An Insight Into the Ecobiology, Vector Significance and Control of Hyalomma Ticks (Acari: Ixodidae): a Review

Accepted Manuscript

Title: AN INSIGHT INTO THE ECOBIOLOGY, VECTOR SIGNIFICANCE AND CONTROL OF HYALOMMA TICKS (ACARI: IXODIDAE): A REVIEW

Authors: M.S. Sajid, A. Kausar, A. Iqbal, H. Abbas, Z. Iqbal, M.K. Jones

PII: S0001-706X(18)30862-3 DOI: https://doi.org/10.1016/j.actatropica.2018.08.016 Reference: ACTROP 4752

To appear in: Acta Tropica

Received date: 6-7-2018 Revised date: 10-8-2018 Accepted date: 12-8-2018

Please cite this article as: Sajid MS, Kausar A, Iqbal A, Abbas H, Iqbal Z, Jones MK, AN INSIGHT INTO THE ECOBIOLOGY, VECTOR SIGNIFICANCE AND CONTROL OF HYALOMMA TICKS (ACARI: IXODIDAE): A REVIEW, Acta Tropica (2018), https://doi.org/10.1016/j.actatropica.2018.08.016

This is a PDF ﬁle of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its ﬁnal form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. AN INSIGHT INTO THE ECOBIOLOGY, VECTOR SIGNIFICANCE AND CONTROL OF HYALOMMA TICKS (ACARI: IXODIDAE): A REVIEW

M. S. SAJID 1 2 *, A. KAUSAR 3, A. IQBAL 4, H. ABBAS 5, Z. IQBAL 1, M. K. JONES 6

1. Department of Parasitology, Faculty of Veterinary Science, University of Agriculture, Faisalabad-38040, Pakistan. 2. One Health Laboratory, Center for Advanced Studies in Agriculture and Food Security (CAS-AFS) University of Agriculture, Faisalabad-38040, Pakistan. 3. Veterinary Research Institute (VRI), Lahore, Punjab, Pakistan. 4. Department of Parasitology, Riphah International University, Lahore, Punjab, Pakistan. 5. KBCMA, College of Veterinary and Animal Sciences (CVAS), Narowal, Sub-campus, UVAS, Lahore-51600, Pakistan. 6. School of Veterinary Science, The University of Queensland, Gatton Queensland 4343, Australia.

Corresponding author: Dr. Muhammad Sohail Sajid Email: [email protected]

Abstract Ticks (Acari: Ixodoidea) are important ectoparasites infesting livestock and human populations around the globe. Ticks can cause damage directly by affecting the site of infestation, or indirectly as vectors of a wide range of protozoa, bacteria and viruses which ultimately lead to lowered productivity of livestock populations. Hyalomma is a genus of hard ticks, having more than 30 species well-adapted to hot, humid and cold climates. Habitat diversity, vector ability, and emerging problem of acaricidal resistance in enzootic regions typify this genus in various countries around the world. This paper reviews the epidemiology, associated risk factors (temperature, climate, age, sex, breed etc.), vector role, vector-pathogen association, and reported control strategies of genus Hyalomma. The various proteins in saliva of Hyalomma secreted into the blood stream of host and the prolonged attachment are responsible for the successfulACCEPTED engorgement of female ticks in spite MANUSCRIPT of host immune defense system. The various immunological approaches that have been tried by researchers in order to cause tick rejection are also discussed. In addition, the novel biological control approaches involving the use of entomopathogenic nematodes and Bacillus thuringiensis (B. thuringiensis) serovar thuringiensis H14; an

endotoxin, for their acaricidal effect on different species and life cycle stages of Hyalomma are also presented. KEY WORDS Ticks, Hyalomma, prevalence, immunization, salivary glands, entomo-pathogenic nematodes 1 | SIGNIFICANCE OF TICKS Ticks (Acari: Ixodoidea) are voracious blood suckers, causing heavy blood losses. The livestock industry all around the globe is under economic threat due to ticks and tickborne diseases (Sajid et al., 2017). Tick infestation has been reported to cause reduction in live weight, affect appetite, body condition, blood composition, respiratory rate of animals, losses in milk production and damage to hides and open wounds leading to secondary infections (Springell, 1974; Rajput et al., 2006; Schroder et al., 2013). Three families of ticks are identified, including the recently identified family Nuttalliellidae, the Ixodidae, or hard ticks and Argasidae, the soft ticks (Guglielmone et al., 2010; 2014). The saliva of ticks contains several pharmacologically active chemicals which help in their blood feeding by modulating inflammation, immunity, and hemostasis of their host. Furthermore, antimicrobial factors are also the part of their saliva among the several other adaptations towards blood feeding (Brossard, 2004; Valenzuela, 2004; Steen, 2006; Hovius, 2008; Francischetti, 2009; 2010). Long hypostome ticks may induce abscesses due to secondary bacterial infections (Ambrose et al., 1999) and those with short hypostomes may cause devaluation of hides and skins (Jongejan & Uilenberg, 2004). As a result, ticks have a direct effect on the availability of good quality hides and skins to the leather industry. Ticks have been reported to cause severe irritation, allergy, toxicosis and paralysis therefore they are global public health problem (Aktaş, 2008; Bursali et al., 2012). Ticks infesting in ears can cause laceration, ear canal abrasion or bleeding leading to otitis externa (Al- Juboori, 2013) and in some cases, canal edema and external ear hyperemia have been reported (Gökdoğan et al., 2016). Ticks are known to cause lowered productivity (Sajid et al., 2007), mortality (Niyonzema & Kiltz, 1986) and can transmit Theileria (T.) spp., Babesia (B.) spp., Coxiella (C.) burnetii, Anaplasma (A.) spp., Rickettsia (R.) spp., and several viruses that causes deadlyACCEPTED diseases (Taylor et al., 2007; Bakheit etMANUSCRIPT al., 2012; Estrada-Peña & de la Fuente, 2014; Gortazar et al., 2014; CDC, 2016). An adult female tick can cause blood loss that can result in reduction in live weight gain of cattle (Pegram & Oosterwijk, 1990), dry matter intake and milk

