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Laboratory assessment of acaricidal activityof Cymbopogon winterianus, Vitex negundo and Withaniasomnifera extracts against deltamethrin resistantHyalomma anatolicum
Nirbhay Kumar Singh • Jyoti • Bhaskar Vemu • Abhijit Nandi •
Harkirat Singh • Rajender Kumar • V. K. Dumka
Received: 26 September 2013 / Accepted: 5 March 2014 / Published online: 20 March 2014� Springer International Publishing Switzerland 2014
Abstract Larval packet test was used for detection of resistance levels against cyper-
methrin and deltamethrin, the most commonly used synthetic pyrethroids, in the multi-host
tick Hyalomma anatolicum collected from district Moga, Punjab (India). Results indicated
the presence of level I resistance against deltamethrin (RF = 2.81), whereas the tick isolate
was susceptible to cypermethrin (RF = 0.2). The aqueous and ethanolic extracts of leaves
of Cymbopogon winterianus, Vitex negundo and Withania somnifera along with roots of
Vitex negundo were assessed for their acaricidal activity against the larvae of deltamethrin
resistant H. anatolicum. The efficacy was assessed by measuring per cent larval mortality
and determination of LC50 values. The various ethanolic extracts produced a concentration
dependent increase in larval tick mortality, whereas the aqueous extracts exhibited a much
lower mortality. The highest mortality (93.7 ± 0.66 %) was observed at the 5.0 % con-
centration of ethanolic extract of leaves of C. winterianus and the lowest LC50 value
(0.011 %) was recorded for ethanolic extracts of leaves of V. negundo. The results indi-
cated that these plant extracts have potential to be developed as herbal acaricides.
Keywords Acaricidal activity � Cymbopogon winterianus � Hyalomma
anatolicum � Vitex negundo � Withania somnifera
N. K. Singh (&) � Jyoti � A. Nandi � H. SinghDepartment of Veterinary Parasitology, College of Veterinary Science, Guru Angad Dev Veterinaryand Animal Sciences University, Ludhiana 141004, Indiae-mail: [email protected]
B. Vemu � V. K. DumkaDepartment of Veterinary Pharmacology and Toxicology, College of Veterinary Science, Guru AngadDev Veterinary and Animal Sciences University, Ludhiana 141004, India
R. KumarDepartment of Agronomy, Punjab Agricultural University, Ludhiana 141004, India
123
Exp Appl Acarol (2014) 63:423–430DOI 10.1007/s10493-014-9791-1
Introduction
Hyalomma anatolicum, a three-host tick species, is widely distributed over most geo-
graphical regions in India infesting cattle, buffaloes, sheep and goat and transmitting
Theileria annulata, T. buffeli, T. lestocardi (T. hirci) (Ghosh et al. 2007). Along with
vectorial potential, the direct effect of this tick on livestock production leads to significant
economic losses. Tick control now is exclusively dependent on a large scale and repeated
use of synthetic acaricides, however, application of these chemical acaricides has had
limited efficacy in reducing tick infestations and is often accompanied by serious draw-
backs, including the development of acaricide resistant ticks, environmental contamina-
tion, and even contamination of milk and meat products with insecticide residues (Graf
et al. 2004). To reduce the utilization of acaricides other approaches including use of hosts
with natural resistance to ticks, use of biological control agents and tick vaccines has been
meagerly evaluated against multi host ticks (Ghosh et al. 2008). In order to offer an eco-
friendly acaricide, there is a need to assess botanical extracts, especially from plants which
are rustic, perennial, easily cultivable and have a better potential of extension, in addition
to their inherent acaricidal properties.
Cymbopogon winterianus (Citronella grass) a tall perennial grass, native to India and
tropical Asia is a medicinal plant from the Poacea family with insecticidal and repellent
activity (Shasany et al. 2000). Vitex negundo L. (Nirgundi) belongs to family Verbenaceae
is a hardy plant, flourishing mainly in the Indian subcontinent and is used as a bio-pesticide
and mosquito repellent (Singh et al. 2011). Withania somnifera (Ashwagandha) is a plant
of Solanaceae family cultivated in many of the drier regions of India with insecticidal
activity (Bansal et al. 2011). Although acaricidal activity against cattle tick Rhipicephalus
(Boophilus) microplus of C. winterianus (Martins 2006; Olivo et al. 2008) and V. negundo
(Kamaraj et al. 2010; Singh et al. 2014) has been reported, the acaricidal activity of these
plants has not been reported against H. anatolicum. Therefore, an attempt has been made in
this study to assess the acaricidal effect of V. negundo, W. somnifera and C. winterianus
against the larvae of H. anatolicum.
Materials and methods
Ticks
Fully engorged females ticks were collected from cattle sheds of Moga, Punjab (India).
