Preplanned Studies: Molecular Identification and Genetic Characterization of Public Health Threatening Ticks — Chongming Island, China, 2021–2022

Ticks pose significant risks to human and animal health due to their capability of transmitting various pathogens, such as viruses, bacteria, and protozoans (1-2). To address this global health issue, the Chongming-based Center for One Health research was established on Chongming Island, China’s third largest island and a crucial component in the ecological preservation of the Yangtze Delta region (3). The presence of ticks, including Haemaphysalis (H.) longicornis and H. flava, was previously confirmed in Dongping Forest Park and Dongtan Park on Chongming Island (4). Nonetheless, the potential risk ticks pose to human and animal populations on the island, along with their evolutionary impacts, remains unexplored.

In the course of 2021 to 2022, we conducted and identified ticks from Chongming Island utilizing 12S rRNA and co1 genes. We not only discovered an elevated prevalence of H. longicornis and H. flava on the island but also identified for the first time two additional tick species — H. doenitzi and H. japonica, both of which have been recognized for pathogenic properties and pose a threat to public health. Genetic diversity and neutrality tests suggested that the tick population was expanding or experiencing genetic hitchhiking. Consequently, there is an urgent need for sustained tick surveillance and targeted research on screening tick-borne pathogens and potential clinical cases to inform public health policies and actions.

In this study, ticks were collected from 18 towns and four protected regions on Chongming Island. Within each town, two to three sites were chosen for sample gathering, which was conducted bimonthly over two consecutive days between the hours of 10:00 AM and 4:00 PM from April 2021 to October 2022 (Figure 1). Each sample collection was performed by three collectors and lasted approximately 60±5 minutes.

Figure 1. 

Locations on Chongming Island where ticks were collected, 2021–2022.

Ticks were selected from two sources: those parasitizing domestic dogs and wild rabbits and free-living ticks collected from vegetation in parks and grasslands. The latter were sourced from areas in proximity to those parasitizing animals or from other external environments using a flag-dragging method.

After collection, ticks were classified as Haemaphysalis species based on their morphological characteristics as determined by microscopic examination. These characteristics include the shape of their prosthetic base, color, capitulum, conscutum, alloscutum, genital aperture, anal groove, anus, and arrangement on the posterior plate (5-6).

However, this study only distinguished between H. flava and H. longicornis, which were the most significantly represented species. Haemaphysalis species generally have eyeless, ciliated palisade, and rectangular basis capitula. H. longicornis is characterized by an abdomen of palpal segment 3 with a long conical spine and coxa II to IV internal spur, slightly larger and extending beyond the posterior (5). H. flava species possess thick and short abdominal spines on the palpal segment 3. Their coxa II to IV are short and triangular, and the scutum is marked with fine, shallow, evenly distributed engraved points (6). In females, the scutum is suborbicular, while in males, it is ovoid.

Microscopic examinations of ticks are depicted in Supplementary Figure S1. Following identification, ticks were each individually preserved at −20 °C in 1.5 mL microcentrifuge tubes until further molecular analysis could be conducted.

Molecular identification and characterization were completed using polymerase chain reaction (PCR) amplification and sequencing of the produced amplicons. Genomic DNAs from a collection of 1,417 ticks were subjected to PCR amplification targeting 12S rRNA and col genes and were subsequently sequenced (7) with primer pairs T1B and T2A and CO1-F and CO1-R (Supplementary Table S1, Supplementary Figure S2). Amplification targeting the 12S rRNA locus proved more successful than that targeting the col locus, with success rates of 1,320/1,417 (93.15%) and 1,085/1,417 (76.57%), respectively. This disparity might be attributed to the limitations encountered in obtaining good-quality sequence reads from the col amplicons.

Analysis of 12S rRNA and co1 amplicons sequenced and verified in GenBank revealed that they predominantly belonged to the Haemaphysalis species, with H. flava constituting the majority at 97.11% (1,376/1,417), followed by H. longicornis at 2.61% (37/1,417), H. doenitzi at 0.21% (3/1,417), and H. japonica at 0.07% (1/1,417) (Figure 2A). When an intraspecific identity comparison based on the 12S rRNA gene was conducted, it was found that the ticks exhibited a high degree of identity with homologs from China and globally, with the exception of H. japonica, which presented an 83.48% degree of homology (Figure 2B).

Figure 2. 