yield (Jonsson et al., 1998). Finally, ticks may cause immunosuppression of host (Inokuma et al., 1993; Ferreira & Silva, 1998) that may facilitate the attack of other microorganisms resulting in disease. According to one estimate, more than one billion cattle of the tropics and subtropics are at risk of tick infestation (Pegram et al., 1993). Hyalomma spp. are the major vectors in the livestock population of Punjab, Pakistan (Durrani et al., 2008). A multi-host tick, Hyalomma (H.) anatolicum, infects the large ruminants and small ruminants acting as a vector for Theileria spp. specifically T. lestocardi (T. hirci), T. annulata and T. buffeli in India (Ghosh et al., 2008). It has been reported from Eurasia and Africa that several human and animal pathogens can be transmitted by Hyalomma ticks (Vial et al., 2016). Facial paralysis after a bite of H. marginatum species has been reported (Campbell, 1977; Gurbuz et al., 2010; Doğan et al., 2012). Tick paralysis in humans is also caused by Hyalomma spp. (Do˘gan et al., 2012). Theileria annulata, vectored by Hyalomma spp. worldwide, causes a disease called bovine tropical theileriosis, from which about 250 million cattle are at risk (Gharbi et al., 2006).

2 | GENERAL BIOLOGY AND LIFE CYCLE OF HYALOMMINE TICKS Hyalomma spp., Amblyomma spp., and Rhipicephalus (Boophilus) spp. are the economically important tick genera belonging to family Ixodidae (Guerrero et al., 2012). Hyalomma spp. can be morphologically identified on the basis specific features which include elongate mouthparts, presence of eyes, irregular festoons, an inornate dorsal shield, a characteristic banding pattern seen on the legs and lastly the spurs on the forecoxae subequal in length (Mathison & Pritt, 2014). The unfed Hyalomma ticks are 5 to 6mm in length including the mouthparts. Striations are present on integument and lateral suture is missing. Mouthparts are anterior. Palp articles 1 and 3 are short while articles 2 are comparatively longer. Angular lateral margins (medium) can be seen on basis capituli. Legs of Hyalomma ticks have these characteristics: slender, presence of pale rings and pulvilli. Brown coloured conscutum is present in the male (a scutum is present in the ACCEPTEDfeamle). Scutum and conscutum do not have MANUSCRIPT any enamel or ornamentation except in case of H. lusitanicum (Figure 1). Convex eyes are not unusual. In males and females, festoons are prominent but fade when females are engorged. Posterior to 4th pair of legs are the larger

spiracular plates which have scattered spiracle goblets. Only males have the ventral plates which are three distinct pairs, usually (Figure 2). Posterior to anus, these is an anal groove. The 4th coxae are of normal size and coxae 1 have equal and large paired spurs (Bowman and Nuttall, 2008; Sonenshine and Roe, 2014; Walker et al., 2014). Hyalommines are moderately-large to large ticks with long mouthparts, and are enzootic in Africa, south-eastern Europe and Asia (Kahn, 2008). Among the Hyalomma spp., most ticks are three host in nature and their larval, nymphal, and adult phases can be existed in free form in surrounding environment standing by for a suitable host. Larval and nymphal forms quest for small mammals including moles, rabbits, rodents and also birds and reptiles. Adult Hyalomma ticks find large vertebrate host including cattle, buffaloes, sheep, goat, dogs and humans. Hyalomma ticks can complete their life cycle in one, two or three variable hosts depending on that one they find. It takes three to four months or more than a year for Hyalomma spp. to complete their life cycle but it depends on the species and climatic parameters and ecological conditions (Robert and Janovy, 2009). For one host tick, all the life stages viz. larva, nymph and adult occur on the same host. After hatching, larvae quest for small animal host to undergo hematophagy. Then after blood feeding the larvae, depending on the nature of tick species, either stay on the host for further molting (two host ticks) or separate from its host to molt (three host ticks). The nymphal forms are either remain on the same host on which molting occurrs (two host ticks) or move to other small vertebrate host (three host ticks). After feeding, the fully engorged nymphs of all Hyalomma spp. leave the host and develop to adults which then attach to a large animal host for blood feeding and matting occurs. After engorgement, females leaves the host and search favorable place for egg laying (Figure 3, 4 & 5). Two host tick spp. include H. schulzei and H. marginatum (Walker et al., 2003) while H. excavatum can be two or three host. Furthermore, H. truncatum, H. impressum, H. nitidum, H. albiparmatum, H. lusitanicum, H. asiaticum, H. impeltatum and H. franchinii are three host ticks. H. scupense, H. dromedarii and H. anatolicum are two or one host ticks (Apanaskevich, 2004; Gharbi, 2014). 3 | EPIDEMIOLOGYACCEPTED OF HYALOMMINE MANUSCRIPT TICKS Prevalence is one of the crucial tools in the study of the epidemiology of ticks and tick-borne parasites. The role of ticks as vectors, their epidemiology, and percentage (qualitative and

