present study when stained with both a-
naphthyl and b-naphthyl acetate. Baffi et al. (2007) reported four esterase bands in larvae
without any correlation with resistance. Similarly, Miller et al. (2007) could not correlate
permethrin resistance with metabolic detoxification via synergistic as well as TPP based
inhibitors study. In a study using heterogeneous Indian field population of R. (B.) micro-
plus naturally exposed to multiple acaricides, Abdullah et al. (2012) identified five esterase
activity bands and were linked with deltamethrin resistance although the results had not
been confirmed in reference homogenous deltamethrin resistant ticks as observed by Baffi
et al. (2005). The IVRI-IV colony was established by repeated selection of deltamethrin
exposure for the last several years and nearly 40th generation having RF of 194.5 was used
in the study. Repeated esterase profiling of the homogenous ticks resulted in identification
of four bands and we never encountered with any additional bands as observed by
Abdullah et al. (2012) while using heterogenous field resistant ticks. The results obtained
by Abdullah et al. (2012) showing an additional band may be due to other factors involved
in the field population and probably not due to deltamethrin resistance alone. In a separate
study, we characterized field ticks collected from six widely separated states of the country
and observed four intensely stained bands in consistent manner in the resistant population
(Ghosh unpublished data).
Esterase proteins have structural similarities and conserved arrangement of residues in
the catalytic site, containing a nucleophilic residue (serine, cysteine or aspartate), an acidic
residue (glutamate or aspartate) and a histidine residue, occurring on the loops formed in
the alpha/beta hydrolase fold. These amino acids are not contiguous in the primary
sequence instead held in juxtaposition in 3D structure by the fold (Montella et al. 2012). In
the present observations different esterases were stained at different intensity following
probing with different inhibitors in comparison to untreated control. The PMSF inhibited
the staining intensity of all the four esterases showing almost no reaction with EST-1 band.
Using copper sulphate, the intensity of all the esterase bands was similar to those observed
in control without inhibitor, thus confirms the absence of arylesterases among the resolved
bands. Although Baffi et al. (2005) reported EST-3 as carboxylesterase after getting sig-
nificant inhibition using both malathion and PMSF, in the present study, the staining
intensity of EST-3 was inhibited using PMSF but was resistant to both malathion and
eserine sulphate. Since EST-2 was inhibited by eserine sulphate, malathion and partially by
TPP while EST-1 strongly by PMSF and partially by eserine sulphate and malathion both
EST-1 and EST-2 were classified as acetylcholinesterase.
Following inhibitor study, it was found that eserine sulphate strongly inhibited EST-2
band while more intense band of EST-1 in comparison to control was observed. However,
Baffi et al. (2005) reported the inhibition of both EST-1 and EST-2 on application of
eserine sulphate in cypermethrin-resistant Brazilian R. (B.) microplus strain and suggested
the presence of this enzyme at a higher copy number in the resistant strain. In contrast,
Temeyer et al. (2013) reported that knock-down of one acetylcholinesterase isoenzyme by
RNA interference resulted in elevated expression and activity of other acetylcholinesterase.
The results of the present study supported the observation of Temeyer et al. (2013) where
inhibition of EST-2 activity resulted in increased activity of EST-1. In tick colony IVRI-
IV, EST-1 is not inhibited by any of the inhibitors used except PMSF, thus the result
indicates that the enzyme could be present at a higher copy number in the highly resistant
deltamethrin tick colony. Accordingly, it may be concluded that EST-1 as
Exp Appl Acarol (2016) 69:239–248 245
123
acetylcholinesterase is involved as one of the mechanisms in resistance development in
IVRI-IV alongwith mutation at S4-5 linker region of sodium channel gene reported earlier
(Kumar Rinesh et al. 2013). The results of the present study were further validated on ticks
collected from wide geographical areas having different level of RF against deltamethrin in
which EST-1 was found predominantly in resistant isolates along with mutation at S4-5
linker region of sodium channel gene (Ghosh unpublished data).
Conclusion
Both metabolic and target site insensitivity mechanisms of resistance are operating in
reference deltamethrin-resistant IVRI-IV colony. The study led to the identification of
EST-1 as one of the monitoring indicators for long term maintenance of the reference tick
line in the laboratory.
Acknowledgments The authors are grateful to the Indian Council of Agricultural Research, New Delhi for
funding through the World Bank funded National Agricultural Innovation Project No. [NAIP/Comp-4/
C2066/2008-09] and National Fund for Basic Strategic and Frontier Application Research in Agriculture
Project No. [NFBSFARA/BSA-4004/2013-14].
