In Vitro Detection of Acaricidal Resistance Status of Rhipicephalus ( Boophilus ) microplus against Commercial Preparation of Deltamethrin in Coastal Areas of South Gujarat, India

A total of 2617 bovines (1682 cattle and 935 buffaloes) of coastal districts (Bharuch, Surat, Navsari and Valsad) of South Gujarat were screened for tick Rhipicephalus ( Boophilus ) microplus, the most economically important ectoparasite of bovines worldwide. Farmers chiefly relied on chemical acaricides (67.52%) to kill the ticks, and under chemical method 44.09, 24.17, 30.50 and 1.24 % animals had received cypermethrin, deltamethrin, ivermectin and fipronil, respectively, thus favouring the resistance. In adult immersion test (AIT), the median lethal concentration (LC 50 ) was 144.23, 93.97, 141.67 and 57.89 ppm for deltamethrin in Bharuch, Surat, Navsari and Valsad district, respectively. Resistance level was I in Valsad and II in other 3 districts for deltamethrin in AIT. In larval packet test (LPT), the median LC 50 was 297.29, 126.81, 127.83 and 93.92 ppm for deltamethrin in Bharuch, Surat, Navsari and Valsad district, respectively. Resistance level was III in Bharuch and II in other district in LPT.


In Vitro Detection of Acaricidal Resistance Status of Rhipicephalus (Boophilus) microplus against Commercial Preparation of Deltamethrin in Coastal Areas of South Gujarat, India
Dharmesh C Patel 1 *, Jayesh B Solanki 2 , Niranjan Kumar 3 IntroductIon T he tick causes economic loss due to lowered milk and meat yield, devaluation of leather, diseases transmission, additional hours of work required, additional facility costs, acaricides application and emergence of resistance against commonly used one (Estrada-Peña and Salman, 2013). Rhipicephalus (Boophilus) microplus is the most prevalent tick species found to infest bovine of South Gujarat, India (Ghosh et al., 2006). Synthetic pyrethroids, deltamethrin and flumethrin are the predominant acaricides used to control ticks all over the world including India (Mathivathani et al., 2011). There are reports regarding failure of the deltamethrin treatment world over against the ticks especially of R. (B.) microplus at the field level due to emergence of resistance (Andreotti et al., 2011;Shakya et al., 2020).
A lack of standardized techniques for diagnosing acaricide resistance appears to be the main difficulty in creating and maintaining a tick resistance monitoring system (FAO, 2004). Acaricide resistance can be detected by both in vivo and in vitro methods. The most widely used tests are bioassays, in vitro exposure of ticks (larvae or engorged females) to a single dose or to several increasing doses of an acaricidal compound (FAO, 2004). The commonly used bioassays for detection of acaricide resistance are larval packet test (LPT), larval immersion test (LIT) and adult immersion test (AIT) (Drummond et al., 1973;FAO, 2004). Detection of acaricides resistance would provide immense help in formulation of different control strategies for management of acaricide resistance. It is quite demanding to assess the resistance status of commonly used deltamethrin from the coastal areas of South Gujarat, hence was studied.

MAterIAls A n d Methods
Systematic survey on ixodid ticks of bovine was undertaken at four coastal districts of South Gujarat, India, viz., Bharuch, Surat, Navsari and Valsad from June 2016 to May 2017. Systemic approach was made to collect owner's response pertaining to the question regarding method of tick control, name of chemical drugs, drug used on the advice of which person, frequency and effectiveness of drugs etc. The female engorged ticks were collected from the cracks and crevices of the animal sheds. For handling field ticks, the recommended method of FAO (2004) was followed. The ticks were identified by studying the standard morphological keys (Miranpuri, 1979). Different concentrations of the commercial deltamethrin (Butox @1.25% EC) prepared in distilled water were used to conduct bioassays to know the status of acaricide resistance. The recommended dose was taken as 25 ppm for deltamethrin. The higher concentration of deltamethrin was used in bioassay, viz., 25, 50, 75, 100 and 125 ppm.

Adult Immersion Test (AIT)
The test was conducted according to Drummond et al. (1973) with some modifications. Ten randomly selected engorged female ticks were weighed and immersed for 2 minutes in 50 ml of prepared graded dose acaricides in glass beakers. As a control, 10 ticks of the same batch were treated similarly in distilled water for 2 minutes. Ticks were observed for their mortality on 14 th day post-treatment (DPT) and laid egg masses were weighed. Dead ticks were confirmed by observing loss of motility and pedal reflex after exposing to light. The regression curve of mortality percentage was plotted against working concentrations of the acaricides in Microsoft Office Excel. The median lethal concentration 50% (LC 50 ) of deltamethrin for field tick isolates were calculated using the regression equation.
Y= mx + C m= slope of the curve, C= y intercept (correlation coefficient), x= concentration of acaricide, Y= probit value of mortality percentage Resistance factors (RF) for field tick isolates were worked out by the ratio between LC 50 of field ticks and LC 50 of susceptible strain of R.(B.) microplus IVRI-I line (13.4 ppm LC 50 of deltamethrin). On the basis of RF, the resistance status in the field population of ticks was classified as susceptible (RF < 1.4), level I resistance (1.5 < RF < 5.0), level II resistance (5.1 < RF < 25.0), level III resistance (26.0 < RF < 40.0) and level IV resistance (RF > 41) (Kumar et al., 2011).