quantitative) infected are very essential tools for a proper tick control program. The epidemiology of Hyalomma ticks has extensively been studied in various parts of the world. Hyalomma spp. are often the most abundant tick parasites of livestock, including camels, in the warm, arid and semiarid, generally harsh lowland and middle altitude biotopes of central and southwest Asia, southern Europe and southern Africa. So far, 30 species of the genus Hyalomma have been reported from various parts of the world (Geevarghese & Dhanda, 1987; Kahn, 2008). Of these, almost 15 species are significantly important for pathogen transmission to livestock and humans. Among the 30 described and accepted Hyalomma spp., H. scupense is the most important tick which is present in Palearctic zoogeographic region on three continents (42 countries) specifically in the humid to arid regions. Apanaskevich et al. (2010) and Hoogstraal (1956) reported Hyalomma ticks in 21 Asian countries (Turkmenistan, Kyrgyzstan, Uzbekistan, Turkey, Tajikistan, Syria, Pakistan, Nepal, Oman, China, Jordan, Israel, Georgia, India, Afghanistan, Iran, Azerbaijan, Armenia, Kazakhstan, Iraq and Lebanon), six African countries (Tunisia, Sudan, Morocco, Libya, Egypt and Algeria), and fifteen European countries (Ukraine, Spain, Serbia, Russia, Romania, Montenegro, Montenegro, Moldova, Macedonia, Italy, Greece, France, Croatia, Bulgaria, Herzegovina, Bosnia and Albania). In India, H. anatolicum is the most abundant among hard ticks infesting dairy animals leading to economic loss (Ghosh et al., 2007), specifically in Punjab (Haque et al., 2011; Singh & Rath, 2013). Prevalence of various Hyalomma spp. reported in Iran by Tajedin et al. (2016) includes H. marginatum (4.9%), H. asiaticum (5.7%), H. anatolicum (11.2%) and H. dromedarii (15.6%). The prevalence of H. detritum infestation in cow was recorded as 84.3% in Tunisia (Bouattour et al., 1996) in comparison with the lower prevalence (51.0%) of H. anatolicum in Pakistan (Sajid et al., 2009) and the low prevalence (<1%) of Hyalomma spp. in the Central Guinea savannah of Cote d’ Ivoire (Knopf et al., 2002). 4 | FACTORS ASSOCIATED WITH PREVALENCE OF HYALOMMA TICKS 4.1 Vulnerable host body parts Ticks attach preferentially to the areas of body which have the following properties; thin skin, nonACCEPTED-accessible to grooming and licking. Therefore, MANUSCRIPT their abundance on animal body depends on the tick species and stage of tick (MacLeod et al., 1977). Rear udder quarters of cows have been observed to harbor more Hyalomine tick populations (41.22% adults and 63.82% nymphs) and

second to udder is thighs harboring 32.08% adult and 13.82% nymphs of H. scupense and other sites of the Hyalommine tick’s attachment on animal body include teats, inguinal region, anterior udder quarters, belly, axilla, neck and interscapular regions (Gharbi et al., 2013). The preferred sites of Hyalomma spp. in cattle are ear, neck, inside of thighs, inside lips of vulva as reported by Mattioli et al. (1998), Al-Araf et al. (2007) and Sajid (2007). Several factors affect the percentage of animals infested with these ticks. Clustering of tick on the body of their host is due to four types of pheromones that have been discovered in ticks so for. These pheromones reduce the distance between the tick individuals resulting in clustering which enhances survival rate (Carde & Baker, 1984). This clustering behaviour has also been observed in H. dromedary (Leahy et al., 1981). 4.2 Availability of host and climate change In Europe, tickborne disease outbreaks are increasing due to factors including a) host population density b) changes in climate c) agricultural practices and d) leisure activities (Gray et al., 2009; Danielova et al., 2010; Godfrey & Randolph, 2011). Much depends upon the circumstances, on the tick species involved, suitability of the local climatic or seasonal conditions and susceptibility of host to infestation. The tick burden of an animal may be affected by season, breed, age and sex of host, lactation stage and nutritional status (Springell, 1974), and even within the hosts of the same genotype, different rates of tick infestation have been reported (Carr et al., 1974; Alexander et al., 1984; Ansell, 1985). Host range for H. anatolicum includes horses, goats, sheep, pigs and some wild animals globally that can be a determinant for more tick population (Luo et al., 2003; Guan et al., 2009). Climate change in the form of increased ambient temperature and varying patterns in rainfall, have great impact in temporal and special distribution of tick vectors (Githeko et al., 2000; Purse et al., 2005). Climate change, particularly increase in temperature, may have deleterious effect on ticks’ habitat and force them to move to new areas. In South Africa, it was a prediction that a 2˚C increase in temperature will disturb the habitat of four ticks’ species namely H. truncatum, Rhipicephalus (R.) appendiculatus, Amblyomma (A.) hebraeum and R. decoloratus (Estrada- Pena, 2003). Climatic conditions e.g. temperature,ACCEPTED humidity and rainfall have been reportedMANUSCRIPT as predominant factors influencing the life cycle, activity and fecundity of tick populations (Mushi et al., 1996; Yeruham et al., 1996; Yakhchali and Hosseine, 2006). Tick phenology differences are due to many factors including