References
Abdullah S, Yadav CL, Vatsya S (2012) Esterase profile of Rhipicephalus (Boophilus) microplus popula-
tions collected from Northern India exhibiting varied susceptibility to deltamethrin. Exp Appl Acarol
58:315–325
Amaury M (2013) Resistance of ticks to the acaricides. http://www.akimoo.com
Baffi MA, Pereira CD, de Souza GRL, Bonetti AM, Ceron CR, Goulart LR (2005) Esterase profile in a
pyrethroid resistant Brazilian strain of Boophilus microplus cattle ticks (Acari, Ixodidae). Genet Mol
Biol 28:749–753
Baffi MA, Pereira CD, de Souza GRL, de Souza CS, Ceron CR, Bonetti AM (2007) Esterase profile in the
postembryonic development of Rhipicephalus microplus. Pesqui Agropecu Bras 42:1183–1188
Baffi MA, de Souza GRL, de Souza CS, Ceron CR, Bonetti AM (2008) Esterase enzymes involved in
pyrethroid and organophosphate resistance in a Brazilian population of Rhipicephalus (Boophilus)
microplus (Acari: Ixodidae). Mol Biochem Parasitol 160:70–73
Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein
utilizing the principle of protein-dyebinding. Anal Biochem 72:248–254
Crampton AL, Baxter GD, Barker SC (1999) A new family of cytochrome P450 genes (CYP41) from the
cattle tick, Boophilus microplus. Insect Biochem Mol Biol 29:829–834
Foil LD, Coleman P, Eisler M, Fragoso-Sanchez H, Garcia-Vazquez Z, Guerrero FD, Jonsson NN, Langstaff
IG, Li AY, Machila N, Miller RJ, Morton J, Pruett JH, Torr S (2004) Factors that influence the
prevalence of acaricide resistance and tick-borne diseases. Vet Parasitol 125:163–181
Ghosh S, Azhahianambi P (2007) Laboratory rearing of Theileria annulata-free Hyalomma anatolicum
anatolicum ticks. Exp Appl Acarol 43:137–146
Ghosh S, Azhahianambi P, Yadav MP (2007) Upcoming and future strategies of tick control: a review.
J Vector Borne Dis 44:79–89
Guerrero FD, Davey RB, Miller RJ (2001) Use of an allele-specific polymerase chain reaction assay to
genotype pyrethroid resistant strains of Boophilus microplus (Acari: Ixodidae). J Med Entomol
38:44–50
Guerrero FD, Lovis L, Martins JR (2012) Acaricide resistance mechanisms in Rhipicephalus (Boophilus)
microplus. Rev Bras Parasitol 21:1–6
He H, Chen AC, Davey RB, Ivie GW, George JE (1999) Characterization and molecular cloning of a
glutathione S-transferase gene from the tick, Boophilus microplus (Acari: Ixodidae). Insect Biochem
Mol Biol 29:737–743
246 Exp Appl Acarol (2016) 69:239–248
123
Jamroz RC, Guerrero FD, Pruett JH, Oehler DD, Miller RJ (2000) Molecular and biochemical survey of
acaricide resistance mechanisms in larvae from Mexican strains of the southern cattle tick, Boophilus
microplus. J Insect Physiol 46:685–695
Jonsson NN, Hope M (2007) Progress in the epidemiology and diagnosis of amitraz resistance in the cattle
tick, Boophilus microplus. Vet Parasitol 146:193–198
Jonsson NN, Mayer DG, Green PE (2000) Possible risk factors on Queensland dairy farms for acaricide
resistance in cattle tick (Boophilus microplus). Vet Parasitol 88:79–92
Kumar S, Paul S, Sharma AK, Kumar R, Tewari SS, Chaudhuri P, Ray DD, Rawat AK, Ghosh S (2011)
Diazinon resistant status in Rhipicephalus (Boophilus) microplus collected from different agro-climatic
regions of India. Vet Parasitol 181:274–281
Kumar S, Sharma AK, Ray DD, Ghosh S (2014) Determination of discriminating dose and evaluation of
amitraz resistance status in different field isolates of Rhipicephalus (Boophilus) microplus in India. Exp
Appl Acarol 63:413–422
Kumar S, Sharma AK, Nagar G, Ghosh S (2015) Determination and establishment of discriminating con-
centrations of malathion, coumaphos, fenvalerate and fipronil for monitoring acaricide resistance in
ticks infesting animals. Ticks Tick Borne Dis 6:383–387
Kunz SE, Kemp DH (1994) Insecticides and acaricides: resistance and environmental impact. Rev Sci Tech
13:1249–1286
Laemmli UK (1970) Cleavage of structural protein during the assembly of head of bacteriphage T4. Nature
277:680–685
Li AY, Davey RB, Miller RJ, Guerrero FB, George JE (2008) Genetics and mechanisms of permethrin
resistance in the Santa Luiza strain of Boophilus microplus (Acari: Ixodidae). J Med Entomol
45:427–438
Lovis L, Guerrero FD, Miller RJ, Bodine DM, Betschart B, Sager H (2012) Distribution patterns of three
sodium channel mutations associated with pyrethroid resistance in Rhipicephalus (Boophilus)mi-
croplus populations from North and South America, South Africa and Australia. Int J Parasitol Drugs
Drug Resist 2:216–224
Lovis L, Mendes MC, Perret JL, Martins JR, Bouvier J, Betschart B, Sager H (2013) Use of the Larval
Tarsal Test to determine acaricide resistance in Rhipicephalus (Boophilus) microplus Brazilian field
populations. Vet Parasitol 191:323–331
Miller RJ, Davey RJ, George JE (1999) Characterization of pyrethroid resistance and susceptibility to
coumaphos in Mexican Boophilus microplus (Acari: Ixodidae). J Med Entomol 36:533–538
Miller RJ, Davey RB, George JE (2007) First report of permethrin-resistant Boophilus microplus (Acari:
Ixodidae) collected within the United States. J Med Entomol 44:308–315
Minjauw B, McLeod A (2003) Tick borne disease and poverty. The impact of tick and tick borne disease on
the livelihoods of small scale and marginal livestock in India and eastern and southern Africa.
