Studies on synthetic pyrethroid resistance in Rhipicephalus (Boophilus) microplus and comparative sequence analysis of carboxylesterase gene
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Date
2013-07
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G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand)
Abstract
Two FAO recommended in vitro bioassays namely Adult Immersion Test (AIT) and Larval Packet Test (LPT) were employed to detect deltamethrin resistance in the tropical cattle tick, Rhipicephalus (Boophilus) microplus collected from tarai and hill regions of Uttarakhand. The AIT revealed the lowest LC50 (0.00085% A. I.; RF= 1) for Reference Susceptible R. microplus tick population. The highest LC50 (0.03842%; RF= 45.17) was observed for Dehradun (Mussourie) population of ticks followed by Nainital I (0.03403%; RF= 40.1), Nainital II (0.00571%; RF= 6.70) and Uttarkashi (0.00114%; RF= 1.29) ticks. In ticks collected from tarai region, the highest LC50 was found for Pantnagar II (0.03361%; RF= 39.53) population followed by Sitarganj (0.00663%; RF= 7.7), Pantnagar I (0.00652%; RF= 7.64), Ramnagar (0.00335%; RF= 3.88) and Tanakpur (0.00252%; RF= 2.94). The LC99 of different populations ranged from 0.3262% to 0.0036%. The R2 values determined from AIT ranged from 0.998 (Susceptible) to 0.847 (Nainital I ticks). The slopes of egg mass of different tick populations were highly negative. The values ranged from -146.95±20.96 (Susceptible) to -40.19±6.86 (Dehradun). The slopes of reproductive indices of different populations of ticks were found to be negative indicating the reduction of fecundity with increased concentration of acaricide. The values of slopes of inhibition of oviposition were positive and the highest value (34.7) was exhibited by Susceptible and lowest (6.92) by Dehradun ticks. In LPT bioassay, an LC50 of 0.00076% A. I. was observed for the Susceptible R. microplus population. Fom hills, Dehradun population exhibited the highest LC50 (0.03454%: RF= 45.39) followed by Nainital I (0.03113%: RF= 40.9), Nainital II (0.00421%; RF= 5.5) and Uttarkashi (0.00084%; RF= 1.1). From tarai region, the highest LC50 concentration was observed for Pantnagar II (0.03091%: RF= 40.65) population of ticks followed by Pantnagar I, Sitarganj, Ramnagar, and Tanakpur population of ticks (0.00493%; RF= 6.44), (0.00460%; RF= 6.05), (0.00250%; RF= 3.28) and (0.00164%; RF=2.10), respectively. The highest LC99 was found for Nainital I (1.8060%) population of ticks and the lowest for Susceptible population (0.0033%). Variations in LC50 values of different populations in AIT and LPT bioassays might be due to the difference in the stage of the tick being tested. The Chi square values ranged from 97.84 (Dehradun) to 18.298 (Nainital II). Six tick populations (Dehradun, Nainital I, Pantnagar II, Pantnagar I, Sitarganj and Nainital II) were found deltamethrin resistant (RF > 5), one tick population (Ramnagar) deltamethrin tolerant (RF 3-5) and two tick populations (Uttarkashi and Tanakpur) deltamethrin sensitive (RF< 3) in both in vitro bioassays. This implies that frequent monitoring of deltamethrin resistance in ticks is required so that timely measures could be taken before resistance to this acaricide becomes established. The Spearman Rank correlation coefficient (rS) was found to be 0.997 between AIT and LPT bioassays. The coefficient of determination (R2) was found to be 0.998. AIT and LPT were found highly correlated. The LPT bioassay takes 6 weeks to give results of acaricide resistance whereas AIT 2 weeks. So, it is suggested that AIT can be used as an effective screening test to detect the acaricide resistance in field populations of ticks. The sequences of amplified partial carboxylesterase gene (372 bp) of R. microplus from tick populations matched 100% with the carboxylesterase gene sequence of R. microplus available in GenBank. The sequence analysis revealed that no population had a nucleotide polymorphism at the position 300. The sequencing of partial carboxylesterase gene revealed four nucleotide changes in Uttarkashi and one in Dehradun population of ticks. The mutations in the nucleotide sequence of Uttarkashi population resulted in two amino acid mutations (Val 66 Gly, Asp 120 Asn) in the translated amino acid sequence. The amino acid sequence deduced from the nucleotide sequence of Dehradun population exhibited one (Glu 123 Arg) mutation. The mutations in Uttarkashi population had no effect in the resistance development as this population was phenotypically susceptible. Further investigations are required to identify role of mutation in resistance development in Dehradun population of ticks. Animal owners use different acaricides and adopt various application methods to control R. microplus. Thus frequent monitoring of acaricide resistance is very important to know the distribution and level of resistance to these acaricides. The baseline information thus generated will enable timely management of spread of acaricide resistance in ticks and hence enhance animal productivity.
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