Reticulinasus salahi (Acarina: Argasidae), a tick of bats and man in the Palaearctic and Afrotropics: review of records with the first pathogens detected

The soft ticks of the genus Reticulinasus Schulze, 1941 (family Argasidae Koch, 1844) are ectoparasites of the fruit bats of the Old World (Pteropodidae). Reticulinasus salahi (Hoogstraal, 1953) is the only representative of this genus that occurs in the western part of the Palaearctic. This unusual distribution reflects the distributon range of its primary host, Rousettus aegyptiacus (Geoffroy, 1810). In this contribution, we present a revised review of records of this tick that were made in two periods, 1951–1966 (records from Egypt, Israel, Jordan, Spain) and 2005–2019 (Cyprus, Iran, Oman), and additionally, we present notes, re-determinations, new records, and summary of hosts of this tick. Besides the primary host, the revised list of hosts comprises two bats (Taphozous perforatus Geoffroy, 1818, Otonycteris hemprichii Peters, 1859) and the human (Homo sapiens Linnaeus, 1758). We also tried to identify pathogens in specimens of this tick collected from R. aegyptiacus in Oman. The DNA of the Mouse herpesvirus strain 68 (MHV-68), of two bacteria, Borellia burgdorferii sensu lato, and Ehrlichia sp. almost identical (98%) with Candidatus Ehrlichia shimanensis was detected in several larvae specimens.


Introduction
In accordance with a recent revision of the Argasidae ticks based on molecular genetics, the subgenus Reticulinasus Schulze, 1941 has to be raised to the genus level (Mans et al. 2021). This genus represents soft ticks parasitizing on the fruit bats (Pteropodidae) of the Old World, and only one species of this genus extends by its distribution to the Palaearctic, the Salah's Egyptian fruit-bat tampan, Reticulinasus salahi ). This range pattern is a direct consequence of the range extent of its primary host, the Egyptian fruit bat, Rousettus aegyptiacus (Geoffroy, 1810); the Palaearctic populations of this bat represent the only geographical offshoot of the fruit bat family (Pteropodidae) out of the tropics.
Records of this tick are rather scarce and most of them were made in the period of 1951-1966 with an additional finding in 2009 (Estrada-Peña et al.1989;Theodor and Costa 1960;Saliba et al. 1990;Benda et al. 2010). The evidence was reviewed by Sándor et al. (2021), who mapped a distribution range stretching from Spain to the Levant and Egypt. The latter authors also suggested a possible distribution extent of the tick based on the known range of its primary host Section Editor: Van Lun Low and added a full list of the recorded hosts besides R. aegyptiacus, i.e. Eptesicus serotinus (Schreber, 1774), Taphozopus perforatus Geoffroy, 1818, andHomo sapiens Linnaeus, 1758. Besides the primary parasitation of the Egyptian fruit bat, R. salahi has been evidenced to be a secondary parasite of humans (cf. Hoogstraal 1953;Lavoipierre and Riek 1955); despite this, only marginal attention has been paid to the distribution and ecology of this thick as well as its potential as a vector of pathogens. In the 1950s, only few attempts were made to find spirochaetes and/or salmonellas; however, these surveys failed in finding any of these pathogens Floyd and Hoogstraal 1956). In other bat ticks of the western Palaearctic and Afrotropics, of the genera Carios Latreille, 1796 and Secretargas Hoogstraal, 1957, the presence of pathogens is enormous. More than twenty species of bacteria and piroplasmids (namely, of the genera Rickettsia Da Rocha-Lima, 1916, Coxiella Philip, 1948, Anaplasma Theiler, 1910/Ehrlichia Moshkovski, 1945, Bartonella Strong, Tyzzer, Brues et Sellards, 1915, Borrelia Swellengrebel, 1907, and Babesia Starcovici, 1893 were reported to be found in two argasid tick species parasitizing bats, which distribution range overlap with the range of R. salahi (Sándor et al. 2021).
Taking the aim of a revision of the current status of R. salahi, we complemented the review by Sándor et al. (2021) with new and/or revised data from the Middle East collected in 2005-2019. A small part of the newly obtained materials was subjected to a survey of bacteria and piroplasmids of the abovementioned genera. Additionally, a possibility of presence of the Mouse herpesvirus strain 68 (MHV-68), recently confirmed in bats of Europe and Central America (Briestenská et al. 2018;Janíková et al. 2020), was tested.

