First record of Alectorobius coniceps (Ixodoidea: Argasidae) and Dermacentor sp. (Ixodoidea: Ixodidae) in Pakistan

Alectorobius species are soft ticks primarily infesting birds, such as swallows, while Dermacentor species are hard ticks mainly infesting mammals, such as small ruminants. This study for the first time reported on the morphological and molecular bases of two tick species, namely A. coniceps and a Dermacentor sp. in Pakistan. The former species was examined in swallows’ nests in Khyber Pakhtunkhwa province, while the latter species was examined in small ruminants in Balochistan province. In total, 25 ticks were collected, with 14 ticks morphologically identified as A. coniceps (males = 9 and females = 5) and 11 ticks identified as Dermacentor sp. (males = 7 and females = 4). Following morphological identification, molecular identification was gained by obtaining 16S rDNA and cox1 sequences for these ticks. The BLAST results for the 16S rDNA and cox1 sequences from A. coniceps shared a maximum identity of 97.46% and 96.49% with the same species from Malta. The BLAST analysis of the 16S rDNA and cox1 sequences from Dermacentor sp. showed maximum identities of 98.42% and 97.45% with Dermacentor pavlovskyi from China. The phylogenetic analysis based on 16S rDNA and cox1 of A. coniceps showed a close evolutionary relationship with the same species. The case of Dermacentor sp., based on 16S DNA and cox1, indicated a close evolutionary relationship with Dermacentor pavlovskyi from China.


Introduction
Ticks are arthropods that fall under Arachnida and are further categorized into three families: hard ticks (Ixodidae), soft ticks (Argasidae), and Nuttalliellidae (1,2).With medical significance, they are obligate ectoparasites of semi-terrestrial and terrestrial vertebrates (3)(4)(5).Usually, hard ticks take a single extended blood meal during each of their life stages, while soft ticks consume multiple brief blood meals during the nymphal and adult stages (6)(7)(8).

Study area
This study was carried out in the districts of Quetta (30°08′55.3″N,66°57′42.1″E)and Charsadda (34°10′17.5″N,71°45′31.7″E) in the provinces of Balochistan and Khyber Pakhtunkhwa, Pakistan, respectively.The geocoordinates of the collection areas were determined using the Global Positioning System (GPS), and the study map was designed using ArcGIS v. 10.3.1 (Figure 1).

Tick collection and preservation
Tick specimens were collected from sheep and nests of swift birds in 2023 from the districts of Quetta and Charsadda, respectively.In order to avoid any external damage to the specimens, the ticks were carefully detached from the host body and nests using tweezers.The specimens were rinsed in distilled water followed by 70% ethanol and subsequently preserved in 100% ethanol in 1.5 mL Eppendorf tubes.

DNA extraction and PCR
A total of 14 ticks including five Dermacentor spp.and nine Alectorobius spp.specimens selected for DNA extraction and were dried in an incubator for 30 min.With the use of sterilized scissors and a micro pestle, the specimens were homogenized in 200 μL of phosphate-buffered saline (PBS).The phenol-chloroform method was used to extract the genomic DNA (46), and 30 μL of "nuclease-free" PCR water was utilized to dilute the extracted DNA pellet.The genomic DNA was measured through NanoDrop (Nano-Q, Optizen, Daejeon, South Korea) and kept at −20°C for further experiments.
Conventional PCR (GE-96G, BIOER, Hangzhou, China) was used to amplify partial fragments of mitochondrial 16S rRNA and cox1 from the extracted genomic DNA of ticks (Table 1).Each PCR reaction mixture was performed in a total 25 μL volume-containing 1 μL each primer-forward and reverse (10 μM), 2 μL of genomic DNA template (100 ng/μL), 8.5 μL of "nuclease-free" PCR water, and 12.5 μL of DreamTaq green MasterMix (2x; Thermo Scientific, Waltham, MA, United States).A positive control (DNA of Argas persicus or Hyalomma anatolicum) and a negative control (PCR water that had been "nuclease-free" instead of DNA) were included in each PCR reaction.Lists of the primers used in the present study along with the thermocycler conditions are shown in Table 1.
The products of the PCR were electrophoresed on a 2% agarose gel and observed under ultraviolet light in a Gel Documentation System (RB Flash Digi, Robus Technologies, United Kingdom).The DNA Clean and Concentrator Kit (Zymo Research, Irvine, CA, United States) was used to purify the PCR-positive samples in accordance with the instructions provided by the manufacturer.