Research report, DFID Animal Health Programme, Centre for Tropical Veterinary Medicine,
University of Edinburgh, UK
Montella IR, Schama R, Valle D, Cruz MIO, Janeiro RD (2012) The classification of esterases: an important
gene family involved in insecticide resistance—a review. Mem Inst Oswaldo Cruz 107:437–449
Nolan J (1990) Acaricide resistance in single and multi-host ticks and strategies for control. Parasitol
32:145–153
Nolan J, Wilson JT, Green PE, Bird PE (1989) Synthetic pyrethroid resistance in field samples in the cattle
tick (Boophilus microplus). Aust Vet J 66:179–182
Oakeshott JG, van Papenrecht EA, Boyce TM, Healy MJ, Russell RJ (1993) Evolutionary genetics of
Drosophila esterases. Genetica 90:239–268
Rinesh Kumar, Nagar G, Sharma AK, Kumar S, Ray DD, Chaudhuri P, Ghosh S (2013) Survey of pyre-
throids resistance in Indian isolates of Rhipicephalus (Boophilus) microplus: identification of C190A
mutation in the domain II of the para-sodium channel gene. Acta Trop 125:237–245
Rodrı
´guez-Vivas RI, Alonso-Dı
´az MA, Rodrı
´guez-Arevalo F, Fragoso-Sanchez H, Santamaria VM,
Rosario-Cruz R (2006) Prevalence and potential risk factors for organophosphate and pyrethroid
resistance in Boophilus microplus ticks on cattle ranches from the state of Yucatan, Mexico. Vet
Parasitol 136:335–442
Rosario-Cruz R, Guerrero FD, Miller RJ, Rodriguez-Vivas RI, Tijerina M, Dominguez-Garcia DI, Her-
nandez-Ortiz R, Cornel AJ, Mc Abee RD, Alonso-Diaz MA (2009) Molecular survey of pyrethroid
resistance mechanisms in Mexican field populations of Rhipicephalus (Boophilus) microplus. Parasitol
Res 105:1145–1153
Sharma AK, Kumar R, Kumar S, Nagar G, Singh NK, Rawat SS, Dhakad ML, Rawat AKS, Ray DD, Ghosh
S (2012) Deltamethrin and cypermethrin resistance status of Rhipicephalus (Boophilus) microplus
collected from six agro-climatic regions of India. Vet Parasitol 188:337–345
Exp Appl Acarol (2016) 69:239–248 247
123
Shyma KP, Kumar S, Sharma AK, Ray DD, Ghosh S (2012) Acaricide resistance status in Indian isolates of
Hyalomma anatolicum. Exp Appl Acarol 58:471–481
Singh NK, Rath SS (2014) Esterase mediated resistance against synthetic pyrethroids in field populations of
Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in Punjab districts of India. Vet Parasitol
204:330–338
Singh NK, Jyoti Haque M, Rath SS (2010) Studies on acaricide resistance in Rhipicephalus (Boophilus)
microplus against synthetic pyrethroids by adult immersion test with a discriminating dose. J Vet
Parasitol 24:207–208
Singh NK, Jyoti Haque M, Singh H, Rath SS, Ghosh S (2014) A comparative study on cypermethrin
resistance in Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum from Punjab (India).