alteration in relative humidity and temperature from year to year, diversity among abiotic factors and local climate variations from one farm to another, lastly the management practices including host breed (Gharbi et al., 2013). 4.3 Reduced immunological response Reduction in T lymphocyte count and proliferation and increase in CD4+/CD8+ ratio as well as in circulating B lymphocyte count after Hyalomma infestation in host have been observed by Boppana et al. (2005). 4.4 Migration of birds/humans It has been reported that the birds also play role in dispersal of ticks infected with Crimean- Congo haemorrhagic fever (CCHF) virus (Ergonul, 2006; Bente et al., 2013; Toma et al., 2014). There is possibility that human can transfer Hyalomma spp. from one continent to other as it is evident from this reported case of import of H. truncatum to United States of America from Ethiopia by a photographer returning from an expedition there (Mathison et al., 2015). 5 | HYALOMMA TICKS AS BIOLOGICAL VECTORS OF MICROORGANISMS 5.1 Bacteria Ticks act not only as potential vectors but also as reservoirs of certain infectious agents (e.g. Pasteurella multocida, Brucella abortus and Salmonella typhimurium) in man and animals (Jongejan & Uilenberg, 2004). Vector roles for Ixodes ricinus and Haemaphysalis punctata under the French farming systems have been reviewed by L’Hostis and Seegers (2002). Quantitative examination of infected ticks revealed that sex plays a significant role in the prevalence (number of infected ticks out of total number of tick population at risk of getting infection) and intensity (number of infected acini of salivary glands per tick) of infection (Dhar et al., 1982; Reid & Bell, 1984; Walker et al., 1983; 1985; Sayin et al., 2003a). Several human infectious diseases are due to pathogens transmitted by Hyalomma ticks (Ergonul, 2006; Portillo et al., 2015). In 1991, R. sibirica mongolitimonae was isolated from H. asiaticum ticks of Mongolia, China (Yu et al., 1993). The R. aeschlimannii is a spotted fever group pathogen first isolated from H. marginatum in Morocco (Beati et al., 1997) and R. s. mongolitimonae and R. aeschlimanniiACCEPTED were detected in H. truncatum andMANUSCRIPT H. rufipes, respectively, from Africa (Parola et al., 2001). Also, H. marginatum has been reported as vector of R. aeschlimannii in Spain (Ferna´ndez-Soto et al., 2003), Croatia (Punda-Polic et al., 2002), Corsica (Matsumoto et al.,

2004). Hyalomma ticks serve both as the vector and reservoir of R. aeschlimannii as both transstadial and transovarial transmission of the bacterium are possible in these ticks (Matsumoto et al., 2004). 5.2 Viruses If the Hyalomma ticks have the suitable receptors then CCHF virus enters the tick cells due to the attachment of viral envelope proteins (Garrison et al., 2013) following blood meal by endocytosis due to the activation of actin-dependent clathrin-mediated endocytic pathway (Simon et al., 2009; Suda et al., 2016). CCHF virus multiplies in midgut lining of the tick where they spread to the hemolymph thereby infesting various tissues. After that, it reaches to the reproductive organs and salivary glands (representing the highest titers) where it undergoes exocytosis to leave the cell. CCHF virus titer increases in the testes, salivary glands and ovaries after the feeding thus enhancing the transmission risk to vertebrate host (Dickson and Turell, 1992). In 1960s, CCHF virus was first found to be present in adult Hyalomma ticks (Kayedi et al., 2015). Gonzalez et al. (1992) reported that H. truncatum can transfer CCHF virus sexually and transovarially. This virus is transmitted by various tick species in wide range of areas globally like in Iran, Pakistan, Turkmenistan, and Tajikistan by H. anatolicum, in central Asia to China by H. asiaticum, in Africa by H. rufipes and in southern Russia, Turkey, and The Balkanand Crimean Peninsulas by H. marginatum (Hoogstraal, 1979; Bakheit et al., 2012; Goddard, 2012). H. marginatum, H. turanicum, H. anatolicum and H. scupense are the major tick species involved in the transmission of CCHF virus in Eurasia (Gargili et al., 2013). It has been reported that in Asia, Africa, and southern Europe, CCHF virus is transmitted by H. marginatum and H. rufipes (Francischetti, 2011; Keshtkar-Jahromi et al., 2013; Kayedi et al., 2015; Brackney & Armstrong, 2016).Significance of the latter increases manifold in the rural areas of resource poor farming community where the CCHF has recently been reported in Pakistan (Dawn, 2016). 5.3 Parasites Tropical bovine theileriosis is the major Hyalomma tick-borne lymphocytotropic protozoan diseaseACCEPTED prevalent in some parts of the world (Uilenbeng,MANUSCRIPT 1981; Robinson, 1982; Sayin et al., 2003b). So far, a number of Hyalomma spp. including H. anatolicum (Dhar et al., 1982; Sangwan et al., 1989), H. detritum (Samish & Pipano, 1978; Bouattour et al., 1996; Grech-