DNA sequencing and phylogenetic analysis
All amplified amplicons of cox1 and 16S rDNA partial fragments were sequenced bidirectionally (Macrogen Inc., Seoul, South Korea) using the Sanger sequencing method.The obtained sequences were cropped through SeqMan v. 5 (DNASTAR, Inc., Madison/WI, United States) to remove poor reading sequences and subjected to Basic Local Alignment Search Tool (BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi) at the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/).After BLAST, high identity sequences were downloaded in FASTA format from the NCBI.The obtained sequences were aligned with the downloaded sequences using ClustalW multiple alignments in BioEdit Sequence Alignment Editor v. 7.0.5 (49).The phylogenetic trees were constructed individually for each gene sequence of the tick, using the maximum likelihood method with the Tamura-Nei model in Molecular Evolutionary Genetics Analysis (MEGA-X), with a bootstrapping value of 1,000 (50).The coding sequences were aligned using MUSCLE alignments (51).

Ticks and their geographic origin
A total of 25 tick specimens were collected and morphologically identified into two distinct species, A. coniceps and Dermacentor sp., both of which were found in different areas.These comprised 14 out Map showing the collection sites of ticks during this survey.

Female
They are broadly rounded posteriorly, obtusely angled anteriorly with a small, bluntly subtriangular hood, and the color is light to dark brownish to black.Approximately 10 setae and large central pores are found on the anterior labium, and irregularly divided longitudinal striations are found on the posterior labium.The coxal and supracoxal folds are conspicuous.The transverse part of the pre-anal groove is small.With comparatively larger disks adjacent to it, the posteromedian groove extends from the anus to the paired organ.The posterior paired organ can be found at approximately posterior 1 out of 7 to 1 out of 9 of the body.The anterior valve is small and finely striated in shape, whereas the posterior valve is wider and less finely striated in shape with coxa IV disks at each apex.The spiracular plates are large and positioned laterally to coxa IV.The capitulum in the camerostome is between the coxa I and the hood.Basis capituli ventral surface is pebbled and approximately two times as broad as long.Legs are narrow and long, and surfaces are pebbled.Tarsi are narrow, elongate, and abruptly tapering distally (Figure 2).

Male
Except for sexual characteristics and size, male ticks are similar to female ticks.The integument around the genital operculum is finely and densely pebbled spiculate, and the posterior integument is transversely rugose.Nymphs resembled adults, except for the lack of external genitalia.Base capituli posthypostomal setae extending to the level of midpalpal segment 2 length.Legs have moderate length and humps (Figure 2).

Morphology of Dermacentor sp. Female
The female tick's body is elongated, oval, and brownish-red in color.Legs and capitulum are lighter.Except for punctations and grooves, the scutum is oval and whitish in color.Eyes are in front of the middle lateral border.The genital opening is level on coxae II and III.The spiracles are well developed.The capitulum is long and hairy with slightly developed whitish patterns dorsally.Cornua formed barely tubercles, and porose regions are subcircular.Palps are twice as long as they are wide; article I is small; article II and III are both well developed; and article II has a slight posterodorsal spine, while article IV is small and cylindrical.Coxa I has long, triangular, close-spaced, and parallel spurs with tapering or narrowly rounded tips; the external spur is usually nearly equal to or slightly shorter than the internal; and both the spurs are directed slightly posterolateral.Each part of coxae II and III has a sharp external spur and a smaller, broadly oval internal spur; coxa IV is enlarged and rounded with a narrow triangular external spur with a tapering apex.Legs are ornamented, hairy, and lack spines.The tarsi are slightly raised (Figure 3).

