Medicina Veterinaria · 2026

Experimental validation of overexpressed transcripts at different stages of the tick's life cycle by qRT-PCR

Ticks are vectors of several pathogens that pose significant threats to human and animal health,making the study of their biology and genetic expression essential for the design of effective control strategies. This research focuses on the transcriptome of Rhipicephalus sanguineus sensu lato (s.l.),a widely distributed tick species of medical and veterinary importance. The study aimed to validate overexpressed transcripts previously identified by differential expression analysis that were sequenced by Next Generation Sequencing (NGS) using different life stages of R. sanguineus s.l. collected from several regions of Colombia. For this validation, we collected new ticks at different instars to extract the RNA. Afterwards, we employed quantitative reverse transcription polymerase chain reaction (qRT-PCR) using primers previously designed according to the gene sequences for each overexpressed transcript. Absolute quantification through standard curve calibration revealed distinct expression patterns across developmental stages: cuticle protein 65-like exhibited the highest overall expression with peaks in engorged females and newly hatched males (>3×10⁸ copies/ng RNA), while cuticle protein 16.5-like was strongly expressed in partially engorged females. Microplusin expression was particularly elevated in newly hatched males, and vitellogenin-1-like showed moderate expression across both sexes. Amplification specificity was confirmed by gel electrophoresis showing single bands at expected sizes (70–178 bp), and functional interaction analysis using STRING revealed enrichment in biological processes related to lipid transport, nutrient reservoir activity, and antimicrobial defence. By identifying and characterizing these stagespecific gene expression patterns, this work sought to uncover critical molecular mechanisms underlying tick development, reproduction, and pathogen transmission. Furthermore, the study explored the functional annotation of these transcripts through Gene Ontology (GO) analysis, providing insights into their potential roles in key biological processes. The identification of stagespecific genes and their associated protein targets offers a foundation for the development of novel interventions, such as vaccines or acaricides, aimed at disrupting essential tick biological functions. This research not only advances our understanding of tick molecular biology but also will contributes to the next goal controlling tick-borne diseases.