Angelini, 2016), H. excavatum, H. marginatum and H. dromedarii (Samish & Pipano, 1983; Bhattacharyulu et al., 1975; Sayin et al., 2003a) have been reported to transmit this disease from cattle to cattle. In a study conducted in Turkey (Aktas et al., 2004), the percentage of H. anatolicum salivary glands infected with T. annulata was determined to be 19.2% and 46.9% in ticks collected from cattle and shelters, respectively. In addition, 2.4% of H. excavatum and 5.6% of H. detritium were reported to carry T. annulata sporozoites in their salivary glands. H. anatolicum has been reported to transmit B. (T.) equi from experimentally infected donkeys, resulting in development of clinical babesiosis (Kumar et al., 2007). In small ruminants, B. motasi and B. ovis are transmitted by Hyalomma spp. (Uilengberg et al., 1980; Friedhoff, 1988; 1997). The seasonal dynamics of tick populations greatly influences the prevalence of tick-borne pathogens (Estrada-Pena, 2001). Seroprevalence of B. ovis has been found to be higher in spring and summer months which are associated with higher activity of the tick vectors (Rodriguez et al., 1989; Trifonov & Ruseve, 1989; Pipano, 1991; Yeruham et al., 1992) including H. anatolicum (Hosein et al., 2007). Recently, H. anatolicum has been reported to transmit a new species of Babesia in sheep of Gansu province of China (Guan et al., 2009). The B. crassa is another valid species of Babesia in small ruminants (Hashemi-Fesharki and Uilenburg, 1981; Hashemi-Fesharki, 1997) but the tick vector responsible for its transmission is still unknown. The T. lestoquardi (the cause of ovine malignant theileriosis) was initially reported to be transmitted by H. excavatum in Iran (Hashemi-Fesharki, 1997), and H. impeltatum has been reported as a potential vector of T. lestoquardi in sheep of Saudi Arabia (El-Azazy et al., 2001). In another study, H. anatolicum was reported and suggested to be focused as a significant vector of T. lestoquardi (Jianxus and Hong, 1997; Razmi et al., 2003). Hyalomma dromerii was found responsible for theileriosis (T. annulata) in camels of Pakistan (Youssef et al. 2015; Karim et al. 2017). Furthermore, the CCHF virus (Tekin et al., 2012), T. annulata, and T. equi (Darghouth et al., 1996), Bhanja virus and C. burnetii (Bakheit et al., 2012) are the pathogen which have been reported to be vectored by H. scupense. Some of the pathogens transmitted by Hyalomma spp. are summarized in Table 1. 6 | SIGNIFICANCEACCEPTED OF SALIVARY GLANDS MANUSCRIPT OF HYALOMMA During blood meal, ticks encounter the problems of coagulation, platelet aggregation and vasoconstriction at the feeding lesion (Francischetti et al., 2010). In order to counter this, tick

salivary glands secrete saliva containing not only large amounts of substances having anti- coagulant, anti-platelet and vasodilatory; but also, compounds with anti-inflammatory and immunomodulatory activities (Francischetti et al., 2009; Chmelar et al., 2012; Kotal et al., 2015; Tan et al., 2015). Wu et al. (2010) identified two immunomodulatory peptides namely hyalomin-A and –B from salivary glands of H. asiaticum and these peptides were found to interfere with the inflammatory response by altering the secretion of cytokines. Ghosh et al. (2015) conducted an experiment on salivary gland proteins of H. anatolicum and identified a novel 28 kDa protein having prominent anti-inflammatory activity. Ticks adopt a variety of strategies to modulate hemostasis and host immunity in order to facilitate their blood feeding. In this regard, various factors that might be associated with a successful prolonged tick attachment and the various mechanisms and pathways have been discussed in detail by Francischetti et al. (2010). Effect of ambient temperatures (15 ◦C, 28 ◦C and 42 ◦C) on the sizes of salivary gland proteins of H. anatolicum has been reported by Nabian et al. (2003). They found significant change in the banding pattern of salivary gland proteins of ticks incubated at various temperatures. In a study conducted by Ribeiro et al. (2017), it has been observed that mucins, glycine-rich proteins, anticoagulants of the madanin family, metalloproteases and lipocalins are the proteins among the most secreted by H. excavatum. Several immunomodulatory molecules have been isolated from tick salivary glands which antagonize host inflammatory responses (Brossard & Wikel, 1997; Wikel & Bergman, 1997). These molecules are polymorphic and have well conserved anti-inflammatory activities (Ribeiro, 1995; Wang et al., 1999). Such type of molecules isolated from Hyalomma ticks include B-cell inhibitory proteins isolated and characterized from H. asiaticum (Yu et al., 2006). Activity of superoxide dismutase, nitric oxide radicals and reduced glutathione (GSH) concentrations in the salivary cocktail of male and female H. anatolicum represents the antioxidant defense which is to tackle the oxidative response presented by host during feeding (Francischetti et al. 2009; Wu et al. 2010; Ghosh et al. 2014; Ghosh et al., 2017). VariousACCEPTED anticoagulants, immunomodulators, biologicallyMANUSCRIPT active proteins and antioxidants, found in salivary cocktail, are altogether compose the tick antioxidant defense system to fight against the oxidative attacks from the host (Ribeiro et al., 2003; Francischetti et al., 2009)