Ticks Tick Borne Dis 5:90–94
Tapia-Perez G, Garcı
´a-Vazquez Z, Montaldo H, George J (2003) Inheritance of resistance to flumethrin in
the Mexican Aldama strain of the cattle tick Boophilus microplus (Acari: Ixodidae). Exp Appl Acarol
31:135–149
Temeyer KB, Davey R, Chen A (2004) Identification of a third Boophilus microplus (Acari: Ixodidae)
cDNA presumptively encoding an acetylcholinesterase. J Med Entomol 41:259–268
Temeyer KB, Pruett JH, Untalan PM, Chen AC (2006) Baculovirus expression of BmAChE3, a cDNA
encoding an acetylcholinesterase of Boophilus microplus (Acari: Ixodidae). J Med Entomol
43:707–712
Temeyer KB, Pruett JH, Olafson PU Jr, Chen AC (2007) R86Q, a mutation in BmAChE3 yielding a
Rhipicephalus microplus organophosphate-insensitive acetylcholinesterase. J Med Entomol
44:1013–1018
Temeyer KB, Olafson PU, Brake DK, Tuckow AP, Li AY, Adalberto A, Leo
´n PD (2013) Acetyl-
cholinesterase of Rhipicephalus (Boophilus) microplus and Phlebotomus papatasi: gene identification,
expression, and biochemical properties of recombinant proteins. Pestic Biochem Physiol 106:118–123
Vatsya S, Yadav CL (2011) Evaluation of acaricide resistance mechanism in the field populations of
Rhipicephalus (Boophilus) microplus collected from India. Int J Acarol 37:405–410
248 Exp Appl Acarol (2016) 69:239–248
123
... For generation of larvae, these ticks were individually placed in glass vials in desiccators containing 10% KOH (85 ± 5% relative humidity) in a BOD incubator at ambient temperature (28 ± 1 °C). The 10-14 days old larvae were used for bioassay (Gupta et al. 2016) as well as stored in liquid nitrogen for biochemical (Surbhi et al. 2019) and molecular assay. ...
... After 24 h of incubation, larval mortality was calculated. Only those larvae capable of walking were considered to be alive while those that moved their appendages but did not walk, were counted as if dead (Gupta et al. 2016). ...
... For esterase pattern analysis, five adult engorged female of H. anatolicum ticks from each collection site were used as per the protocol mentioned by Gupta et al. (2016). Briefly, a longitudinal incision was made in the abdomen of each adult female tick, excess blood was removed with distilled water and was used to prepare homogenate for native PAGE. ...
Article
Full-text available
To study the acaricide resistance status and possible mechanisms of action in conferring resistance to commonly used acaricides (deltamethrin and coumaphos), Hyalomma anatolicum ticks were collected from 6 dairy farms of Hisar and Charkhi Dadri districts of Haryana. By using standard larval packet test, H. anatolicum tick larvae of Charkhi Dadri isolates were found to be susceptible (100% mortality) to both the acaricides. Level-I resistance against coumaphos was recorded from four isolates, whereas, level-II was observed in only one isolate, collected from Hisar. One isolates (Kaimri) from Hisar also showed level-I resistance against deltamethrin. Biochemically, the ticks having higher values of resistance factor (RF) against coumaphos were found to possess increased enzymatic activity of α-esterase, β-esterase, glutathione-S-transferase (GST) and mono-oxygenase enzymes, whereas, the monoamine oxidase did not show any constant trend. However, the RF showed a statistical significant correlation with GST only. Native PAGE analysis of H. anatolicum ticks revealed the presence of nine types of esterases (EST-1 h to EST-9 h) by using napthyl acetate as substrate. In the inhibitory assay, esterases were found to be inhibited by PMSF, indicating the presence of serine residue at catalytic triad. The partial cds of carboxylesterase and domain II of sodium channel genes were sequenced to determine any proposed mutations in resistant isolates of H. anatolicum ticks, however, no mutations were observed in either gene, indicating that increased expression of detoxification enzymes as a possible mechanism for resistance development, in the current study.
... Using a tissue homogenizer, adult ticks were homogenized in 200 μl of cold 0.1 M sodium phosphate buffer, pH 6.5, containing 20% sucrose (IKA T10 basic Ultra-Turrox). Following centrifugation of the homogenate at 15,000 X g for 15 min at 4 • C, the supernatant obtained was filtered through a 0.22 μ syringe filter (MIL-LEX-GV), and esterase activity was estimated as per the previously mentioned protocol (Gupta et al., 2016). After staining, the gels were washed with water and analyzed using the gel documentation system (Syngene, UK). ...
... Ticks use a detoxifying enzyme system to break down or sequester a wide range of acaricides. According to reports, metabolic resistance confers resistance to ticks through enhanced activity of detoxifying enzymes such as esterases, glutathione S-transferases (GST), and cytochrome P450 monoxygenases (Gupta et al., 2016;Ghosh et al., 2017;Chigure et al., 2018). Our result corroborated the previously reported high esterase activity in acaricideresistant R. microplus ticks in Haryana, India (Gaur et al., 2017). ...
... Further, EST-5b showed inhibition in the presence of eserine sulphate, which is a reversible inhibitor of acetylcholineaserases (AChE), indicating that the increased activity of acetylcholineaserases is probably responsible for emergence of coumaphos and deltamethrin resistance in ticks. These finding were in agreement with the earlier findings of the researchers (Gupta et al., 2016;Gaur et al., 2017;Ghosh et al., 2017). Further, three types of acetylcholineaserases (AChE1, AChE2, and AChE3) are linked with acaricide resistance in R. microplus ticks . ...
... It also exhibits anticholinesterase activity [26,27]. Current research on AchE in ticks mainly focuses on resistance to commercial insecticides and essential oils derived from plants [28,29,30,31]. Our study identi ed four AchEs in one tick species and demonstrated that HL-AchEs were signi cantly in uenced by cinnamaldehyde stimulation. ...