After identification the ticks were kept individually in labelled plastic tubes covered with
muslin cloth and kept in desiccators placed in BOD incubator maintained at 28 ± 1 �C and
85 ± 5 % relative humidity for oviposition. The eggs laid were allowed to hatch to larvae
under similar conditions of incubation.
Acaricides
Technical grade (100 % pure) cypermethrin and deltamethrin (AccuStandard�, USA) were
used for conducting Larval Packet Test (LPT). The acaricides were dissolved in methanol
for preparation of stock solutions and different concentrations of cypermethrin and del-
tamethrin were prepared in distilled water from the stock solutions for testing against H.
anatolicum.
424 Exp Appl Acarol (2014) 63:423–430
123
Larval packet test (LPT)
The LPT was conducted according to FAO (1971) guidelines with minor modifications.
Briefly, 0.5 ml of different concentrations of cypermethrin (50, 100, 200, 400 and
800 ppm) and deltamethrin (12.5, 25, 50, 100 and 200 ppm) in water were used to
impregnate 7 9 7 cm filter paper (541 Whatman). The aqueous solution of acaricide was
dried by keeping the filter paper for 30 min in incubator at 37 �C. The filter papers were
then folded in half diagonally and sealed on one side with adhesive tapes, forming an
open-ended triangular packet to place tick larvae. After insertion of approximately 100
larvae, the open end of each packet was sealed with adhesive tape and the packets were
placed in a desiccator placed in BOD incubator maintained at 28 ± 1 �C and 85 ± 5 %
RH. For each concentration of acaricide the test was conducted in triplicate and in
control group distilled water was used. The packets were removed after 24 h, and larval
mortality was calculated.
Estimation of resistance status
Dose response data were analyzed by probit method (Finney 1962) using GraphPad Prism
4 software. The lethal concentration for 50 % (LC50) and 95 % (LC95) values of cyper-
methrin and deltamethrin against H. anatolicum were determined by applying regression
equation analysis to the probit transformed data of mortality. Resistance factors (RF) was
worked out by the quotient between LC50 of field ticks and LC50 of susceptible line of H.
anatolicum (Castro-Janer et al. 2009). The LC50 values of cypermethrin and deltamethrin
against acaricides susceptible reference IVRI-II line of H. anatolicum were used as per
Shyma et al. (2012) and were 245.91 and 11.7 ppm, respectively. On the basis of RF, the
resistance status was classified as susceptible (RF \1.4), level I (RF = 1.5–5.0), level II
(RF = 5.1–25.0), level III (RF = 25.1–40) and level IV (RF[40.1) (Sharma et al. 2012).
Plant material
The leaves of V. negundo, W. somnifera and C. winterianus along with roots of V. negundo
were collected from Herbal garden, Department of Agronomy, Punjab Agricultural Uni-
versity, Ludhiana, India.
Preparation of plant extract
The leaves of V. negundo, W. somnifera and C. winterianus (both tender and old) along
with the roots of V. negundo were cleaned, shade dried at room temperature and were
finely pulverized using a grinder. The plant extraction was carried out by maceration
wherein 100 g each of the powdered material was added with the solvent in the ratio of
1:20 (distilled water) and 1:10 (ethanol) separately. The flasks were agitated for frequent
mixing over a period of 24 h. The mixtures were filtered using muslin cloth followed by
filter paper (No. 5 B—Advantec�) and the filtrates were completely air dried at 40 �C and
yields were measured. The required quantity of aqueous and ethanolic extracts were
weighed and dissolved in distilled water and 10 % DMSO, respectively for preparing the
working dilutions of 0.1, 1 and 5 % to test their acaricidal potential against H. anatolicum.
Exp Appl Acarol (2014) 63:423–430 425
123
Bioassay
For evaluation of anti-tick activity of these extracts LPT as described above was adopted.
Various concentrations (0.1, 1.0 and 5.0 %) of aqueous extracts of leaves of C. winterianus
(CLA), W. somnifera (WLA), V. negundo (VLA) and roots of V. negundo (VRA), as well
as ethanolic extracts of leaves of C. winterianus (CLE), W. somnifera (WLE), V. negundo
(VLE) and roots of V. negundo (VRE) were used and the larval mortality in H. anatolicum
was determined against each concentration after exposure of 24 h.
Analysis of data
Dose response data were analyzed by probit method using GraphPad Prism 4 software and
LC50 values of various plant extracts against H. anatolicum were determined.
Results
Resistance status
The ticks collected were identified as H. anatolicum. The slope, LC50, LC95, RF values and
the level of resistance to cypermethrin and deltamethrin are shown in Table 1. Results
indicate presence of level I resistance to deltamethrin (RF = 2.81), whereas the tick isolate
was susceptible to cypermethrin (RF = 0.2).
