A qPCR assay for sex identification and sex-ratio estimation in Ascaris suum.

Abstract

Natural selection is expected to maintain a stable 1:1 sex ratio in randomly mating populations. However, obligate parasites, especially nematodes with complex host-migratory life cycles, often show pronounced departures from this equilibrium. Ascaris suum, a globally distributed nematode with an XX/XO sex-determination system, exhibits an obvious female-biased sex ratio in adults, making it an ideal model to investigate when and how sex-ratio bias arises. Progress has been limited because, at present, no practicable method exists to identify worm sex at stages other than adulthood. Here, we aimed to establish a valid approach designed to be applicable, in principle, for precise sex identification and sex-ratio estimation across all stages of A. suum. Using a chromosome-level A. suum genome assembled in our laboratory (CNP0003380), we identified single-copy sequences on autosomes and the X chromosome and selected 3C (Chr3) as an autosomal marker and 5X (Chr5X) as an X-linked marker. We developed a qPCR assay that infers sex or sex ratio from the 3C/5X copy-number ratio, with theoretical values of 1 for XX females and 2 for XO males. Validation with 30 adult females and 30 adult males yielded 3C/5X ratios of 1.009 ± 0.042 and 1.995 ± 0.054, respectively, with no significant deviation from theoretical expectations (p > 0.05). The relative abundance of X-bearing to O-bearing sperm measured by qPCR was 0.980 ± 0.093, compared with 1.081 ± 0.163 by flow cytometry. Although the mean values differed significantly between the two methods (paired Students t-test, t = -2.886, d.f. = 29, p = 0.0073), the qPCR-based estimates showed lower variability than those obtained by flow cytometry. These results indicate that this qPCR assay provides an accurate tool for sex identification and quantification of the relative abundance of X-bearing to O-bearing sperm in A. suum.