Rhipicephalus (Boophilus) annulatus ticks are adept in transmitting a large variety of infectious diseases such as anaplasmosis and babesiosis and causing huge impediments in cattle industry. R.(B) annulatus occupy large areas of Southern India [10, 41]. R. (B) annulatus voraciously sucks blood meal, inject toxins, transmits pathogens, and finally modifies immune responses in cattle, which negatively impacts cattle productivity and results in significant economic losses [57, 58]. Tick control through acaricide treatment remains a basic strategy, although progress toward vaccine development or alternative tick control tools has been made. Amitraz, a formamidine pesticide predominantly used in ectoparasite control in several geographic areas of South India due to its inhibitory effect on tick fecundity and the impairment of oocytes [40, 41]. However, the high efficacy of amitraz against ticks has diminished due to the development of acaricidal resistance. Consequently, R. annulatus with a high resistance level is insensitive to certain amitraz formulations, rendering this acaricide ineffective for tick control. Thus, it is necessary to understand the mechanism through which R. (B) annulatus counteract the effect of insecticide. Ongoing research and development initiatives are geared to find a successful management strategy for this pest.
Previous studies for the underlying resistance mechanism in ticks led to the finding that behavioral, biochemical and genetic mechanisms are involved in neutralizing acaricide [59, 60, 31]. The genetic mechanisms accelerating resistance evolution that may involve target site insensitivity or increased metabolic enzymes such as cytochrome P450s, GSTs, CCEs, and ABC transporters, which sequester, catalyze or aid the elimination of the acaricide molecules, thus inhibiting them from binding their target . The extensive scientific background on metabolic enzymes [62, 63, 40] encouraged us to assess the expression levels of metabolic detoxifying enzymes in response to amitraz. In this study, the transcriptomics approach was employed to obtain the differential expression level of genes related to metabolic detoxification. In addition, using qRT-PCR the expression level of CYPs and GSTs in R.(B)annulatus at the sublethal doses of flumethrin and amitraz was examined.
In-vitro bioassays such as larval packet test and adult immersion test was done to compare the acaricidal effects of amitraz and flumethrin against R. (B) annulatus. Larval toxicity experiments revealed that R.(B)annulatus showed slight susceptibility difference against Amitraz and flumethrin, indicating that flumethrin is comparatively more effective in larval mortality than amitraz. The LC50 value observed for amitraz was 100 ppm and 80 ppm for flumethrin against R. (B) annulatus. Based on this result, flumethrin seems to be comparatively effective than amitraz. This result is in parallel with the results of acaricidal efficacy of flumethrin in R. microplus collected from Northern India, which showed high efficacy compared with other compounds . The present result and previous data advocate that a comparatively lower concentration of flumethrin is required to produce 50% mortality than amitraz. This might be due to the late introduction of flumethrin into the Indian market . Several authors have studied the efficacy of amitraz and flumethrin and revealed different spectrum of susceptibility in tick populations [65, 66, 41]. This susceptibility may be influenced by many factors such as application method, dose, and frequency of acaricides, genetic variation of the tick, geographic locations, defence mechanism in cattle and breed of cattle [67, 68]. Keeping these factors into consideration, livestock farmers can make a logical decision to adopt a particular chemical for tick control. To date, several studies have assessed the acaricidal efficacy of different compunds against R. (B) annulatus [40, 41, 64]. Yet, research headways in understanding the defence mechanism at the genetic level have been few and far between due to the lack of genomic information of this ectoparasite. Therefore, to better understand their defence molecular mechanism and association of the metabolic enzymes with acaricide resistance, we conducted a comparative transcriptome analysis of Amitraz treated and untreated R. (B) annulatus. We used larval stage of R. (B) annulatus for de novo sequencing, assembly, annotation and downstream analysis. According to the annotated results, 70 genes were found to be involved in the detoxification mechanism in R. (B) annulatus. Among the 70 genes, 44 were annotated as CYP450, 23 as GSTs and 3 as ESTs. Considering the abundance of detoxifying genes, more than half of the genes belonged to the Cytochrome P450 monooxygenase family. Further, to investigate gene expression changes associated with the amitraz treatment in R. (B) annulatus, differentially expressed contigs were compared using DEseq. There was a total of 16635 transcripts exhibited upregulation, 15539 exhibited down regulation. Among results obtained with Deseq analysis, we compared differentially expressed detoxifying genes upon amitraz treatment. Of these, 29 upregulated (11 CYP450 and 18 GST) and 12 (11 CYP450 and 1 GST) downregulated detoxifying genes were identified. Further, a total of nine differentially expressed genes obtained from the Illumina analysis were selected to examine the expression changes in various developmental stages of R. (B) annulatus mediated by amitraz and flumethrin by qRT-PCR. Among the 9 genes, 3 CYP genes (CYP41222; CYP27576 & CYP 309890) and 1 GST gene (GST34065) were highly expressed in amitraz treated adult and larva of R. (B) annulatus. Similarly, in the case of flumethrin treated samples, 3 CYP genes (CYP38633; CYP51361 & CYP30980) were significantly upregulated in both adult and larvae. Cytochrome P450 Monooxygenases are remarkable in their ability to oxidize widely diverse substrates and are capable of producing an array of molecular reactions . The existence of the cytochrome P450 system has been established in different arthropod species including ticks. Study on metabolic resistance showed that highest expression of cytochrome P450-like transcript assists in growing coumaphos resistant Mexican populations of R. (B) microplus . Different expression patterns or expressional variation of transcripts encoding CYP450 genes in multiple tissues and in different developmental stages of an insect indicate the CYP450 catalytic activity . As a result, populations of cattle ticks, R. (B) microplus, have developed tolerance to nearly all synthetic and chemical acaricides over the past decades due to their intensive use . Comprehensive analysis of the expression of P450 cytochrome oxidases in R. microplus showed the association of pyrethroid acaricide resistance with the level of CYP expression . Studies [73, 40, 62, 63] have also reported the association of acaricide resistance in ticks with the increased level of the expression of P450s genes and their role in xenobiotic detoxification. Apart from CYP protein, high expression of GSTs have also been identified, which play a role in conferring resistance to ticks [74, 75]. In our study, the high expression of CYP and GST genes in R. (B). annulatus during acaricide stressed condition may implicate their role in resistance development and increased survival rate of R.( B) annulatus. Those genes could be used as targets for pest control by inhibiting their action through sequence-specific gene silencing via RNA interference . Also, the downregulation of certain genes in response to these acaricides could be due to other novel feedback mechanisms. Understanding these pathways could open up new avenues to improve tick management strategies.
The larval insensitivity to flumethrin may indirectly highlights the efficiency as a drug to target the larval stage compared to Amitraz. This is consistent with the larval packet and adult immersion tests where flumethrin that caused more lethality than Amitraz. This could be attributed to the unaltered gene expression pattern of various genes involved in the metabolic resistance in the larval stages of the ticks. This provides an evidence that overexpression could confer acaricide resistance, possibly in the absence of target site insensitivity.
Overall, our research exploited a combination of transcriptome analysis, differential gene expression along with gene expression validation for the study of resistance mechanism. The results from this study showed that our approach relying on the Illumina Hi-Seq platform enabled a comprehensive representation of the R. (B) annulatus transcriptome. Identified gene targets from this study may be used as valid targets for evolving gene-based management strategies. These outcomes are geared to evaluate the possible molecular mechanisms involved in tick-borne diseases, thus contributing positively to the benefit of medical and veterinary professionals, farmers, and the common public. The knowledge creation under the aegis of this study provides ample scope for practical application, especially in generating important insights for developing new strategies to control this menacing pest.