Argas vulgaris (Filippova, 1961)

Wang, Yi-Fei, Zheng, Jia-Jing, Zhang, Ming-Zhu, Du, Li-Feng, Cui, Xiao-Ming, Han, Xiao-Yu, Tian, Di, Cheng, Nuo, Wang, Ning, Gao, Wan-Ying, Wang, Bai-Hui, Shi, Xiao-Yu, Jiang, Jia-Fu, Jia, Na, Sun, Yi, Shi, Wenqiang & Cao, Wu-Chun, 2024, The complete mitogenome of Argas vulgaris (Filippova, 1961) and its phylogenetic status in subgenus Argas (Acari: Argasidae), International Journal for Parasitology: Parasites and Wildlife 23, pp. 100912-100912 : 100912-

3.1. Molecular-phylogenetic relationships of Ar. vulgaris

We obtained the first representative complete mitochondrial genome sequence of Ar. vulgaris through short-read sequencing and assembly ( Fig. 1 ). The complete genome of Ar. vulgaris was 14,479 bp in length. Additionally, the circular and double-stranded DNA structure of the mitochondrial genomes of this species was successfully predicted. The gene arrangement of the mitochondrial genomes included 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, and 2 ribosomal RNA (rRNA) genes, similar to other argasid ticks ( Burger et al., 2014). Four of the 13 PCGs (ND5, ND1, ND4L, and ND4), nine tRNAs (trnF, trnC, trnQ, trnP, trnH, trnV, trnY, trnL2, and trnL1) and two rRNAs (16 S rRNA and 12 S rRNA) were coded on the light strand (L-strand), while the other 22 genes (trnI, trnT, trnS2, trnD, trnM, trnG, trnW, trnK, trnA, trnR, trnN, trnE, trnS1, ATP8, ATP6, ND6, CYTB, COIII, ND3, ND2, COI, and COII) were transcribed on the heavy strand (H-strand). The overall nucleotide composition of the complete mitochondrial genome was listed in Table 1. The nucleotide sequence of the Ar. vulgaris mitochondrial genome included approximately adenine = 5600 (38.67%), thymine = 4850 (33.50%), guanine = 1338 (9.24%), and cytosine = 2691 (18.59%). The proportion of adenine + thymine (72.17%) was substantially higher than that of guanine + cytosine (27.83%). GC skew ranged from – 0.633 (for ND4L) to – 0.15 (for COI), while AT skew ranged from – 0.125 (for ND2) to 0.323 (for ND1). The whole AT skew and GC skew in the entire mitochondrial genome of Ar. vulgaris was 0.072 and – 0.336, respectively.

Phylogenetic analysis was then performed with available sequences of other argasid species deposited in GenBank. In terms of phylogenetic relationships within the genus Argas , the subgenus Persicargas was intricately nested within the subgenus Argas as reported in previous studies ( Mans et al., 2019; Estrada-Pena ˜et al., 2010), albeit with low bootstrap support value ( Figs. 2–4 ). Among species from these two subgenera, a range of 69.96–70.83% nucleotide identity was observed in the complete mitogenomes, while the 16 S rRNA gene exhibited identities of 69.23–87.78%, and the COI gene showed identities of 75.92–79.67% (Supplementary Tables 1 –3). Morphologically, the two subgenera were diagnosed by the combination of the absence or presence of postpalpal setae and the shape of marginal cells.

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Abbreviations: PCG, protein-coding gene; rRNA, ribosomal RNA; tRNA, transport RNA.

Notably, a closely related species of Ar. vulgaris was identified in the phylogenetic analysis. In the phylogenetic tree constructed from complete mitogenomes ( Fig. 2 ), the sequence of Ar. vulgaris obtained in this study clustered with the sequence of Ar. japonicus (MT371799), showing a nucleotide identity of 98.56% (Supplementary Table 1). Based on the 16 S rRNA sequences ( Fig. 3 ), Ar. vulgaris in this study clustered with sequences of both Ar. japonicus and Ar. vulgaris from GenBank. This cluster could be divided into two clades. In the first clade, the 16 S rRNA

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of Ar. vulgaris obtained in this study displayed 100% similarity with Ar. japonicus Aj-A from Japan (AB819156.1) and 99.51% with Ar. japonicus Aj-C (MH782636.1). In the second clade, the 16 S rRNA of Ar. vulgaris from the Kyrgyzstan ( Dabert et al., 1999) (AVAF001404.1) in GenBank and Ar. japonicus Aj-B from Japan (AB819157.1) were grouped, showing 100% identity to each other (Supplementary Table 2). The phylogeny based on the COI gene was concordant with the topology based on 16 S rRNA. The COI gene sequence of Ar. vulgaris clustered with Ar. japonicus

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( Fig. 4 ), and the two sequences showed 98.69% nucleotide identity with each other (Supplementary Table 3). These results suggest that Ar. vulgaris and Ar. japonicus cannot be distinguished merely based on sequences of 16 S rRNA, COI and even the whole mitogenome.

