Description of the male and the larva of Ixodes collaris Hornok, 2016 with drawings of all stages

Background Ixodes collaris Hornok, 2016 is a recently discovered tick species associated with bats in Asia. This study provides the description of the male and the larva, as well as high quality drawings of all stages. Methods Ticks were collected from cave walls and bats in Phia Oac (Vietnam). DNA was extracted from one individual of each stage/sex, while another was morphometrically analysed. Based on two genetic markers, all ticks were identified as I. collaris. Results The male of I. collaris has long legs (i.e. the length of Haller’s organ exceeds the maximum diameter of tarsus I), unlike the male of I. simplex Neumann, 1906, but similarly to males of I. vespertilionis Koch, 1844 and I. ariadnae Hornok, 2014. The lateral and medial edges of the palpi of male I. collaris are both convexly curved, unlike in I. ariadnae and I. simplex, but similarly to I. vespertilionis. The male of I. collaris has long palpal setae (up to 210 µm), unlike the males of I. ariadnae (30–100 µm) and I. simplex (20–80 µm), but similarly to I. vespertilionis (100–200 µm). Males of I. collaris have sparse distribution of long palpal setae (vs dense in I. vespertilionis) and posteriorly diverging, sclerotized trapezoid ridge dorsally on the basis capituli (posteriorly convergent, U-shaped and less evident in I. vespertilionis). The larva of I. collaris has long legs (unlike the larva of I. simplex, but similarly to I. vespertilionis and I. ariadnae), elongated club-shaped palpi (240 × 70 vs 200 × 90 µm in I. ariadnae, 200 × 70 µm in I. vespertilionis; and 140 × 60 µm in I. simplex:), pentagonal scutum, which is longer than broad (different from I. ariadnae and I. simplex, but similar to that of I. vespertilionis). The larva of I. collaris has strongly concave caudolateral margin of ventral basis with perpendicular angle (vs slightly concave, with obtuse angle in I. vespertilionis) and a prominent, dark sclerotized edge, “collar” (absent in I. vespertilionis). Conclusion Several features allow to distinguish the male and the larva of I. collaris morphologically from those of other bat-associated ixodid tick species.


Background
In a recent Eurasian survey on ixodid ticks infesting bats, high degree of mitochondrial gene heterogeneity of Ixodes vespertilionis Koch, 1844 was reported, postulating that it is actually a species complex, within which hitherto unknown or formerly not distinguished bat tick species might exist [1]. Accordingly, I. collaris Hornok, 2016, has been described, based on material from the intermediate horseshoe bats (Rhinolophus affinis) in Vietnam. However, the description of this new species was based only on the female and the nymph [2], because at that time male bat ticks were not seen on cave walls and larvae could not be collected from the only known host species.
This study provides the description of the male and the larva of I. collaris, in order to complete the description of the species. In addition, because detailed drawings of the female and the nymph of I. collaris have not been

Open Access
Parasites & Vectors *Correspondence: hornok.sandor@univet.hu available, high quality drawings of its all stages are also provided here.

Sample collection
Ixodes sp. ticks (two individuals per stage or sex) were collected at a cave located in the buffer zone of Phia Oac, Phia Den National Park (22.563611N, 105.874167E), Cao Bang Province, Vietnam (i.e. the type-locality of I. collaris) on June 1, 2016 and November 17, 2017. Male ticks were removed from cave walls, whereas females, nymphs and larvae were collected from R. affinis. All specimens were stored in 96% ethanol. After microscopical evaluation of their conspecificity, DNA was extracted from one individual of each stage/sex, while the other was morphometrically analysed and described. Specimens of I. vespertilionis (used for comparison) were collected in Leány cave, Hungary.
Pictures and measurements were made with a VHX-5000 (Keyence Co., Osaka, Japan) digital microscope.

