This study aimed to identify,characterize,and functionally analyze the CYP450 gene fam-ily within Hyalomma asiaticum unfed nymphs(HasiUN)under regular physiological conditions,laying the groundwork for further study into the biosynthesis and metabolism of exogenous sub-stances associated with the CYP450 gene in HasiUN.Through our methods,we identified and characterized HasiUNCYP450 genes.We then annotated them using the gene ontology(GO)and kyoto encyclopedia of genes and genomes(KEGG).This was achieved with the help of bioinforma-tics,based on the transcriptome of 2-week-old HasiUN.Further on,we conducted a preliminary a-nalysis of tissue expression,distribution,and potential function with qRT-PCR and network inter-action predictions.Our analyses showed that 87 HasiUN CYP450 genes,ranging in size from 1089 to 1692 bp,encoded predominantly basic,hydrophilic,and unstable protein sequences.These se-quences had nearly identical numbers of transmembrane regions,both with and without a signal peptide.Phylogenetic and domain analyses indicated that HasiUN CYP450,having a complete CYP450 domain,were distributed among various clans,with most members found in clan 3.CYP20 clans,present in 13 species from 2 families and 7 genera,required either one-host or three-hosts to undergo their full life cycle.MEME analysis results hint at different clans having variations in the amount and combination of motifs.Expression level analysis showed that HasiUN CYP450 was expressed at different levels,with the highest distribution of normally expressed genes in clan 3 and low expression of all mito clan genes.Functional annotation revealed HasiUN CYP450 key as-sociation with biological processes,metabolism,and organismal systems according to the GO and KEGG annotations.Distribution results showed that HasiUN CYP20a1 was expressed in specific tissues,including salivary glands and ovaries.Functional analysis points to CYP20a1's role in UV-induced toxic metabolism,endocrine disruptor metabolism,and hazardous substance metabolism in the nervous system.Our research presents the first analysis of HasiUN CYP450 gene's biological characteristics and expression pattern under normal physiological conditions.Additionally,investi-gations into the distribution and function of a new category,HasiUN CYP20a1,were embarked upon.These findings establish a theoretical foundation for continued functional study of HasiUN CYP45 gene.

This study aimed to identify,characterize,and functionally analyze the CYP450 gene fam-ily within Hyalomma asiaticum unfed nymphs(HasiUN)under regular physiological conditions,laying the groundwork for further study into the biosynthesis and metabolism of exogenous sub-stances associated with the CYP450 gene in HasiUN.Through our methods,we identified and characterized HasiUNCYP450 genes.We then annotated them using the gene ontology(GO)and kyoto encyclopedia of genes and genomes(KEGG).This was achieved with the help of bioinforma-tics,based on the transcriptome of 2-week-old HasiUN.Further on,we conducted a preliminary a-nalysis of tissue expression,distribution,and potential function with qRT-PCR and network inter-action predictions.Our analyses showed that 87 HasiUN CYP450 genes,ranging in size from 1089 to 1692 bp,encoded predominantly basic,hydrophilic,and unstable protein sequences.These se-quences had nearly identical numbers of transmembrane regions,both with and without a signal peptide.Phylogenetic and domain analyses indicated that HasiUN CYP450,having a complete CYP450 domain,were distributed among various clans,with most members found in clan 3.CYP20 clans,present in 13 species from 2 families and 7 genera,required either one-host or three-hosts to undergo their full life cycle.MEME analysis results hint at different clans having variations in the amount and combination of motifs.Expression level analysis showed that HasiUN CYP450 was expressed at different levels,with the highest distribution of normally expressed genes in clan 3 and low expression of all mito clan genes.Functional annotation revealed HasiUN CYP450 key as-sociation with biological processes,metabolism,and organismal systems according to the GO and KEGG annotations.Distribution results showed that HasiUN CYP20a1 was expressed in specific tissues,including salivary glands and ovaries.Functional analysis points to CYP20a1's role in UV-induced toxic metabolism,endocrine disruptor metabolism,and hazardous substance metabolism in the nervous system.Our research presents the first analysis of HasiUN CYP450 gene's biological characteristics and expression pattern under normal physiological conditions.Additionally,investi-gations into the distribution and function of a new category,HasiUN CYP20a1,were embarked upon.These findings establish a theoretical foundation for continued functional study of HasiUN CYP45 gene.