Adult ticks were collected on vegetation from 8 counties in Haidong city (102°09′-102°47′ E and 36°16′-36°40′ N, average elevation of more than 2,500 m above sea level) of Qinghai Province (89°35–103°04′ E and 31°36′-39°19′N) on the Qinghai-Tibetan Plateau (73°19′-104°47′ E and 26°00′-39°47′ N) in northwestern China from March to April 2021 (Figure 1). All ticks were placed in sterile glass bottles, sent to Qinghai University within one day, and reared in an incubator at 25–30°C and 60–90% humidity.

Microscopy identification of tick species and sex was performed at Qinghai University. After morphological identification (46), 200 males and 200 females of D. nuttalli were selected for laboratory rearing.

To characterize the life-cycle of D. nuttalli, New Zealand white rabbits (specific-pathogen-free, female, 9-weeks-old, purchased from Yifengda, Xi'an, China) housed in the animal facility of Qinghai University at a temperature of 25 ± 2°C and humidity of 40% under controlled lighting (i.e., period of light from 6:00 to 19:00 h) were used to feed the adult ticks. After a few days, engorged ticks were weighed, placed in a sterile glass bottle, and reared in an incubator at 25 ± 2°C and 80% humidity. After tick eggs were laid and hatched, a total of 500 larvae were allowed to refeed on the ears of rabbits within 3–4 days after hatching all eggs. The engorged ticks were collected for incubation until molting as nymphs and then allowed to feed on the rabbit ears again within 12–14 days after molting all larvae. During these periods, the processes of egg laying, hatching, and molting and the viability of the ticks were regularly observed. All animal procedures were carried out according to the ethical guidelines of Qinghai University (2020017 and SL-2022007).

In this study, after morphological identification of the field-collected D. nuttalli, we randomly selected five females and males to extract DNA for molecular identification of ticks. And for the first-laboratory generation, we also randomly selected five males and five females for molecular identification. DNA from the field-collected and first-laboratory generation adult males and females was extracted using the QIAamp DNA Mini Kit (QIAGEN, Germany) according to the manufacturer's manual. The DNA concentration was determined using a NanoDrop 2000 (Thermo Fisher Scientific, USA), and the DNA was stored at−80°C until further use. To further confirm the tick species, PCRs based on the 12S ribosomal RNA (12S rRNA), 16S ribosomal RNA (16S rRNA), cytochrome c oxidase subunit I (COI), and internal transcribed spacer 2 (ITS2) genes were conducted, respectively. All primers are listed in Table 1. The PCR assay volume was 10 μl including 2 μl of DNA, 0.5 μl of each forward and reverse primer (100 μM), 0.2 μl of deoxyribonucleotide triphosphate (200 μM, New England BioLab, USA), 1 μl of 10× ThermoPol Reaction Buffer (New England BioLab, USA), 0.1 μl of Taq polymerase (0.5 U, New England BioLab, USA) and enough double-distilled water to reach a final volume of 10 μl. Double-distilled water was used as a negative control and D. nuttalli DNA stored in our laboratory was used as a positive control.

To identify SFG Rickettsia in the midgut and salivary glands in both field-collected and first-laboratory generation males and females of D. nuttalli in the Qinghai-Tibetan Plateau area, organ DNA from 100 original males, 100 original females, 100 first-laboratory generation males and 116 first-laboratory generation females chosen randomly was extracted and screened using previously established PCR assays based on the citrate synthase gene (gltA). To further confirm SFG Rickettsia species, all samples positive for the gltA gene were tested for the outer membrane protein A (ompA) gene, outer membrane protein B (ompB) gene, and surface cell antigen 4 (sca4) gene. All primers for the four genes are listed in Table 1. The PCRs were performed as described above.

For the confirmation of ticks and detected pathogens, at least one positive sample of each tissue per sex was selected for sequencing and molecular characterization. PCR products of the positive samples were purified using a QIAquick Gel Extraction Kit (QIAGEN, Germany). Purified PCR products was cloned into E. coli DH5α using the PMDTM 19-T Vector Cloning Kit (TaKaRa, Japan). At least three positive clones were sequenced by Sangon Biotech (Shanghai) Co., Ltd. The obtained sequences were confirmed by a BLASTn search in GenBank.

Phylogenetic trees were constructed using the maximum likelihood statistical method and bootstrap analysis with 500 replications in MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for larger datasets (51).