7 | APPLICATION OF EXPRESSED SEQUENCE TAGS (ESTS) IN HYALOMMA SPECIES The genomic resources for ectoparasites, which currently are limited to express sequence tags (ESTs), have been reviewed. The largest EST dataset has been reported for Ixodes scapularis. However, despite huge global economic significance of H. anatolicum, only a few ESTs which originated from ticks infected with T. annulata mentioned in a bovine macrophage library are available (Jensen et al., 2006). 8 | IMMUNITY AGAINST TICKS WITH REFERENCE OF GENUS HYALOMMA Trager’s statement of development of tick resistance in guinea pigs exposed to Dermacentor variabalis (Trager, 1939) and the emerging problem of acaricidal resistance (De la Fuente et al., 1998) are major thrusts for scientists to search for some immunological control for arthropods. In the tropics and sub-tropics, cross-bred cattle (Bos indicus x Bos taurus) are high milk yielding but have lower resistance to ticks and tick-borne diseases (Sran et al., 1996). This has attracted many researchers into the hunt for immunological tools to control the ticks and their associated diseases rather than changing the breeding program. Immunological control of ticks can help prevent environmental contamination as a result of frequently used acaricides (De la Fuente et al., 1998; Trimnell et al., 2002) and selection of acaricide-resistant tick populations (De la Fuente & Kocan, 2003). A number of experiments have so far been conducted using various antigens of different developmental stages of ticks (Willadsen, 1980; 2001; Pipano et al., 2003). Gill (1986) reported an immediate type hypersensitivity reaction against three polypeptides extracted from the SDS electrophoresed saliva of H. anatolicum in rabbits. Later, immunization studies were conducted against natural infestation (Singh et al., 1991) and artificially administered salivary gland extract (SGE) of H. anatolicum in cattle (Banerjee et al., 1990; Singh, 1993). Repeated infestations of H. anatolicum have also been reported as a successful tool for rejection of tick larvae and nymphs without affecting the adult stage on young cross bred (Bos indicus x Bos taurus) calves (Singh et al., 1991). IgE type basophil homocytotropic antibiodies mediating a cutaneous type hypersensitivity (ITH)ACCEPTED response is one of the probable mechanisms MANUSCRIPT of rejection of ticks (Fivaz & Norval, 1990; Matsuda et al., 1990). In another study, administration of Ascaris (Nematoda: Ascariidae) extract, as immunopotentiator of IgE response, in combination with SGE of H. anatolicum ticks

caused abnormal feeding of larvae and nymphs (Sran et al., 1996). In tick-immune hosts, vasoactive amines, chiefly histamine, play a significant role in the rejection of ticks at the biting sites (Kemp & Bourne, 1980) and the impairment of their feeding behavior (Paine et al., 1983). 9 | RECENT ADVANCES IN HYALOMMA GENETIC RESEARCH

A study was conducted on H. dromedarii collected from one humped camels in India which was focused on the amplification of whole nucleotide sequences of the genes encoding Internally Transcribed Spacer Region 2 (ITS-2) and Calreticulin. Complete nucleotide sequences for ITS-2 and Calreticulin genes were found to be 1285 bp and 1408 bp, respectively. These genes of H. dromedarii from India shared the similarity with those of H. dromedarii from China and H. excavatum. This study could help in the phylogenetic analysis of hard ticks based on ITS-2 and Calreticulin encoding genes (Sivakumara et al. 2018). A study has been reported about the identification of novel miRNAs in H. anatolicum using combined approach of real-time PCR analysis, deep sequencing and bioinformatics. The miRNAs expressed included miR-92a, miR- 1-3p and miR-275-3p. These miRNAs are necessary for the complex life style of these parasites. This miRNAs approach could help in the understanding of this tick biology which would help in constructing novel control strategies (Jin et al. 2015). 10 | CONTROL OF HYALOMMA TICKS The control of Hyalomma tick can be planned based on the objectives including elimination of transmission of tickborne disease causing agents, reduction of direct impact of ticks like skin lesions, pruritis and anemia, and breaking life chain of the tick to eradicate it from a specific animal and human populated area. 10.1 | Chemical control There are various ways through which ticks and tickborne diseases can be controlled. The main method mostly used for control of ticks and tickborne diseases is through the use of chemical acaricides (Spickett & Fivaz, 1992; Pound et al., 2009). These acaricides are used to control tick population on the animals as well as in the environment in way that minimum adverse effects in the ACCEPTEDform of harm to host or applicator and environment MANUSCRIPT will occur (Drummond, 1983). Acaricides can have some negative impacts, like presence of residues in milk, meat and the development of chemical resistant tick strains, as well as harmful effects on humans, animals and the environment (Willadsen et al., 1988; Nolan, 1990; García-García et al., 2000). This has led to