Male
They are oval and brownish-red in color.The scutum is partially curved, and the whole scutum is white except for the grooves and punctations.The cervical grooves are twisted outward, while the marginal grooves are deep.The outside festoons are wider than the inner ones.The punctations can be seen in some short hairs, some of which are dispersed.Eyes are on a level on coxa II.The genital aperture is parallel to coxa II, and the genital grooves extend almost parallel to coxa IV.They have thin and well-developed spiracles.The cornua is tapered to large spines.The palps are twice as long as the hypostome.Articles II and III are well developed, article II has a tiny spine postero-dorsally, article III is triangular dorsally, and article IV is small and cylindrical.Legs are similar to or resemble those of the female (Figure 3).

Molecular analysis
The BLAST analysis of the 16S rDNA sequence belonging to morphologically identified A. coniceps showed 97.46% maximum identity with the same species.In the phylogenetic tree, the 16S rDNA sequence for A. coniceps was clustered with the same species reported from Malta (MK946450) and grouped in a sister clade with Alectorobius capensis (KU946450) and Alectorobius sawaii (MK606017).The BLAST analysis of the 16S rDNA sequence for Dermacentor sp.showed 98.42% maximum identity with Dermacentor The BLAST analysis of the mitochondrial cox1 partial sequence obtained for A. coniceps showed 96.49% maximum identity with the same species.In the phylogenetic tree, the cox1 sequence for A. coniceps clustered with the corresponding sequence was reported from Malta (MK946447).While the BLAST analysis of the obtained cox1 sequence for Dermacentor sp.showed 97.45% maximum identity with D. pavlovskyi followed by 93.85% with D. raskemensis, 92.09% with D. nuttalli, 91.92% with D. sinicus, and 91.39% with D. marginatus, D. silvarum, and D. niveus.In the phylogeny, the cox1 sequence for Dermacentor sp.clustered with the D. pavlovskyi species was reported from China (OK489456) and grouped in a sister clade with D. raskemensis, D. nuttalli, D. sinicus, D. marginatus, and D. silvarum (Figure 5).The obtained cox1 sequences for A. coniceps and Dermacentor sp. were deposited to the GenBank under accession numbers OR660126 and OR643819, respectively.
The sequences were used in the phylogenetic analysis for Alectorobius spp.(Tables 2, 3) and Dermacentor spp.(Tables 4, 5), and their identities with the species of the corresponding genus are shown in Table 2.