Preprint
Full-text available
Background The control and prevention of ticks and tick-borne diseases relies on chemical insecticides and repellents. Plant-derived compounds potentially represent new and safer repellents. Cinnamaldehyde, a component of cinnamon oil, exhibits antibacterial, anti-inflammatory, acaricidal and repellent activity against ticks. Here we studied the molecular mechanism of the repellent effects of cinnamaldehyde on ticks. Methods Assessment of cinnamaldehyde as a tick repellent was conducted using a Y-tube olfactometer, transcriptomics and metabolomic analyses. Nymphs were exposed to cinnamaldehyde for 30 min, and the tick acetylcholinesterase (AchE) family was cloned and characterized. The role of AchE in cinnamaldehyde repellency was elucidated through the specific activity of the enzyme, electrophysiology, RNA interference and repellency tests. Results A 2% cinnamaldehyde treatment resulted in more than 90% nymph repellency within 6 h. Nymphs were exposed to cinnamaldehyde for 30 min, and subsequent transcriptome and metabolome analyses revealed the involvement of AchE in the response process. The HL-AchE family was cloned and functionally explored. AchE was transcribed in all tick developmental stages and tissues. Following cinnamaldehyde treatment, the transcript and protein levels of AchE were altered, and the specific activity of the enzyme significantly increased. RNAi was used to validate these findings. Following RNAi, electroantennography (EAG) tests demonstrated a significant decrease in response to various repellents as well as a significant decrease in repellency. Conclusions AchE mediates cinnamaldehyde-induced tick repellency, and the results provide insights into the mechanism of plant-derived tick repellents.
... Chlorpyrifos-treated ticks, on the other hand, showed a significant decrease in GSH, an increase in MDA and a significant inhibition in AchE. Gupta et al. (2016) reported an inhibition in AchE in R. microplus ticks treated with deltamethrin, and they ascribed the ticks' resistance to deltamethrin to this inhibition. AchE is also suppressed in the chlorpyrifos-treated ticks. ...
Article
Full-text available
The most economically significant ectoparasites in the tropics and subtropics are ixodid ticks, especially Rhipicephalus annulatus and Rhipicephalus sanguineus. Years of extensive use of the readily available acaricides have resulted in widespread resistance development in these ticks, as well as negative environmental consequences. Benzyl alcohol (BA) has been frequently used to treat pediculosis and scabies, and it may be an effective alternative to commonly used acaricides. The main aim of the present study was to evaluate the acaricide activity of BA and its combination with the regularly used chemical acaricides against R. annulatus and R. sanguineus. Different concentrations of BA alone and in combination with deltamethrin, cypermethrin and chlorpyrifos were tested in vitro against adult and larvae of both tick species. The results showed that BA is toxic to R. annulatus and R. sanguineus larvae, with 100% larval mortality at concentrations of ≥50 mL/L, and LC50 and LC90 attained the concentrations of 19.8 and 33.8 mL/L for R. annulatus and 18.8 and 31.8 mL/L for R. sanguineus, respectively. Furthermore, BA in combination with deltamethrin, cypermethrin and chlorpyrifos exhibited synergistic factors of 2.48, 1.26 and 1.68 against R. annulatus larvae and 1.64, 11.1 and 1.14 against R. sanguineus larvae for deltamethrin + BA, cypermethrin + BA and chlorpyrifos + BA, respectively. BA induced 100% mortality in adult R. annulatus at concentrations of ≥250 mL/L with LC50 and LC90 reached the concentrations of 111 and 154 mL/L, respectively. Additionally, BA had ovicidal activity causing complete inhibition of larval hatching at 100 mL/L. The combination of BA with deltamethrin and cypermethrin increased acetylcholinesterase inhibition, whereas the combination of BA with chlorpyrifos decreased glutathione (GSH) activity and malondialdehyde levels. In the field application, the combination of BA 50 mL/L and deltamethrin (DBA) resulted in a significant reduction in the percentage of ticks by 30.9% 28 days post-treatment when compared with groups treated with deltamethrin alone. In conclusion, BA causes mortality in laboratory and field studies alone and in combination with cypermethrin or deltamethrin. BA can be used for control of ticks of different life stages, that is, eggs and larvae, through application to the ground.
... Nandi et al., 2015; Ghosh et al., 2015;Gupta et al., 2016; Ghosh et al., 2017; Chigure et al., 2018; Fular et al., 2018). Regarding esterases, CaE catalyze the hydrolysis of esters and are classified in the serine hydrolase superfamily, involved in detoxification and playing an important physiological role in lipid metabolism(Ran et al., 2009 ...