a search for alternative methods of tick control as development process for a new drug is a laborious and lengthy process (Graf et al., 2004). Regassa (2000) has reported that natural compounds, such as herbals, also have some efficacy in controlling ticks. The most commonly used acaricides, for tick control on the body of animals, include chlorinated hydrocarbons, organophosphates, synthetic pyrethroids, carbamates and arsenic (George, 2000; 2004). Arsenic was the first acaricides used for the control of ticks in South Africa in 1893 (Bekker, 1960) and it is water soluble, cheap, stable and considered most effective agent before resistance became a problem (Drummond, 1983; George, 2000). Chlorinated hydrocarbons are synthetic in nature and being used extensively after development of resistance in many tick species against arsenic (Mathewson & Baker, 1975; Graham & Hourrigan, 1977; Angus, 1996). After development resistance to chlorinated hydrocarbons, organophosphates took their place as a chemical acaricides to control ticks (Shanahan & Hart, 1966). But it has been reported by Wharton & Roulston (1970) that several tick species have developed resistant to organ phosphorous acaricides. Resistance to acaricides has also been reported in isolates of H. anatolicum in 20 areas of three agro climatic zones in India and these acaricides were diazinon, deltamethrin and cypermethrin (Shyma et al., 2012). The ticks, H. anatolicum, were also found resistant to deltamethrin and diazinon using adult immersion test in India (Gaur et al., 2016). Photosensitizers as novel acaricides have been used to check the efficacy against ticks (H. dromederi). Photosensitizers cause death of the tick when exposes to visible light because of their accumulation in the body of the tick. Examples include tetramethrin and Safranin (Luksiene et al., 2007). 10.2 | Immunological control through vaccines To overcome the increasing resistance pressure to acaricides and to minimize their environment unfriendly impacts, recombinant vaccines were introduced in early 1990s. Immunization of animals with tick antigens reduced the number of ticks on cattle leading to formulation of vaccines against wide range of tick species (Fuente et al., 2006). There are two types of tick antigensACCEPTED including exposed and concealed (Kiss MANUSCRIPT et al., 2012). The major constraint in successful development of the vaccine against ticks is the selection of right antigens. Concealed antigens can be accessed by host antibody taken during blood feeding.

The exploration of tick genome and with the help of the bioinformatics, mutagenesis, transcriptomics, proteomics, immunomapping, expression library immunization (ELI) and ribonucleic acid interference (RNAi) has made it possible to open new pathway to tick vaccine discovery (Fuente et al., 2006). The suppression of subolesin (SUB), cathepsin L-like cysteine proteinase (CathL) and calreticulin (CRT) genes of H. anatolicum using RNAi resulted in the reduced engorgement of ticks to some extent than control (Kumar et al., 2017). The data about the Hyalomma tick genome is lacking and more research in this area is needed. Various antigens have been undergone trials against several tick species (Willadsen, 2006). In a study, cattles were immunized using antigens Hd86 and Bm86 of H. scupense and R. microplus, respectively (Galaı¨ et al., 2012). The commercially available anti-tick vaccines were first used to immunize cattle against R. microplus in Latin America (GavacTM) and in Australia (TickGARDTM and TickGARDPLUSTM). The result of these study showed that there has been reduction of about 59.19% in the number of H. scupense nymphs observed which were on the cattle vaccinated with Hd86 while no result of Bm86 was observed against H. scupense nymphs. But none of Bm86 or Hd86 antigens was able to provide absolute protection of cattle against adult ticks of H. scupense. In the case of Hd86, the low protective efficacy against adult ticks might be due to expression of Hd86 gene only in nymph forms (Ben Said et al., 2012; 2013). Antigens of B. microplus can also immunize the host against H. dromedarii (de Vos et al., 2001). El Hakim et al. (2011) reported the reduction in egg hatch rates due to GLP antigen of Hyalomma dromedarii. Bm86 antigen have been used to partially control the H. anatolicum and H. dromedarii (Rodriguez-Valle et al., 2012). The rHaa86, an orthologue of Bm86 could be effective in controlling H. anatolicum (Jeyabal et al., 2010) 10.4 | Biological control of Hyalomma ticks Ticks are susceptible to entomopathogenic steinernematid and heterorhabditid nematodes (Samish & Glazer, 2011). During the last decade, various trials of the biological control of Hyalomma ticks using entomopathogenic nematodes (EPNs) of various strains have been reported. A sharp increase in IgG level of mice infested with H. dromedarii and EPNs have been observedACCEPTED indicating penetration of EPNs through MANUSCRIPT cuticle of engorging female ticks (Saad et al., 2006). Steinernema spp. S1 and Heterorhabditis spp. IS-5 have been reported as most virulent strains of nematodes for H. dromedarii and H. excavatum, respectively (El-Sadawy et al., 1998;