Study area and materials
The study area covers the distribution range of the Egyptian fruit bat, Rousettus aegyptiacus, in the Middle East and north-eastern Africa, at the transition area of two zoogeographic regions, the Palaearctic and Afrotropics. Politically, it comprises wholes or parts of the following countries: Turkey, Cyprus, Syria, Lebanon, Jordan, Egypt, Sudan, Yemen, Oman, United Arab Emirates, and Iran. In these countries, the fruit bats were caught by standard methods, using mist or hand nets. All the body parts of the captured fruit bats (pelage, face, ears, wing membranes) were examined for the parasite presence, and all the ticks found were removed directly in the field by using tweezers and preserved in 96% ethanol. Some additional tick specimens were obtained secondarily, by examinations of the host specimens deposited in the collection of the National Museum, Prague, Czech Republic (NMP). In addition, one record from the Cave at the Sâsân Springat Bishapur, Iran, was realized by a random collection from the cave bottom under the fruit bat colony. Five specimens of Reticulinasus salahi were selected for a detailed examination and mounted onto slides using the Swan's embedding medium (Swan 1936).

Literature sources
All published records of Reticulinasus salahi were summarized, starting in 1953 when the species was described , till mid-2022 (Table 2). Taxonomy and nomenclature of Reticulinasus salahi follow the revision of the family Argasidae by Mans et al. (2021).

Images
The images of the larvae specimens of Reticulinasus salahi from Oman (Fig. 1a, b) were taken by an Olympus XC30 digital camera installed on the Nikon E600 light microscope, where the bright-field and interference contrasts (Nomarsky-DIC) were applied. For processing the photos, analysis Docu v. 5.1 and Corel Photopaint X5 were employed. The photos of an adult female of R. salahi from Iran ( Fig. 2a, b) were made using a Canon EOS 30D digital camera and Canon MP-E 65/2.8 Macro lens in multiple layers, stacked using Helicon Focus and edited in Corel Photopaint X3.

Depositories
The list of depositories containing specimens of Reticulinasus salahi species is included. The following abbreviations are used: BMNH, Natural History Museum (formerly, British Museum of Natural History), London, UK; FNHM, Field Museum of Natural History (formerly, Chicago Natural History Museum), Chicago, USA; CMŠ, private collection of Martin Ševčík, Nitra, Slovak Republic; CRBH, collection of Dr. R. B. Heisch, Nairobi, Kenya; DVSO, Division of Veterinary Services, Onderstepoort,

Pathogen screening and phylogenetic analyses
Selected tick specimens (Table 1) were washed with fresh 70% ethanol, then with sterile water, dried, transferred individually to tubes, and crushed with a sterile Carbon Steel Surgical Scalpel Blade (Surgeon, JAI Surgicals Ltd., India). The DNA from the samples was isolated using QIAamp DNA Mini Kit (Qiagen, Germany) according to the manufacturer's instructions. The concentration and purity of the DNA were measured by NanoPhotometer Pearl (Implen, Germany). The DNA samples were stored at −20 °C and later used as templates for the PCR amplifications. Tick samples were screened by PCR-based methods for the presence of the MHV-68 virus, bacteria Rickettsia spp., Anaplasma/Ehrlichia spp., Borrelia burgdorferi sensu lato, Bartonella spp., and piroplasms Babesia spp. (Table 3). The PCR amplicons were purified and analyzed by sequencing in both directions in Macrogen Inc. (Amsterdam, The Netherlands). The DNA sequences were compared with those available in GenBank using the Basic Local Alignment Search Tool (Blast) (http:// blast. ncbi. nlm. nih. gov). The new sequence generated in this study was submitted to GenBank under accession number OQ466707. Hoogstraal (1953: 256) reported the first record of Reticulinasus salahi from Israel; he mentioned the specimens collected in Jerusalem by O. Theodor. An additional locality of this tick from Israel was mentioned by Theodor and Costa (1960: 381), who found it on Rousettus aegyptiacus "in a cave in Herzliah together with specimens of Ornithodorus tholozani in 1951." However, Theodor and Costa (1960: 381) also commented Hoogstraal's (1953) report as follows: Table 1 New and corrected published records of Reticulinasus salahi arranged by country and date of collection Explanations: *redetermination specimen, first record from country; **new and first record from the country; † the released bats are not mentioned; 1 3 specimens from this collection used in pathogens study; 2 2 specimens from this collection used in pathogens study; 3 1 specimen from this collection used in pathogens study  Benda et al. (2010) "Hoogstraal, in a footnote in his paper on O. salahi [= Reticulinasus salahi] mentions that it has been found in Jerusalem. This is not correct, the only locality in which it has been found so far is Herzliah in the coastal plain. This mistake has been taken over by Leeson (1955Leeson ( [= 1956]) in his second paper on the distribution of species of Ornithodorus." A similar situation appeared, when Hoogstraal (1962: 185) discussed the distribution of the genus Reticulinasus in Lebanon as follows: "Members of this subgenus are […] [Ornithodoros (Reticulinasus)] salahi Hoogstraal of Egypt, Lebanon, and Palestine." Nevertheless, we did not find any record of this tick from Lebanon (for a review, see Benda et al. 2016). Thus, we are unsure whether this note refers to an unpublished record or represents just an assumption based on the range of its primary host, R. aegyptiacus, in the Middle East. Benda et al. (2012: 530) reported a single record of Ornithodoros sp. from Iran originally published with the following note: "An adult female of the tick Ornithodoros sp. was sampled from the bottom of the cave at the Sasan spring at Bishapur (Fars) where colonies of Rousettus aegyptiacus, Rhinopoma microphyllum, R. muscatellum, Myotis blythii, and Miniopterus pallidus were found." We examined the specimen in detail and clearly identified it as a nymph of Reticulinasus salahi (Fig. 2a, b).