Article
Full-text available
The ixodicidal activity of the methanolic extracts of Artemisia ludoviciana (Astereceae), Cordia boissieri (Boraginaceae) and Litchi chinensis (Sapindaceae) against two field populations of Rhipicephalus (Boophilus) microplus from the state of Nuevo Leon (NL) and Veracruz (VER) was evaluated. The extract of L. chinensis in the concentration of 150 mg/ml showed efficacies of 100% and 99% against engorged females and mortalities of 98% and 99% against larvae. C. boissieri in the same concentration showed efficacies of 71% and 37% against engorged adults and mortalities of 33.04% and 10.33% against larvae and A. ludoviciana had efficacies of 94% and 83% in adults and mortalities of 89.39% and 89.21% against larvae in both populations respectively. The enzymatic activity of Acetylcholinesterase (AChE), Carboxylesterase (CaE), Glutathione-S-Transferase (GST) and Alkaline Phosphatase (ALP) was measured in both populations of ticks. As a result, a significant difference between both populations was shown, being the VER population the one that exhibited a higher enzymatic activity (p ≤ 0.05). It can be concluded that the methanolic extract of the seed of L. chinensis shows potential ixodicidal activity and can be used as an alternative source of tick control, however, prior characterization, toxicity and formulation studies are necessary.
... From previous research, CE are one of detoxification enzymes are involved in resistance to pyrethroids and organophosphate in many insects [41][42][43]. Delorme et al [41] described that the S. exigua resistant strain has an enhanced esterase activity toward chromogenic substrates, such as naphthyl acetate or methylumbelliferyl acetate, the level depending on the substrate used. It is likely that activity toward chromogenic substrates and the hydrolysis of deltamethrin are related. ...
Article
Full-text available
Background Plant secondary metabolites or mixtures in extracts or essential oils are well known to enhance the activity in binary mixtures. The present study is the first to report that thymol synergistically or additively enhances the activity of P. ribesioides extracts and isolated compounds against S. exigua larvae at sublethal doses. Results Thymol was synergistic when are mixed with hexane extract; however, if the hexane extract level was higher (LD30) than the thymol level (LD10), the reaction was antagonistic. CH2Cl2 extract and thymol were more toxic than the extract or thymol alone, and EtOAc extract was synergized by thymol if the components were combined at similar levels (1:1 thymol:EtOAc extract at the LD10 or LD30). MeOH extract individually had moderate insecticidal activity, but all combinations with thymol were synergistic as binary mixtures. Isolated compounds, piperine, phenethyl cinnamamide and cinnamic acid represented synergistic, additive, and antagonistic action after combining with thymol (1:1 at the LD10 or LD30). Detoxification enzymes after exposure of insects to treatments showed isolated compounds + thymol could inhibit CE, GST and AChE reaction of S. exigua exceptional being piperine + thymol, which induced detoxification enzyme activity. Conclusion The synergistic activity was extract- and dose-specific. The impact on detoxification enzymes was variable and dependent on the composition of the extract and the doses of extract and thymol used in a binary mixture. In this metabolic model, the major insect compound in an extract may become detoxified, whereas a minor compound will act unimpeded, showing a lower LD50 than acting alone. This model suggests that thymol synergizes with extract components differently, which could depend on the specific metabolites in the extract and the dose applied. Such studies will help design effective insecticides based on natural plant mixtures and a synergistic compound. Graphical abstract
... Trichlorfon and deltamethrin were tested in-vitro on engorged female ticks to check the number of oviposition inhibited, the reproductive index, LC50, LC95 as well as the mortality percentage. Same study was conducted by Gupta et al. (2016). The comparison of mortality rate for trichlorfon and deltamethrin in zone 1 and zone 2 were the lowest mortality against trichlorfon on two and tenfold dilutions i.e. (7-58 percent), while the highest mortality rate was (25-83 percent) against deltamethrin . ...
Article
Full-text available
Boophilus microplus is a major cattle tick specie causing great economic loss to the dairy industry throughout the globe including Pakistan. Trichlorfon and Deltamethrin are used to control bovine ticks, and their sprays are also used in other pest control programs that exert pressure on ticks to gain resistance. This study is aimed to examine the resistance level of Rhipiciphalus microplus against trichlorfon and deltamethrin. The engorged ticks were collected from two ecological regions of Khyber Pakhtunkhwa, KPK Pakistan i.e., Swat & Dir (zone-1), and Charsadda & Nowshera (zone-2). Four concentrations of acaricides in two-fold and ten-fold ppm with three replicates for each were used in both bioassays. Egg hatch assay and adult immersion tests were used to assess the resistance status. The probit analysis of egg hatch assay showed the highest hatching percentage in zone 1 on both dilutions (67-76% on two-fold and 68-88% on ten-fold dilution) while lethal concentration (LC95) was found to be 2.187 ppm and discriminating dose (DD) as 4.374 ppm for trichlorfon. In zone 2, hatching percentage was 73-84 on two-fold and 72-91% on ten-fold dilution while LC95 was recorded as 0.599 ppm and DD as 1.198 ppm. The same parameters were studied for deltamethrin and in zone 1 the hatching percentage was found as 38-56% on two-fold dilution and 37-80% on ten-fold dilution while LC95 was recorded as 0.001 ppm and DD as 0.002 ppm. In zone 2, the hatchability was recorded as 42-58% on two-fold and 43-85% on ten-fold dilution. The values for LC95 was recorded as 0.001 ppm and DD as 0.002 ppm. Further, analysis of adult immersion test against trichlorfon revealed the values of LC50 as 2.85 ppm and LC95 as 4.71 ppm in zone 1 and in zone 2 as 3.14 ppm and 5.28 ppm, respectively. Similarly, LC50 and LC95 against deltamethrin was recorded as 0.79 ppm & 1.71 ppm in zone 1 and 0.45 ppm & 4.325 ppm in zone 2, respectively. Based on the findings of this study, the isolated Rhipicephalus microplus was found to be more resistant to the widely used acaricides i.e., trichlorfon than deltamethrin. In order to maintain the efficacy of acaricides at country level, the study recommends continuous monitoring of resistance.
