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

Abstract

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.

Keywords: RADseq; Y chromosome; black–legged tick; deer tick; preprandial; sex identification.

Fig. 1.

Bioinformatics workflow used to identify male-specific DNA regions of Ixodes scapularis.

Fig. 2.

Dorsal view of an adult Ixodes scapularis female (left); and male (right). Adult females are larger than males. In addition, adult females have a non-sclerotized alloscutum (arrow) that the males lack and paired porose areas at the posterior of the basis capituli (arrowheads). Anterior is up and scale bar represents 0.75 mm.

Fig. 3.

Molecular sexing method (duplex PCR) for Ixodes scapularis visualized on an agarose gel; primers described in Table 1. Autosomal marker primer set (Lanes 1 to 6): female samples have a 406 bp band; male samples have a 406 bp band; and positive control plasmid has no band. Male-specific marker primer set (Lanes 7 to 12): female samples have no band; male samples have a 326 bp band; and positive control plasmid has a 326 bp band. Two markers in duplex (Lanes 13 to 18): female samples have a single 406 bp band; male samples have double bands (406 and 326 bp); and positive control plasmid has a single 326 bp band. Genomic DNA from I. scapularis female (Lanes 1, 2, 7, 8, 13, and 14) and male (Lanes 3, 4, 9, 10, 15, and 16) sourced from the northeastern population at the Centers for Disease Control and Prevention (Lanes 1, 3, 7, 9, 13, and 15) or the southern population at the Oklahoma State University (Lanes 2, 4, 8, 10, 14, and 16). Plasmid (P) positive control (Lanes 5, 11, and 17). No template (NT) negative control (Lanes 6, 12, and 18).

Fig. 4.

Mating status of adult female Ixodes scapularis that were paired with males in the laboratory compared with females that were never paired, using the molecular sexing method (duplex PCR) visualized on an agarose gel. Primers described in Table 1. Eleven females (Lanes 1 to 11), plasmid (P) positive control (Lane 12), and no template (NT) negative control (Lane 13). A) Paired females (without legs) used as template. All samples have the expected single 406 bp band, however, 5 samples have a second band at 326 bp (Lanes 2, 3, 7, 9, and 11). The plasmid positive control has the expected single band at 326 bp. B) Legs of the paired females used as template. All samples have the expected single 406 bp band and the plasmid positive control has the expected single band at 326 bp. C) Unpaired females used as template. All samples have the expected single 406 bp band and the plasmid positive control has the expected single band at 326 bp.

Fig. 5.

Field-collected ticks that potentially have mated. Determination of the mating status of adult female Ixodes scapularis using the molecular sexing method (duplex PCR) visualized on an agarose gel. Primers described in Table 1. A) Off-host samples used as template. All female samples have the expected single 406 bp band, however, 5 samples have a second band at 326 bp (Lanes 1, 3, 4, 5, and 6). The plasmid positive control has the expected single band at 326 bp. Male samples have the expected double band at 406 and 326 bp. Six females (Lanes 1 to 6), plasmid (P) positive control (Lane 7), no template (NT) negative control (Lane 8), and 6 males (Lanes 9 to 14). The positive control plasmid has the expected single band at 326 bp. B) On-host samples used as template. All female samples have the expected single 406 bp band; however, one sample has a second band at 326 bp (Lane 6). Male samples have the expected double band at 406 and 326 bp. The plasmid positive control has the expected single band at 326 bp. Females (Lanes 1, 2, 5, 6, 7) and males (Lanes 3, 4, 8, 9, 10) sourced from bears (Lanes 1 to 4) or from deer (Lanes 5 to 10). Plasmid (P) positive control (Lane 11). NT negative control (Lane 12).