Results
Based on the amplified fragment of their cox1 and 16S rRNA genes, all ticks described here were identified as I. collaris, having 100% sequence identity to formerly reported female and nymph specimens of this tick species (cox1 gene: KR902756, 16S rRNA gene: KR902771). The newly generated sequences were submitted to the GenBank database under the accession

Differential diagnosis
Prior to its discovery, I. collaris may have been misidentified as I. vespertilionis in Southeast Asia (e.g. in [3]). Therefore, the differential diagnosis (see Figs. 1, 2, 4 and 6) focuses on the latter species, as redescribed in [4] and [5]. Apart from data taken from the latter source on larvae of I. simplex Neumann, 1906, the redescription of the male of I. simplex was also taken into account [6], together with measurements (including those of I. ariadnae Hornok, 2014) in [7].
The male of I. collaris has long legs (i.e. the length of Haller's organ exceeds the maximum diameter of tarsus I), unlike the male of I. simplex. The palpi of I. collaris male have convexly curved lateral and medial edges, unlike the males of I. ariadnae (where the lateral edge is straight) and I. simplex (where the lateral edge is bent at an angle). The palpal setae are long (up to 210 µm), unlike in the males of I. ariadnae (30-100 µm) and I. simplex (20-80 µm). The most important features to distinguish males of I. collaris from those of I. vespertilionis are on the gnathosoma: (i) more elongated palpi (length to breadth ratio 2.52 vs 2.0-2.1) (Fig. 1d vs 1c); (ii) the sparse distribution and low number (< 10) of long anterior and lateral palpal setae, which show dense distribution and higher number (> 20) in I. vespertilionis (Fig. 1c); and (iii) the posteriorly diverging (trapezoid), sclerotized ridge dorsally on the basis capituli, which is much less conspicuous and posteriorly converging (U-shaped) in I. vespertilionis (Fig. 1c). In addition, (iv) the idiosoma of male I. collaris is more elongated than the idiosoma of male I. vespertilionis (length to breadth ratio 1.61 vs 1.35, Fig. 1b vs 1a) and (v) ventrally the ivory coloration anterior to the anus is oval, elongated in I. collaris, whereas this is less apparent in I. vespertilionis (Fig. 2c).
The larva of I. collaris has long legs, unlike the larva of I. simplex (in which the legs are short, i.e. the length of Haller's organ does not exceed the maximum diameter of tarsus I). The palpi of I. collaris larva are elongated, club-shaped, with a length to breadth ratio above three (length × width: 240 × 70 µm), unlike in I. vespertilionis (Fig. 6a:  concave, with an obtuse angle, in I. vespertilionis (Fig. 6a); and (ii) at/behind the maximum breadth of ventral basis, the presence of a conspicuous, long, dark sclerotized edge ("collar") also makes I. collaris larvae different from I. vespertilionis larvae, where this structure is less apparent ( Fig. 6b vs 6a). In addition, (iii) caudal setae on the idiosoma are considerably longer than 100 µm in I. collaris larvae, unlike in I. vespertilionis larvae (≤ 100 µm, see Figs. 4a and 6c); and (iv) the medial edge of coxa I is much shorter than that of coxa II in I. vespertilionis larvae (Fig. 6c), as contrasted to I. collaris larvae.

Discussion
With the morphological characters, high resolution pictures and drawings provided in the present study, descriptions of all stages of I. collaris are now complete. Adding to already reported genetic differences between I. vespertilionis, I. collaris, I. ariadnae and I. simplex [1], these ixodid bat tick species show different morphology in all stages, as shown formerly for females and nymphs [2] and here for males and larvae. In summary, short legs distinguish all stages of I. simplex from I. collaris. Ixodes ariadnae has laterally straight and short palpi in all stages, unlike I. collaris. The male of I. vespertilionis has dense long palpal setae (sparse in I. collaris), whereas its females, nymphs and larvae lack the ventrolateral collar of gnathosoma (characteristic of I. collaris).

Conclusions
Based on the descriptions above, several features allow to distinguish the male and the larva of I. collaris morphologically from those of other bat-associated ixodid tick species.