Pathogen Profiling and Molecular Evaluation Along With In Silico Modeling of Voltage‐Gated Sodium Channel Gene in Deltamethrin Exposed <i>Haemaphysalis longicornis</i> Ticks

Haemaphysalis longicornis is associated with the transmission of 30 various types of pathogens, having medical and veterinary importance. Hence, this study was designed to investigate the prevailing pathogen in Zhangjiachuan county, Gansu province, China. For the said purpose, we performed a molecular investigation of Rickettsia and Piroplasms pathogens in morpho-molecularly confirmed H. longicornis tick species, collected from cattle in the study area. Furthermore, the efficacy of deltamethrin was evaluated using larval packet tests (LPTs) against this tick species, followed by molecular and in silico analysis of voltage-gated sodium channels (VGSCs) gene. Moreover, their amplified VGSCs genes (Domain II and a part of its linker region with Domain III) were comparatively aligned with insecticide-susceptible Rhipicephalus (Rh) microplus (AF134216.2, OQ349192.1, KM073932.1), Rh. sanguineus (KU886031.1) and resistant Rh. microplus (MH986341.1, KM073928.1, KM073930.1, KM073931.1, KM073933.1), Rh. annulatus (MN535171.1), and Rh. appendiculatus (MH053429.1). Our findings revealed 149/469 (31.77%) and 92/469 (19.62%) prevalence rates of single infection for Candidatus Rickettsia jingxinensis (via 17-KDa and ompB genes) and Theileria orientalis (via 18S rRNA gene), respectively. Additionally, LPTs results expressed diverse mortality rates of 8.17%, 17.63%, 22.26%, 43.18%, 73.46%, and 87.51% via the application of 0.00006, 0.0006, 0.006, 0.06, 0.6, and 6 mg/mL deltamethrin concentrations, respectively, indicating the presence of reduced susceptible larvae in the targeted region. Comparative alignment of VGSCs gene (domain-II) showed 29 single nucleotide polymorphisms (SNPs) including 5 nonsynonymous (NS) SNPs. All five NS-SNPs were in the linker regions, playing a crucial role in protein channels (VGSCs) formation. Whereas comparison of the obtained partial domain-III of VGSCs gene with Rh. microplus and Rh. sanguineus tick species showed 100% conservativeness as no SNP was identified in its domain-III. In silico analysis regarding the 3D structure of VGSCs protein revealed suitability for attachment of deltamethrin and recorded binding affinity was -524.58 kcal/mol. This study presented the first ever molecular report of VGSC gene of H. longicornis tick species which will be helpful in their management as well as to mitigate their negative impacts.