Molecular identification of ticks from Chongming Island (2021–2022) based on the 12S rRNA gene. (A) Distribution of species derived from Chongming Island. (B) Species confirmed through comprehensive sequence analysis, coupled with intraspecies validation, utilizing multiple alignments of the 12S rRNA gene amplicons.

Abbreviation: H.=Haemaphysalis.

Phylogenetic relationships were ascertained using both the neighbor-joining (NJ) and maximum likelihood (ML) methods. These relationships were established based on the correlation with publicly accessible homologous and orthologous sequences of the 12S rRNA and col genetic loci (Supplementary Table S2). From this examination, a dendrogram for 12S rRNA suggested the existence of four main groups. These included H. japonica and H. concinna in the second cluster, H. doenitzi and H. cornigera in the third, and H. longicornis and H. hystricis in the fourth. Conversely, the first cluster distinctly comprised H. flava (Figure 3A). A comparison of the topological structure between the col and 12S rRNA phylogenetic trees yielded similar results (Figure 3B). Phylogenetic relationships derived from the ML method are available in Supplementary Figure S3.

Figure 3. 

Comparison of genetic and geographical relationships between Haemaphysalis species from Chongming Island, 2021–2022, and those on record in GenBank. (A) Neighbor-joining tree based on 12S rRNA sequences. (B) Neighbor-joining tree based on col sequences.

Note: Percentage values on the tree branches denote bootstrapping values from 1,000 replicates. The identification process for each Haemaphysalis species sequence involved its accession number and geographical location (country). The gene sequences relevant to the species identified in this study are highlighted in blue. For the 12S rRNA and col phylogenetic trees, Argas reflexus and Argas persicus were used as outgroups, respectively. The scale bar signifies nucleotide substitutions per site.

Abbreviation: H.=Haemaphysalis.

Genetic divergences were assessed at various taxonomic levels among tick species, utilizing Kimura’s 2-parameter (K2P) distances based on the 12S rRNA locus (Supplementary Table S2). The highest within-species K2P distance was exhibited by H. japonica (0.0946), while the intraspecific distances for both H. cornigera and H. hystricis were zero. The zero intraspecific distance in these cases infers identical genetic sequences among populations within each species, offering potential insights into population history and overall biodiversity origins. The maximum interspecific K2P distance was identified between H. hystricis and H. japonica (0.1960). Conversely, the smallest interspecific distance of 0.0723 was observed between H. cornigera and H. doenitzi, indicating a potential genetic similarity between these species, as also depicted in the phylogenetic tree. An intermediate genetic distance was found between H. hystricis and H. longicornis (0.0900), comparable to the distance observed between H. flava and H. japonica (0.0903), both of which are in alignment with the phylogenetic topology.

The mapping of K2P genetic distances revealed that H. japonica and H. longicornis maintained notable genetic differentiation compared to other tick species on Chongming Island. In particular, H. japonica presented the highest intraspecific and interspecific K2P distances. Its intraspecific distance, notably, exceeded the lowest interspecific distance (between H. doenitzi and H. cornigera) by 0.0946 (Figure 4A). Collectively, distinct interspecific boundaries were evident in more than 96% of the 12S rRNA fragments examined. We conducted genetic diversity indices and neutrality tests for these tick species using the 12S rRNA locus as a reference (Figure 4B). Among the species examined, the greatest number of polymorphic loci was found in H. japonica (n=56), while H. flava reported the lowest number (n=1). In terms of nucleic acid diversity (Pi), H. japonica was the most diverse (0.08333), while H. doenitzi exhibited the highest haplotype diversity (0.714). Neutral tests signified consistent negative values for Fu and Li’s D and Tajima’s D for the four species, yet they reported positive values for H. concinna. These findings suggest that there has been a recent expansion in global populations of H. longicornis, H. doenitzi, H. flava, and H. japonica ticks, including those based in Chongming.

Figure 4. 

Analysis of Kimura’s two-parameter distance, genetic diversity, and neutrality tests for Haemaphysalis ticks collected from Chongming Island, 2021–2022. (A) Plot of the K2P distance of the Haemaphysalis species using NJ-K2P distances. (B) Genetic diversity indices and neutrality tests based on the 12S rRNA gene in the Haemaphysalis species.

Note: The red solid line in panel A represents the minimum interspecific distance between H. cornigera and H. doenitzi.

Abbreviation: n=the number of species; S=the number of polymorphism loci; Pi=nucleic acid diversity; h=haplotype number; Hd=haplotype diversity; H.=Haemaphysalis.