Comments on records
On the other hand, we regard the reported finding of R. salahi from Eptesicus serotinus in Prat de Llobregat (Barcelona), Spain (Estrada-Peña et al. 1989, also in Cordero del Campillo et al. 1994, as doubtful, requiring a revision. The authors mentioned the deposition of the specimen at the Parasitology Unit of the Faculty of Veterinary Medicine of Zaragoza; however, we were not able to obtain it for a revision. Regarding the geographic distance to other record sites and the reported host species, we consider the species identification of the tick as erroneous (at least temporarily), until other records supporting such geographically and ecologically extraordinary findings are available. Estrada-Peña et al. (1989) considered this record unusual and accidental. In the western part of the Mediterranean, the bat species of the genus Eptesicus are primary hosts of other tick species, Secretargas  transgariepinus (Beaucournu and Clerc 1968;Médard et al. 1997; own unpublished data from Morocco). Finally, Saliba et al. (1990: 164) reported a record of Reticulinasus salahi (as Ornithodoros salahi) from Jordan being found on "Myotis sp., […] Azraq-Shishan, 2.v.1966." However, the bat specimens originally identified as Myotis sp., collected in May 1966 in Azraq-Shishan, represent in fact Otonycteris hemprichii Peters, 1859 (see Atallah (1967) and Benda et al. (2010)).
Newly collected specimens of Reticulinasus salahi were found on Rousettus aegyptiacus examined in two countries of the Middle East, Cyprus, and Oman (Table 1, Fig. 1a, b).
The morphologic characters by Dumbleton (1959) and Sonenshine et al. (1966) mentioned as additional for description and identification of the larvae of R. salahi, i.e., the shape of dorsal plate and/or capsule of Haller's organ, are very variable in the Omani specimens (Fig. 1b).

Pathogens
DNA samples were screened for the presence of MHV-68 virus by nested PCR targeting the ORF50. Of the six analyzed samples (3 larvae from Al Hoota cave, 2 larvae from Ain Sahnawt and 1 larva from Wadi Hannah, Oman; Table 1) of the genomic DNA isolated from the larvae of Reticulinasus salahi, the presence of the ORF50 sequence of the MHV-68 virus was found in one sample (Al Hotta Cave, Oman). The obtained sequence of the ORF 50 nested PCR fragment showed 100% homology (position from 68,219 to 68,799 nucleotides) only with the major lytic transactivator protein, which is specific for this virus, and 85% homology with wood mouse herpesvirus. In addition, the temperature profile of the PCR reaction was designed in such a way that amplification of partially homologous sequences does not occur. The presence of the MHV-68 strain has been documented for the first time in this part of the Middle East.
On the other hand, all the analyzed samples of Reticulinasus salahi larvae were negative for the presence of the DNA of Rickettsia, Bartonella, and Babesia spp. One tick sample (Al Hotta Cave, Oman) was positive for Borellia burgdorferi by real-time PCR. However, since the ct value was > 36, it was not successfully sequenced. The DNA extract of the R. salahi larva collected from Rousettus aegyptiacus in Wadi Hannah (Oman) was PCR positive for the presence of the Anaplasma/Ehrlichia 16S rRNA. In this sample, the sequences (GeneBank Accession Number OQ466707) were identical for 97.8% to the Candidatus Ehrlichia shimanensis (GeneBank Accession Number AB074459).

