Duplex PCR assay to determine sex and mating status of <i>Ixodes scapularis</i> (Acari: Ixodidae), vector of the Lyme disease pathogen

Ticks are a major health threat to humans and other animals, through direct damage, toxicoses, and transmission of pathogens. An estimated half a million people are treated annually in the United States for Lyme disease, a disease caused by the bite of a black-legged tick (Ixodes scapularis Say, 1821) infected with the bacterial pathogen Borrelia burgdorferi. This tick species also transmits another 6 human-disease causing pathogens, for which vaccines are currently unavailable. While I. scapularis are sexually dimorphic at the adult life stage, DNA sequence differences between male and female I. scapularis that could be used as a sex-specific marker have not yet been established. Here we identify sex-specific DNA sequences for I. scapularis (male heterogametic system with XY), using whole-genome resequencing and restriction site-associated DNA sequencing. Then we identify a male-specific marker that we use as the foundation of a molecular sex identification method (duplex PCR) to differentiate the sex of an I. scapularis tick. In addition, we provide evidence that this molecular sexing method can establish the mating status of adult females that have been mated and inseminated with male-determining sperm. Our molecular tool allows the characterization of mating and sex-specific biology for I. scapularis, a major pathogen vector, which is crucial for a better understanding of their biology and controlling tick populations.