Copy Number Variation-Based Molecular Sexing of Ixodes scapularis and Rhipicephalus microplus Immature Stages Using qPCR and ddPCR Approaches

Accurate sex identification of immature ticks is essential for understanding sex-specific ecological dynamics, pathogen transmission, and reproductive biology. However, tick larvae and nymphs lack morphological sexual dimorphisms, limiting the studies. Here, we report the development and validation of copy number variation (CNV)-based molecular sexing approach for two important hard tick species, Ixodes scapularis, a major public health vector of human pathogens, and Rhipicephalus microplus, a pest responsible for significant economic losses in cattle industry. Using newly published chromosomal-level genome assemblies and whole-genome resequencing data, we identified female-enriched CNVs in two genes, calcium/calmodulin-dependent 3’,5’-cyclic nucleotide phosphodiesterase 1A-like (NPD) and rap guanine nucleotide exchange factor 2-like (RAPGEF2), and developed SYBR-Green based and probe-based qPCR, and droplet digital PCR (ddPCR) assays using DNA extracted non-destructively from a single leg, preserving ticks for continued feeding, molting, and behavioral analysis. In I. scapularis, we were able to assign the sex of 87% of nymphs based on results from at least two molecular tests, and these assignments were confirmed when the nymphs later molted into adults. In R. microplus, two qPCR assays revealed clear, sex-linked differences in gene copy number, despite the species having an XX: XO sex system. Together, these findings indicate that CNV-based markers are a reliable and widely applicable method for sex determination in immature ticks.