In vitro efficacy of aqueous extracts
Yield, mortality, slope and LC50 values of aqueous extracts of leaves of C. winterianus, W.
somnifera, V. negundo and roots of V. negundo against deltamethrin resistant H. anatol-
icum are shown in Table 2. The efficacy of various extracts against H. anatolicum larvae
were assessed by estimating mortality. Larval mortality caused by the aqueous extracts of
different plants varied from 0.0 to 28.97 %, when tested at concentrations ranging from 0.1
to 5.0 %. Further, because of the low mortality the LC50 values of aqueous extracts against
H. anatolicum were impracticable except WLA with LC50 value of 20.3 %.
In vitro efficacy of ethanolic extracts
Yield, mortality, slope and LC50 values of ethanolic extracts of leaves of C. winterianus,
W. somnifera, V. negundo and roots of V. negundo against deltamethrin resistant H.
anatolicum are shown in Table 3. The larval mortality caused by the ethanolic extracts
showed a concentration dependent increase and for different extracts varied from 9.7 to
93.7 % at concentrations of 0.1–5.0 %. Among all ethanolic extracts the best acaricidal
Table 1 The results of larval packet test to various acaricides performed on Hyalomma anatolicum
Acaricide Slope (95 % CL) R2 LC50 (ppm)
(95 % CL)
LC95 (ppm)
(95 % CL)
RF RL
Cypermethrin 2.87 ± 0.39 (1.62–4.13) 0.946 49.26 (48.77–49.75) 183.21 (176.50–190.17) 0.2 S
Deltamethrin 3.56 ± 0.75 (1.17–5.95) 0.882 32.89 (32.40–33.38) 94.9 (92.13–97.74) 2.81 I
RF resistance factor, RL resistance level
426 Exp Appl Acarol (2014) 63:423–430
123
property was exhibited by the VLE with the minimum LC50 values of 0.011 %, followed
by CLE as 0.14 %, VRE as 1.27 % and maximum for WLE as 10.12 % (Table 3).
Discussion
The problem of ticks and tick borne diseases is particularly relevant in India because of the
congenial environmental conditions for tick survival throughout the most parts of the year
and maintenance of susceptible cross bred animals to improve the production of milk and
other animal products. Among the various ixodid ticks H. anatolicum is one of the
important ticks infesting dairy animals of India (Ghosh et al. 2007) particularly Punjab
state (Haque et al. 2011; Singh and Rath 2013). The use of acaricides as the principal
means for tick control has resulted in the selection of chemical resistant ticks. Presence of
wide spread resistance to cypermethrin and deltamethrin in H. anatolicum by LPT has been
recently reported from different places of India (Shyma et al. 2012; Singh et al. 2013). In
bioassays (LPT), technical grade cypermethrin and deltamethrin were selected over
commercial formulation as commercial products are prepared with many proprietary
ingredients and it is difficult to assess the responses due to active ingredients (Shaw 1966).
Further, the stock solutions were prepared by dissolving in 100 % methanol and the
working concentrations were prepared with distilled water. Use of organic solvent facili-
tates adsorption of compound over the surface area of target biological materials and also
enhances penetration of active ingredients of the acaricide across the exoskeleton (Sharma
et al. 2012).
Acaricides of herbal origin stand out as an important possible alternative as future
acaricide (s) as they are environment friendly and the problem of resistance development is
minimal. During the last few years, many plant extracts have been tested for their acari-
cidal effects against R. (B.) microplus (Chungsamarnyart and Jiwajinda 1992; Martins
2006; Olivo et al. 2008; Kamaraj et al. 2010; Singh et al. 2014). However, studies on
Table 2 The effect of various aqueous plant extracts on larvae of Hyalomma anatolicum
Plant Yield(%)
Conc.(%)
Mortality (%)(mean ± SE)
Slope (95 %CL)
R2 LC50 (%)
Vitex negundo (root) 8.97 0.1 5.06 ± 0.81 0.103 ± 0.091(-1.05 to1.26)
0.562 7.61 9 1010
1.0 7.84 ± 1.96
5.0 7.11 ± 1.22
Vitex negundo (leaves) 18.17 0.1 6.26 ± 2.41 0.194 ± 0.054(-0.50 to0.89)
0.926 3.82 9 1018
1.0 9.69 ± 4.93
5.0 11.24 ± 2.55
Withania somnifera(leaves)
20.48 0.1 0 ± 0 1.035 ± 0.130(-0.61 to2.68)
0.984 20.25
1.0 6.85 ± 2.23
5.0 28.97 ± 1.7
Cymbopogonwinterianus (leaves)
16.32 0.1 0 ± 0 0.630 ± 0.455(-5.16 to6.42)
0.656 985.4
1.0 0 ± 0
5.0 12.28 ± 1.17
Control (DW) – – 0 ± 0 – – –
Exp Appl Acarol (2014) 63:423–430 427
123
herbal acaricides against H. anatolicum tick are limited in number (Abdel-Shafy and Zayed
2002) and as per our knowledge no published reports are available on the effect of C.
winterianus, W. somnifera, V. negundo extracts on deltamethrin resistant H. anatolicum
ticks.