Samish et al., 1999; 2000). A study on the comparative virulence of EPNs of families

Steinernematidae and Heterorhabditidae in various tick species and life cycle stages indicated that preimaginal stages of ticks including H. excavatum and R. bursa are more resistant to EPNs. However, unfed and engorged adult stages of both tick species were equally susceptible to EPNs (Samish et al., 1999). In other studies, H. excavatum has been reported as less susceptible to EPNs than Rhipicephalus spp. (Samish et al., 2000). In contrast to findings of Samish et al. (1999), El-Sadawy et al. (2008) found higher biological control activities of heterorhabditid strains than those of stienernematid strains in engorged female H. dromedarii ticks. Recently, Bacillus thuringiensis serovar thuringiensis H14; an opportunistic insect pathogen first discovered in 1992 (Gill et al., 1992), was induced in H. dromedarii ticks. This resulted in severely damaged ultrastructural characteristics of hemoplasts of tick hemolymph suggesting its acaricidal mechanism involves damaging the cellular immune system of ticks (Habeeb & El- Hag, 2008). Alternative control of tick is use of entomopathogenic fungi (EPF) to overcome the resistance developed against chemicals (Maniania et al., 2007; Castro-Janer et al., 2010). Three mycoacaricides are commercially available for the control of hard ticks and these are based on Metarhizium anisopliae (Faria & Wraight, 2007). A study conducted by Sun et al. (2011) shown that Beauveria bassiana, a fungi, suspension was able to cause 100% mortality of H. anatolicum at the concentration of 108 conidia/mL. Tanada and Kaya (1993) found six out of many entomopathogenic fungi genera as pathogenic to various tick species. Fungal biopesticides were found efficacious against ticks represented by many studies (Polar et al., 2005; Leemon & Jonsson, 2008). 11 | CONCLUSIONS Hyalomma ticks are endemic in various regions of the world, playing significant roles in direct and indirect damage to the livestock sector. Immunization against various proteins of various developmental stages has been found fruitful in reducing the tick burden. Targeting salivary gland proteins may also be useful in hampering the process of tick feeding. Furthermore, interruptionACCEPTED of host pathogen interaction and otherMANUSCRIPT biological mechanism of ticks by gene silencing using RNA interference (RNAi). The novel approach of the use of EPNs may also

provide effective control strategies of Ixodid ticks. However, more research is still needed in these new concepts of tick control. AUTHOR CONTRIBUTION MSS – did the elaboration and correction of the draft, contributed the abstract, control of Hyalomma ticks and conclusions, AK – contributed the significance and epidemiology of ticks, factors associated with prevalence of Hyalomma ticks portion of the draft; AI - contributed the Hyalomma ticks as biological vectors of diseases and significance of salivary glands of Hyalomma; HA - general biology and life cycle of Hyalommine ticks and control of Hyalomma ticks portion of draft; ZI - application of expressed sequence tags (ESTs) in Hyalomma species and immunity against ticks with reference of genus Hyalomma; MKJ - review the manuscript both grammatically and technically.

ACKNOWLEDGEMENTS

We would like to thank Professor Malcolm Jones, Director of Students and Professor of Parasitology, School of Veterinary Science, The University of Queensland, Gatton Queensland 4343, Australia for reviewing the manuscript both grammatically and technically. I am also thankful to all the co-authors for their contribution in the manuscript.

ACCEPTED MANUSCRIPT

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FIGURE LEGENDS:

FIGURE 1 Hyalomma excavatum (A) female; (B) male (Walker et al., 2014)

FIGURE 2 Hyalomma truncatum male, ventral (Walker et al., 2014)

FIGURE 3 Life cycle of one host Hyalomma sp.

FIGURE 4 Life cycle of two host Hyalomma sp.

FIGURE 5 Life cycle of three host Hyalomma sp.

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TABLE 1 List of pathogens transmitted by Hyalomma spp. Tick Species Tick borne Disease Selected references Pathogens Hyalomma T. annulata bovine tropical Bhattacharylu et al., 1975; spp. theileriosis Parmar, 1984; Dhar et al., 1987; Bouattour et al., 1996 T. lestoquardi Theileriosis in cattle Hashemi-Fesharki, 1997 B. (T.) equi equine babesiosis or Chaudhuri et al., 1969; Schein, theileriosis 1988; Ali et al., 1996; Kumar et al., 2007 B. caballi equine babesiosi Blouin & de Waal, 1989 Trypanosoma Trypanosomiasis Morzaria et al., 1986 theileri-like flagellates R. aeschlimannii Rickettsiosis Jiang et al., 2012; Gargili et al., 2012 H. a. CCHF virus Crimean-Congo Hoshmand-Rad & Hawa, 1973 anatolicum hemorrhagic fever in Iran H. CCHF virus Crimean-Congo Gonzalez et al., 1992 truncatum hemorrhagic fever in Iran

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