Larval Packet Test (LPT)
Fifteen ticks were incubated at 28°C and 75-85% RH in labelled glass bottle covered with muslin cloth for oviposition. The eggs were further incubated at same condition in the BOD incubator for 18-25 days to hatch into larvae. The LPT was performed using 10-12 day old hungry larvae as recommended by FAO (2004) with some minor modifications. Approximately 0.6 ml of prepared acaricide solution was poured on Whatman filter paper number 1. After saturation of the compound the filter paper was dried in incubator at 37°C for 30 minutes. Treated and dried parallelogram of paper was folded in half forming equilateral triangular packet and sealed on the sides with adhesive tapes forming an open ended packet. After placing approximately 100 larvae with the help of paint brush, the open side of each packet was sealed with adhesive tape. The sealed packet was then placed in a desiccator having 50 ml of 10% KOH solution in the bottom and incubated in BOD. incubator at 28°C and 75-85% RH. After 24 hours, the packets were opened, and mortality percentage was calculated on the basis of live and dead larvae count. The regression curve of larvae mortality % was prepared in the same manner as in above test. Resistance factors (RF) for field tick's larvae isolates were worked out by the ratio between LC 50 of field tick's larvae and LC 50 of larvae of susceptible strain R.(B.) microplus IVRI-I line (111.8 ppm LC 50 of deltamethrin) (Castro-Janer et al., 2009). On the basis of RF, the resistance level in the field population of tick's larvae was classified as described in AIT.

Statistical Analysis
The statistical analysis of the data was done using software IBM-SPSS 20 and Micro Soft Excel-2010. The differences in mean values of entomological data amongst the groups were analyzed by one way ANOVA.

results A n d dIscussIon
Animal owners' response (n=2617) to the questionnaire asked about the control measures adopted against the tick infestation in the area under study is illustrated in Table 1. Three major treatment approaches, mechanical by hand picking/ rearing poultry, chemical, and herbal/ no treatment were adopted by the animal owners in the studied areas to minimize the tick infestation of the animals. The farmers applied mechanical, chemical and herbal/ no treatment strategies on 21.40, 67.52 and 11.08% animals, respectively (Table 1).
Under chemical method adopted on 1767 animals, i.e., 67.52% animals exposed to chemical acaricides, 24.17, 44.09, 30.50 and 1.24 % animals had received deltamethrin, cypermethrin, ivermectin and fipronil, respectively (Table  1). Effective and non-effective treatment/ control strategies were observed on 50.25% and 49.75% bovines, respectively. As per recent reports, among all the methods, deltamethrin is commonly used as an acaricide by animal owners for tick control (Kumar et al., 2019). Hlatshwayo and Mbati (2005) observed that only few participants applied commercial acaricides (16%) on their animals. However, many farmers (70 %) perceived the use of commercial acaricides as beneficial, citing economical constraints as the main reason for their reduced level of participation.

Adult Immersion Test (AIT) with Deltamethrin
The rate of mortality in the current study showed increasing trend with the increasing concentrations of the acaricide with decreasing amount of the egg mass (Table 2) and it varied significantly (p < 0.05) from the control group. In Navsari district, the Reproductive Index (RI) was 0.66±0.02 and 0.34±0.01 to 0.08±0.01 in the control and treated groups, respectively, with 48.25±1.93 to 88.60±1.33% of Inhibition of Oviposition (IO). The slope of the mortality curve was 0.24. The calculated median LC 50 was 141.67 ppm from the equation with 10.57 RF, thus indicated level II resistance in Navsari (Table 2; Fig. 1). The RI, slope of mortality curve, median LC 50 and RF value of other three districts were as mentioned in Table 2. The present study noted level II resistance in Bharuch (RF=10.76) / Surat (RF=7.01)/ Navsari (RF=10.57) and level I in Valsad (RF=4.32) district (Table 2; Fig. 1). The targeted 4 districts are in close proximation and have almost same agroclimatic conditions, but this variation in the level of resistance can be due to variation in animal management practices, treatment and control strategy adopted by the local farmers. The farmers of Valsad relied mainly on the mechanical/ herbal method to minimize or destroy the ticks population present on their bovines. Such type of area-wise varying resistance level was observed by Sharma et al. (2012)

Larval Packet Test (LPT) with Deltamethrin
The mortality percentage in the larvae of ticks gradually increased with increasing concentrations of the acaricide ( In conclusion, the area-wise acaricides resistance detection study is essentially required to develop long term control measures against the ticks. The present study involving coastal districts of south Gujarat observed varying level of resistance to deltamethrin. This information must be utilized while instituting treatment and control strategy against the bovine ticks in this area.