National Distribution of Bisexual and Parthenogenetic Haemaphysalis longicornis of Japan, and a Real-Time PCR-Based Method to Distinguish the Two Reproductive Groups.

The Asian longhorned tick, Haemaphysalis longicornis, is a well-known vector of several zoonoses including SFTS and Japanese spotted fever. This species consists of two different reproductive forms, bisexual and parthenogenetic, which are suggested to have differential roles in disease transmission. In Japan, the distributions of these forms have been suspected from sex bias in each population. Meanwhile, recent insights about the origin of the parthenogenetic form suggest the possibility of individual-level diagnosis using mitochondrial phylogeny. To reassess the current distribution of those reproductive forms and potential changes over time in Japan, 1750 H. longicornis collected across 23 prefectures of Japan were investigated. The COI barcoding region was used to distinguish the mitochondrial haplogroup of each individual. Additionally, we developed real-time PCR probes to distinguish the two mitochondrial haplogroups representing each reproductive form. As previous studies have suggested, the parthenogenetic mitochondrial haplogroup was present throughout the country, while the bisexual mitochondrial haplogroup was found only in central and western Japan. Interestingly, populations consisting of either one or both haplogroups were found in neighboring areas, highlighting the need for more detailed and fine-scaled investigations in order to fully reveal the distribution of the reproductive groups. The newly developed real-time PCR method successfully differentiated all tested haplotypes. Furthermore, alignment of 173 COI haplotypes revealed that the diagnostic nucleotide selected for the method was consistent across all haplotypes, including those outside of Japan. This suggests that this method can accurately distinguish between the two haplogroups of H. longicornis in both Japan and other countries. This one-step method will serve as a rapid tool to distinguish bisexual and parthenogenetic H. longicornis, making further investigations, particularly into their role in disease transmission and shifts in distributions, possible.