The chi-squared test was used to compare proportions of detected sample positivity in different regions and among different animals via Prism 8 software. Observed differences were considered to be statistically significant when the resulting P-values were <0.05.

In this study, D. nuttalli ticks were preliminarily morphologically identified. As shown in Figure 2, the dorsal view of the female D. nuttalli is about 5.1 × 3.0 mm with an elliptical scutum of the specimen, which only occupies a small part of the front of the body (Figure 2A). And the genital pore of the female has a wing-like protrusion (Figure 2B). The male D. nuttalli is about 5.8 × 4.0 mm. Its scutum on the back almost covers the entire back of the body and has a lighter enamel color on its back (Figure 2C). In the ventral view of male D. nuttalli, the paraproct is very obvious (Figure 2D). Moreover, the capitulum of the male and female D. nuttalli is rectangular and the overall color is dark brown. The anal groove presents an inverted “U” shape in front of the anus.

Furthermore, the species identify was confirmed by molecular analysis of the PCR assay based on the 12S rRNA, 16S rRNA, COI, and ITS2 genes. The sequencing of positive samples showed that the length of the obtained tick sequences varied from 386 to 1,210 bp (Table 2). The BLASTn analysis of two D. nuttalli 12S rRNA sequences in this study showed identities ranging from 98.7 to 99.5% with reference sequences in China from GenBank (Table 2). The 16S rRNA sequences of D. nuttalli obtained in this study showed 99.8% identity with each other and 98.2–99.0% identity with the sequences from Dermacentor spp. in China. Sequence analysis revealed that the nucleotide sequence of COI in this study shared 99.0 to 99.7% identity with Dermacentor spp. sequences from China and 99.0–99.7% identity with Dermacentor spp. sequences from other countries from GenBank. Moreover, the BLASTn analysis of the ITS2 gene revealed that the three sequences obtained in this study had 98.7 to 99.2% identity with each other, and 97.5–99.2% identity with the sequences of Dermacentor spp. from China. Additionally, the phylogenetic analysis of Dermacentor spp. based on the 12S rRNA, 16S rRNA, COI, and ITS2 genes revealed that tick sequences obtained from this study grouped with the D. nuttalli clades (Figure 3).

For laboratory-rearing of D. nuttalli and detection of Rickettsia colonization in first-laboratory generation D. nuttalli, 200 D. nuttalli males and 200 females were randomly chosen to generate next-generation adults by blood-biting in rabbits and rearing in an incubator at 25 ± 2°C and 80% humidity. The results showed that the first laboratory-generation of adults was completed within an average of 86.1 days (Figure 4; Table 3). The average weight of engorged females was 656.0 mg, which was 74.5 times the weight of unfed females (Table 4). No significant difference in average weight was found between field-collected and first-laboratory generation female adults, while the first-generation males produced in the laboratory weighed 2 mg more than the original male adults (Table 4).

In this study, 100 field-collected D. nuttalli males and 100 females collected were chosen randomly to determine Rickettsia colonization. For first laboratory-generation ticks, 100 males and 116 females were screened by randomly selecting one or two males or females for each female mother tick. SFG Rickettsia in the midgut and salivary glands of the adult D. nuttalli males and females was determined using a PCR assay based on the gltA gene. As shown in Table 5, high colonization rates of Rickettsia were found in both the midgut and the salivary glands of both field-collected D. nuttalli males and females, and no positivity was found in single tissue colonization. However, low rates (4.0–6.0%) of Rickettsia colonization in the midgut or salivary glands of first-laboratory generation D. nuttalli were detected (Table 5). The positivity rates of SFG Rickettsia in the same generation of ticks showed no significant difference depending on either sex and organ.

All positive samples for the gltA gene were confirmed by PCR of the ompA, ompB and sca4 genes, and the GenBank numbers of these sequences with lengths ranging from 209 to 813 bp are listed in Table 6. BLASTn analysis of the four genes showed that the Rickettsia spp. detected in this study had 98.6 to 100% identities with either R. slovaca or R. raoultii sequences from Dermacentor spp. from China. The phylogenetic analysis of Rickettsia sequences based on the gltA, ompA, ompB and sca4 genes revealed that the sequences obtained from D. nuttalli in this study grouped with the R. slovaca and R. raoultii clades of isolates from Dermacentor spp., Homo sapiens, animals and other tick species from China, Russia, France, Pakistan, the United States, the Netherlands, Turkey, Romania, Austria, and Italy (Figure 5).