Discussion
Ticks of the genus Alectorobius and Dermacentor have medical and economic importance worldwide (20,34,52).However, they are among the most neglected tick genera in the Oriental region, including Pakistan.Importantly, research in Pakistan has considerably focused on the exploration of ticks and tick-borne pathogens in the last halfdecade (4, 5,9,39,[53][54][55][56][57][58][59][60][61][62][63][64][65].This study provides the first morphological and molecular record of A. coniceps and Dermacentor sp. from the Oriental region, including Pakistan. Alectorobius spp.are distributed globally possibly due to their association with birds, facilitating an efficient distribution (6,13,20).Among Alectorobius ticks, A. coniceps has been collected from various locations such as caves, crevices, cliffs, ravines, nests, stables, wells, and lofts in both the Palearctic and Oriental regions (20,21,66).In this study, A. coniceps ticks were collected from swallows' nests in Khyber Pakhtunkhwa, which is located at the junction of the Palearctic and Oriental regions.Moreover, the larval stages of Alectorobius ticks may feed on the same bird species, and therefore, they are relatively well understood from the Palearctic region (13,21,67).With this tendency, the adults and nymphs of A. coniceps were collected in this study.Future studies should also prioritize the investigation of larval stages from the Oriental region, as, to the best of our knowledge, this stage has not been described in the Oriental region.
Dermacentor spp.are believed to have evolved in central Asia, and this region exhibits the highest diversity of Dermacentor spp.(26, 68, 69).Among Dermacentor ticks, D. pavlovskyi has been documented infesting goats and sheep in mountainous regions, as reported in earlier studies (29,31,32).Similarly, the area (Balochistan), where Dermacentor sp., a closely related tick to D. pavlovskyi, was collected in the present study, is situated adjacent to the Palearctic region and has a mountainous terrain with an approximate elevation of 5,500 feet.Other than open areas, D. pavlovskyi ticks have been found on wild animals in nature reserves and national parks.For instance, these ticks were collected by the Republican Tropical Station in 1941-1955 in the Aksu-Dzhabagly nature reserve in Kazakhstan (31).Interestingly, the current study area is located near the renowned national park, "Hazarganji-Chiltan. " It could be assumed that D. pavlovskyi or closely related ticks have an affinity for wild goats and sheep in such protected areas from where they could invade domestic animals.The tendency of larval Dermacentor ticks to infest small animals has resulted in a limited understanding of this stage.Consequently, this study collected adult and nymphal stages of Dermacentor sp.ticks, and no larval stage of this species was collected.
Molecular-based analysis is pivotal for comprehending the debated systematics and taxonomy of tick species, including the genus Alectorobius and the genus Dermacentor (19,37,70,71).Consequently, Maximum likelihood phylogenetic tree based on partial mitochondrial 16S ribosomal DNA sequences for Alectorobius spp.and Dermacentor spp.The 16S rDNA sequence of Argas persicus was used as an outgroup.The levels of bootstrap support (>65%) for phylogenetic groupings are given at each node; the accession numbers are followed by the species names, hosts, and locations (if applicable).The obtained sequences are shown in boldunderlined font.
A. coniceps and Dermacentor sp.ticks of the current study were subjected to molecular-based analysis involving 16S rDNA and cox1 sequences.The analysis revealed that the A. coniceps ticks from this study displayed variations of 2.54 and 3.51% with their respective species from Malta, based on 16S rDNA and cox1 sequences, respectively.Similarly, Dermacentor sp.ticks exhibited variations of 1.68% and 2.58% with D. pavlovskyi from China as determined by 16S rDNA and cox1 sequences, respectively.At present, although ticks having this range of variations are considered a single species (72, 73, 4), Dermacentor sp. could not be validated as D. due to some morphological variations.In contrast to D. pavlovskyi and Dermacentor montanus (28,29), Dermacentor sp.examined in this study lacks prominent spines on their legs.In comparison to female ticks of D. montanus and D. pavlovskyi, Dermacentor sp.exhibited a genital aperture with a more wing-like shape.Furthermore, other morphological differences among these species were observed in punctuations, cervical grooves, and lateral grooves.However, further comprehensive studies such as mitochondrial genome sequencing of these ticks are essential to gain an accurate understanding of their genetic structure.Furthermore, these intraspecific variations can be attributed to various factors, including tick population size, ecology, and geographic isolation (37, 74).In addition to its closest evolutionary relationship with the same species, A. coniceps displayed proximity to A. capensis, confirming their classification into the same species complex (6,13).Although the closest evolutionary relationship of Dermacentor sp. with D. pavlovskyi was observed, however, its Maximum likelihood phylogenetic tree based on partial mitochondrial cox1 sequence for Alectorobius spp.and Dermacentor spp.The cox1 sequence of Argas walkerae was used as an outgroup.The levels of bootstrap support (>65%) for phylogenetic groupings are given at each node; the accession numbers are followed by the species names, hosts, and locations (if applicable).The obtained sequences are shown in bold-underlined font.

Conclusion
This study for the first time presented both morphological and molecular data on poorly known ticks, A. coniceps, and Dermacentor sp., closely related to D. pavlovskyi, from the junction of the Palearctic and Oriental regions in Pakistan.The geographic, morphological, and genetic data of these tick species may aid future studies on tick systematic and taxonomy.Furthermore, the study suggests that the study area, showcasing a combination of traits from two different zoogeographic regions, could harbor a notable diversity of ticks.

FIGURE 2
FIGURE 2 Male (A: dorsal and B: ventral) and female (C: dorsal and D: ventral) views of Alectorobius coniceps collected in this study from nests of swift birds in district Charsadda, KP.

TABLE 1
List of primers that were used to amplify the ticks' targeted DNA.56%; 9 males and 5 females) A. coniceps from three different nests of swift birds in the district of Charsadda, Khyber Pakhtunkhwa and 11 out of 25 (44%; 7 males and 4 females) Dermacentor sp. from sheep in the district of Quetta, Balochistan.

TABLE 5
Obtained cox1 (OR643819; <649 bp) sequence identities with the diversity of Dermacentor species.with other species of the same subgenus, such as D. montanus, could not be verified due to the lack of authentic genetic data in GenBank. closeness