Transcriptomics insights into the functional role of tick Ixodes ricinus midgut proteins metalloprotease and antigen p23

Ticks are hematophagous ectoparasites that impact human and animal health worldwide. Tick saliva contains multiple biomolecules capable of disrupting host immunity, occasionally causing emergent tick-borne allergies like the alpha-Gal syndrome (AGS). Current results highlight a knowledge gap in characterizing tick salivary biomolecules, without alpha-Gal modifications, that influence host response to alpha-Gal and may be implicated in the AGS. This study aimed to functionally characterize biomolecules antigen p23 (A0A0K8RKR7) and metalloprotease (A0A0K8RCY8) based on their role in Ixodes ricinus tick feeding and reproduction, determined after gene knockdown by RNA interference and further transcriptomics analysis. Silencing the expression of antigen p23 and metalloprotease did not result in any significant differences in tick engorgement and egg batch weights compared to the GFP control group. Transcriptomics analysis revealed a total of 438 differentially expressed genes (DEGs) were found in the antigen p23-silenced group, including 149 upregulated and 78 downregulated genes. GSEA analysis following antigen p23 gene knockdown identified significantly upregulated pathways associated with protein production and suppressed routes mostly correlated with ion transport, lipid metabolism, catalytic activity, protein modification, and G-protein activity. Partially silencing metalloprotease in tick midguts led to the identification of 182 DEGs, comprising 104 upregulated and 78 downregulated genes. Ablation of endogenous metalloprotease led to the upregulation of several biological and functional pathways associated with RNA splicing and significantly suppressed routes connected with detoxification, protein modification, catalytic activity and molecule binding. These findings provide valuable insights into tick physiology, providing a foundation for further research on tick-host interactions and AGS pathogenesis. Overall design: Total RNA from 3 groups (antigen p23, metalloprotease and GFP control) was submitted to RNA-seq anaysis. Samples included the midguts of nine Ixodes ricinus female ticks per group divided into three biological replicates, each containing three ticks per pool.