Discussion
Specimens of the tick Reticulinasus salahi were collected in two separate periods. In 1951-1966, the species was described and the first data on its natural history were gathered. Several species of hosts were documented at that time: Rousettus aegyptiacus, Eptesicus serotinus, Taphozous perforatus, Myotis sp. [= Otonycteris hemprichii], and Homo sapiens. The records were made mainly in Egypt and additionally also in Israel, Jordan, and Spain Theodor and Costa 1960;Estrada-Peña et al. 1989;Saliba et al. 1990;Benda et al. 2010). In 2005-2019, new records are available only from one host species, Rousettus aegyptiacus. The latter records findings come from specialized trips organized to investigate the bat fauna of the Middle East (Benda et al. 2007(Benda et al. , 2010(Benda et al. , 2012 (Fig. 3). However, the records of R. salahi from the northern Levant (Israel and Lebanon) remain uncertain. On the other hand, we identified a finding from Iran, which represents new extension of the species distribution range. The westernmost record of R. salahi, reported from Eptesicus serotinus collected in Spain (Estrada-Peña et al. 1989), does not conform to other records considering the host species as well as the distribution area. We suggest to consider it as dubious until it is revised and the species identification confirmed without doubts. Collections of ticks in free habitats (off the hosts) and checks the tick presence in various habitats still miss, despite the records made in recent time. Considering the primary host range, the available records of R. salahi come from just a fragment of the expected distribution range. Based on his personal records,  regarded R. salahi to be by far the most common tick parasite of bats in the downtown of Cairo; its density and abundance thus could be very high. A factor influencing the occurrence of R. salahi could be the size colony of the Egyptian fruit bats, the primary host. Already, Hoogstraal (1953: 260) reported that in the course of 2 years, he searched for this tick in tens of potential roosts (caves and artificial spaces) in the Cairo region and surrounding areas of Lower Egypt, but he found of R. salahi only in the proper area of Cairo in three sites (Mohammed Ali Mosque in the Citadel, Sultan Hassan Mosque, Fom el Khalig). In all cases, the sites of findings were roosts of very large colonies of R. aegyptiacus. Another important factor influencing the obvious presence of ticks in the bat roost could be the day period; Hoogstraal (1953: 261) noted as follows: "Engorged larvae can easily be found among moist bat droppings on the floor at each site where fruit bats rest. Nymphs and adults rest among bat droppings, under rocks, or in lower wall crevices. They commence crawling upwards on walls toward midday." Benda et al. (2011) summarized the records of colonies R. aegyptiacus throughout its Palaearctic range; this review could be used for searching of the tick occurrence.
The evidence of the MHV-68 virus in specimens from Oman (Al Hoota Cave) includes R. salahi among possible vectors of this pathogen among ticks; the DNA of this virus was detected already in the ixodid species Dermacentor reticulinasus and Ixodes ricinus (Kúdelová et al. 2015(Kúdelová et al. , 2017(Kúdelová et al. , 2018. Besides ticks, one of the main reservoirs of this virus is rodents (Blaškovič et al. 1980;Mistríková and Blaskovic 1985;Hricová and Mistríková 2008). However, according to the results of laboratory experiments, R. salahi does not parasitize other vertebrates (Lavoipierre and Riek 1955), and this fact suggests that another reservoir of the MHV-68 virus could be bats and/ or humans (primates), the only two known groups of hosts of R. salahi. In both groups of hosts, this virus was already confirmed (Briestenská et al. 2018;Janíková et al. 2020;Wágnerová et al. 2015). Our new finding of this virus in Oman represents the southernmost known occurrence spot of this pathogen in the Old World. Our results also support the hypothesis that the MHV-68 virus is a globally widespread herpesvirus capable of inter-species transmission, using one of the suitable vectors available on the site. Now is clear that R. salahi is another tick species which could serve as a reservoir of the virus and play a certain role in its ecology and epidemiology.
One larva of R. salahi collected in Oman (Al Hotta Cave) was positive for Borellia burgdorferi s.l. by real-time PCR. Among the soft ticks parasitizing bats, the presence of B. burgdorferii s.l. was documented only in Carios vespertilionis Latreille, 1796 (Hubbard et al. 1998). This tick species was collected from Rousettus aegyptiacus at Lokwi in South Sudan (Hoogstraal 1956), so the parasitation of this bat by C. vespertilionis cannot be excluded also in other parts of Fig. 3 Map of the records of Reticulinasus salahi (star dots) against the Palaearctic distribution range of its principal host, Rousettus aegyptiacus (dark gray areas); the gray dots denote the records of R. aegyptiacus out of its continuous range. For the parasite records, see text and Tables 1 and 2; the host range is reconstructed after Benda et al. (2011Benda et al. ( , 2012, Judas et al. (2018), and Benda and Ševčík (2020). The alleged record of R. salahi from Spain is not depicted (see text for details) 1 3 its range, including the Palaearctic-this tick is a common parasite of the vespertilionid bats in the latter region (Sándor et al. 2021).
The presence of the Candidatus Ehrlichia shimanensis DNA was discovered in a larva of R. salahi from Rousettus aegyptiacus collected at Wadi Hannah in Oman. The Candidatus E. shimanensis has been known only from the temperate zone of Central and East Asia, found in game species and small rodents, and also in the hard tick Haemaphysalis longicornis Neumann, 1901(Kawahara et al. 2006Rar et al. 2008). The vectors of Ehrlichia sp. are hard ticks and any connection with the soft ticks has been unknown (Socolovschi et al. 2012). Further studies are needed to describe in detail these agents and determined whether R. salahi could really represent their vector and/or reservoir.