... Several studies documented that ticks developed resistance against various acaricides i.e.,amitraz and chlorfenvinphos (Chitombo et al.,2021), cypermethrin (Klafke et al., 2017;, ivermectin (Rodriguez-Vivas et al., 2017;Rodríguez-Vivas R et al., 2014;Cruz et al., 2015), fluazuron (Recket al., 2014;Maciel et al., 2016), amitraz (Murigu et al.,2016;Stone et al., 2014;Muyobela et al., 2015;Singh et al., 2015), deltamethrin (Gupta et al., 2016;Jyothimolet al., 2014), pyrethroid (Ziapouret al., 2016Wyket al., 2016), amitraz & deltamethrin (Petermannet al., 2016, fipronil (Lopes et al., 2014), amitraz & cypermethrin (Fernández-Salas et al., 2012b) & permethrin & amitraz (Li et al., 2007deltamethrin and cypermethrin (Godara et al., 2019). Feyereisen (1995) reviewed effective insecticides & concluded that the list is rapidly shrinking; meanwhile introduction of new insecticides in the market became almost negligible, largely because of the high costs associated with research, development & registration of the new products. ...
Article
Full-text available
International Journal of Zoology and Applied Biosciences (IJZAB) is a scholarly, Peer-reviewed and open access e-journal (ISSN: 2455-9571). IJZAB aims to publish original, quality research and review articles in the latest fields of Zoology, and Applied Biosciences. The journal is published bi-monthly by Rishan Publications.
Article
Haemaphysalis longicornis (Acari: Ixodidae) is an important vector of numerous pathogens and poses a great threat to veterinary and public health. Commercially available tick repellents are extensively used and primarily comprise synthetic molecules; however, there are concerns over their safety and environmental impacts. Biologically based acaricides, particularly the plant-derived essential oils (EOs), may constitute an appealing alternative. We screened 20 different EOs by packet tests of unfed H. longicornis nymphs, and found that EOs of cinnamon, clove and chamomile were the most toxic (mortality > 80 %). Cinnamon EO had the most competitive acaricidal activity, with lethal concentration 50 (LC50) rates of 0.4530 %, 0.2316 % and 0.0342 % (v/v) for unfed adults, nymphs and larvae, respectively. Furthermore, 5.00 % (v/v) cinnamon EO showed reproductive inhibition against H. longicornis, with significantly higher rates of oviposition reduction (53.19 %) and hatching reduction (46.21 %) compared with the negative control group. Composition analysis of cinnamon EO by gas chromatography–mass spectrometry (GC-MS) revealed that the major chemical compounds were trans-cinnamaldehyde (72.21 %) and cinnamic acid (19.45 %), with the former showing similar levels of acaricidal activity and oviposition inhibition as cinnamon EO. This study has demonstrated the potential of cinnamon EO and trans-cinnamaldehyde as natural acaricides against H. longicornis, and is the first to characterize their oviposition inhibition activity.
Article
Full-text available
Discriminating concentrations (DCs) of malathion, coumaphos, fenvalerate and fipronil were determined to monitor acaricide resistance in field conditions. The LC99 values with 95% confidence interval for malathion, coumaphos, fenvalerate and fipronil were 5126.8 (5011.5-5240.7), 131.0 (120.4-142.5), 2257.5 (2198.1-2318.4) and 6.2 (5.87-6.55), respectively. The narrow confidence intervals in LC50 and LC99 of adult immersion test (AIT) and larval packet test (LPT) affirming the homogeneity of IVRI-I line. Variation in LPT based LC50 and LC99 values of malathion (55.9ppm) and coumpahos (28.4ppm) compared to those obtained in AIT indicating that larvae were more susceptible to these chemicals. The DCs for malathion, coumaphos, fenvalerate and fipronil against adults were determined as 10253.6, 262.0, 4515.0 and 12.4ppm while against larvae the values were 111.8, 56.8, 4014.0 and 9.6ppm, respectively. The working efficiency of DCs was successfully tested in field tick isolates. Establishment of country specific DCs of commonly used insecticides for monitoring of resistance in field ticks is emphasized for establishing tick control strategies. Copyright © 2015 Elsevier GmbH. All rights reserved.