Various species of the genus Cymbopogon (Poaceae) showed high acaricidal activity
against the larvae and adult females of R. (B.) microplus (Chungsamarnyart and Jiwajinda
1992; Olivo et al. 2008), Amblyomma cajennense (Soares et al. 2010), R. sanguineus
(Heimerdinger et al. 2006). Further, C. winterianus has also been investigated against R.
(B.) microplus (Martins 2006; Singh et al. 2014). These properties are attributed to the
presence of volatile substances in its leaves such as citronellal, eugenol, geraniol and
limonene, among others, denominated in general as monoterpenes (Shasany et al. 2000).
Results of the current study indicate that the ethanolic extracts of leaves of C. winterianus
(LC50 = 0.14 %) can prove to be a promising herbal acaricide against deltamethrin
resistant H. anatolicum.
The leaves, flowers, fruit, roots and bark of V. negundo have great medicinal value and
has proven insecticidal property against various agricultural pests. The acaricidal property
of leaf extracts of V. negundo against cattle tick R. (B.) microplus has been studied
(Kamaraj et al. 2010; Singh et al. 2014). The current study report a LC50 value of 0.011 %
against deltamethrin resistant H. anatolicum. The acaricidal property may be due to the
number of constituents that are present in the plant as terpenoids, irridoids, steroids,
phenolic compounds, lignane derivatives, amino acids, fatty acids and aliphatic alcohol
(Singh et al. 2011). Similarly, in recent past W. somnifera has also been evaluated for its
larvicidal property against larvae of Anopheles stephensi, Aedes aegypti and Culex quin-
quefasciatus (Bansal et al. 2011). The acaricidal property against R. (B.) microplus has also
been reported (Singh et al. 2014) but effects against H. anatolicum has not been reported
and the current study reports LC50 values of 20.25 and 10.12 % of the aqueous and
ethanolic extracts of leaves of W. somnifera against deltamethrin resistant H. anatolicum.
The acaricidal activity may be because of the presence of biologically active chemical
constituents as alkaloids (isopelletierine, anaferine), steroidal lactones (withanolides,
Table 3 The effect of various ethanolic plant extracts on larvae of Hyalomma anatolicum
Plant Yield(%)
Conc.(%)
Mortality (%)(mean ± SE)
Slope (95 %CL)
R2 LC50 (%) (95 %CL)
Vitex negundo (root) 1.9 0.1 9.74 ± 2.76 1.045 ± 0.314(-2.94 to5.03)
0.917 1.27 (1.21–1.33)
1.0 58.48 ± 14.48
5.0 66.66 ± 12.27
Vitex negundo(leaves)
9.34 0.1 66.01 ± 1.60 0.381 ± 0.131(-1.28 to2.04)
0.894 0.011 (0.010–0.013)
1.0 73.05 ± 22.11
5.0 85.66 ± 4.81
Withania somnifera(leaves)
10.46 0.1 11.23 ± 1.81 0.566 ± 0.241(-2.49–3.62)
0.846 10.12 (9.21–11.11)
1.0 36.81 ± 7.08
5.0 37.78 ± 2.22
Cymbopogonwinterianus(leaves)
6.35 0.1 40.57 ± 9.42 1.064 ± 0.149(-0.83 to2.95)
0.980 0.14 (0.13–0.15)
1.0 84.82 ± 4.05
5.0 93.68 ± 0.66
Control (10 %DMSO)
– – 4.05 ± 0.48 – – –
428 Exp Appl Acarol (2014) 63:423–430
123
withaferins), saponins containing an additional acyl group (sitoindoside VII and VIII), and
withanolides with a glucose at carbon 27 (sitoindoside IX and X) (Mishra et al. 2000).
The beneficial medicinal effects of plant materials typically result from active com-
pounds present in the plant and can be due to effects of a single compound or combination
of one or more. The chemical constituents of the plants may exert acaricidal effects in
different ways; therefore, the development of resistance seems difficult against botanical
acaricides in combination. Further, studies are needed to identify the active ingredients
present in these plant that caused the mortality of tick larvae. Analyzing the data obtained
in the present study, it can be concluded that the ethanolic extract of leaves of V. negundo
and C. winterianus possesses acaricidal properties which may provide an effective eco-
friendly herbal formulation for the control of deltamethrin resistant tick infestation on
animals.
Acknowledgments Authors are thankful to the Director of Research, GADVASU, Ludhiana for providingfacilities to carry out the research work.
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