3.2. Diagnostic characters of Ar. vulgaris and notes with Argas species relevant

The ticks collected in this study were morphologically recognized as Ar. vulgaris ( Fig. 5a and b ) according to the following features, including the invisible postpalpal setae ( Fig. 5c ), marginal cell fenced with a narrow and long rugose ridge ( Fig. 5d ), and the anterior lip of genital aperture without setae ( Fig. 5e ) ( Sun et al., 2019). In addition, the anal of Ar. vulgaris was situated posteriorly, far from the middle level of the body ( Fig. 5f ).

We further compared the morphological traits of Ar. vulgaris to that of Ar. japonicus , which was a closely related species to Ar. vulgaris based on the phylogenetic analysis, revealing several distinctive differences between the two species. Ar. japonicus exhibited numerous short frayed setae on the anterior lip (Yamaguti et al., 1968), whereas the setae of Ar. vulgaris were located on the posterior lip ( Fig. 5e ). Meanwhile, the basic capitula wide/long ratio as well as the positions of the genital aperture and anus of these two species were also different. Additionally, Ar. japonicus was rectangular with a wide/long ratio of 1.7 ( Hu et al., 2021), while Ar. vulgaris in this study had a partial round shape with a wide/- long ratio of 1.5 ( Fig. 5g ). The posterolateral setae on the basis capitula of Ar. vulgaris exceeded 6 pairs, whereas 3–4 pairs were observed in Ar. japonicus (Yamaguti et al., 1968) . The genital aperture and anus of Ar. vulgaris situated closer to the anterior margin of the body compared to Ar. japonicus ( Hu et al., 2021) . In terms of the palpal segment, Ar. vulgaris exhibited a decreased length from segments I to IV, with segments III and IV being nearly equal in length ( Fig. 5f ). The ossification ring at the base of the marginal sensilla was either smaller than or equal to that of the bell-shaped sensilla ( Teng, 1983). Furthermore, in Ar. japonicus , segments I and II were of equal length, shorter than segments III and IV, which were also equal in length. The ossification ring at the base of the

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body-marginal sensilla of Ar. japonicus was larger than that of the bell-shaped sensilla ( Sun et al., 2019). The terminal portion of Tarsus I in Ar. vulgaris exhibited a sloping gradient, whereas in Ar. japonicus , it was curved apically on a fleshy projection at its end (Yamaguti et al., 1968). However, other distinguishing features such as the shortness of eyes and arrangement of anal setae were same in both species. Besides, the postpalpal setae in both Ar. japonicus and Ar. vulgaris were invisible on ventral view of basic capitula (Yamaguti et al., 1968) ( Table 2).

Ar. assimilis was similar to Ar. japonicus , in that the integumental ridges were relatively narrower and markedly raised (integumental ridges were thick and not markedly raised in Ar. japonicus ); peripheral integumental ridges were narrower and elongated and regularly arranged (peripheral ridges are thick and short, irregularly arranged in Ar. japonicus ); the hypostome of females extending to the mid-length of palpal article 3rd (extended to mid-length of palpal article 2nd in Ar. japonicus ); article 3rd was shorter than article 4th (article 3rd is equal to article 4th in Ar. japonicus ); the basic capitula of Ar. assimilis was rectangular with a wide/long ratio of 1.8, and the anterior lip of the genital aperture with setae ( Teng, 1983; Teng and Song, 1983). Ar. beijingensis also closely resembled Ar. vulgaris , but could be distinguished by the combination of the following characteristics. The anus of adults was slightly posterior to the center of the venter (much more separated from the middle of the ventral body surface in Ar. vulgaris ), peripheral integumental ridges were short and sinuous (relatively narrower and longer in Ar. vulgaris Fig. 5d ) ( Chen and Liu, 2022), the dental formula was 2|2 for the first three rows and approximately 3|3 backward (2| 2 type of hypostome dental formula, and each row has 7 to 9 teeth in Ar. vulgaris Fig. 5h ). The palpal segments were cylindrical, widest in segment I, narrowed markedly in segment II, and progressively narrower in segments III and IV. The basic capitula of Ar. beijingensis was rectangular with a wide/long ratio of 2, and the anterior lip of the genital aperture had setae ( Teng, 1983).

Figures

Fig. 1. The assembled mitogenome of Argas vulgaris. All the annotated genes are plotted in the outer circle and the inner tract shows the GC content.

Fig. 2. Evolutionary relationships among ticks of the Argasidae families. The phylogenetic tree of complete sequences of mitochondrial genomes. Numbers at the nodes are bootstrap values of the ML analysis. The GenBank accession numbers are listed after the species names.

Fig. 3. Phylogenetic tree of Argas species based on the 16S rRNA gene contained in the mitochondrial genome. Numbers at the nodes are bootstrap values of the ML analysis. The GenBank accession numbers are listed after the species names.

Fig. 4. Phylogenetic tree of Argas species based on the COI gene contained in the mitochondrial genome. Numbers at the nodes are bootstrap values of the ML analysis. The GenBank accession numbers are listed after the species names.

Fig. 5. Key morphological characters of female Ar. vulgaris. (a) Dorsal view of female Ar. vulgaris. (b) Ventral view of female Ar. vulgaris. (c) Posthypostomal seta, ventral view. (d) Details structure of the marginal cells between the dorsal and ventral surfaces. (e) Genital aperture of female Ar. vulgaris. (f) Ventral view of anus location. (g) Ventral view of capitulum. (h) Ventral view of hypostome of the dentition formula.