Article
Full-text available
Field tick isolates of Rhipicephalus (Boophilus) microplus were collected from eleven districts located in the northern and eastern states of India to access the resistance status to "Amitraz". Adult immersion test was optimized using laboratory reared acaricide susceptible IVRI-I line and minimum effective concentration was determined as 487.7 ppm with 95 % confidence interval of 455.8-521.8. The discriminating concentration was determined as 975.4 ppm and was tested on female ticks collected by two stage stratified sampling from organized dairy farms and villages. Based on three variables, viz., mortality, egg masses and reproductive index, the resistance level was categorized. Resistance to amitraz was detected at level I in 3 isolates (RF = 1.56-5.0), at level II in 6 isolates (RF = 9.3-23.3) and at level III in 1 isolate (RF = 27.3) whereas one isolate was found susceptible. The highest resistance was found in the SKR isolate (RF = 27.3) and minimal resistance was detected in the N-24P isolate (RF = 1.56). These experimental data will help in designing tick control strategy which is suffering from acaricide failure and to overcome development of resistance in ticks.
Article
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Article
The objective of this work was to analyze the pattern of esterase activity in the development stages of Rhipicephalus microplus by nondenaturing polyacrylamide gel electrophoresis using specific staining for esterase. The electrophoretical results revealed the presence of nine regions displaying esterase activity, stained with both alpha-naphthyl acetate and beta-naphthyl acetate, and classified as alpha-beta-esterase. Stage-specific esterases were found, with the first nymphal and larval stages showing the greatest esterase activity throughout the development. An esterase called EST-4 was detected only in males and was considered sex-specific. There are differences in the esterase profile among the different postembryonic development stages of R. microplus.
Article
The southern cattle tick Rhipicephalus (Boophilus) microplus (Bm) is a vector of bovine babesiosis and anaplasmosis. Tick resistance to organophosphate (OP) acaricide involves acetylcholinesterase (AChE) insensitivity to OP and metabolic detoxification. Sequencing and in vitro expression of Bm genes encoding AChE allowed biochemical characterization of three BmAChEs expressed in tick synganglion. rBmAChE1, rBmAChE2 and rBmAChE3 exhibited substrate preference for acetylthiocholine, high substrate inhibition and sensitivity to AChE-specific inhibitors. OP-insensitivity mutations were demonstrated in rBmAChE1 and rBmAChE3. Gene silencing suggested functional complementation of BmAChEs in vivo. BmAChE genes were amplified in copy number and multiple transcript polymorphisms were expressed in individual tick synganglia for each of the BmAChEs, suggesting allelic diversity within individuals. Studies also identified a gene encoding AChE of the sand fly, Phlebotomus papatasi, a vector of leishmaniasis in humans and animals. Expression of recombinant P. papatasi AChE (rPpAChE) enabled biochemical characterization and identification of effective inhibitors that selectively target rPpAChE.
Article
Detection of resistance levels against cypermethrin and deltamethrin, the most commonly used synthetic pyrethroids (SP), in Rhipicephalus (Boophilus) microplus collected from thirteen districts of Punjab (India) was carried out using adult immersion test. The regression graphs of probit mortality of ticks plotted against log values of concentrations of drugs were utilized for the determination of slope of mortality, lethal concentration for 50% (LC50), 95% (LC95) and resistance factor (RF). On the basis of the data generated on variables (mortality, egg mass weight, reproductive index and percentage inhibition of oviposition) the resistance levels were categorized. Against cypermethrin RFs of 1.48-11.22 were recorded in 12 isolates whereas, one isolate was susceptible. Resistance factors against deltamethrin were 2.4-38.54 and all 13 isolates were found to be resistant. Quantitative analysis of general esterase activity (measured by the production of the metabolite naphthol) revealed a range of 3.34±0.30-13.75±1.33 and 1.31±0.15-8.09±0.68μmol/min/mg protein for α and β-esterase activity, respectively in different field isolates. Further, multiple pairwise comparisons of the mean values with susceptible strain (Tukey, P=0.05) revealed significant elevated levels of both α-esterase and β-esterase in nine tick isolates resistant to both deltamethrin and cypermethrin. The data generated on acaricide resistant status and esterase mediated mechanism in ticks will help in formulating tick control strategy for the region.
Article
Ticks and tick-borne diseases cause major economic losses affecting many domestic animals in tropical and subtropical countries including India. Synthetic pyrethroids particularly deltamethrin and cypermethrin have been used extensively to control Rhipicephalus (Boophilus) microplus in most parts of Punjab, India for the past decade leading to tick populations resistant against both the acaricides. Ticks (R. microplus) were collected from dairy farms at Haibbowal Dairy Complex, Ludhiana, Punjab and were subjected to adult immersion test with a discriminating dose (AITDD) against deltamethrin and cypermethrin. Results revealed a very high degree of resistance against deltamethrin (96.67%) and cypermethrin (93.33%) indicating need for adoption of alternative tick control strategy.