New species based on the biological species concept within the complex of Lariophagus distinguendus (Hymenoptera, Chalcidoidea, Pteromalidae), a parasitoid of household pests.

The pteromalid parasitoid Lariophagus distinguendus (Foerster) belongs to the Hymenoptera, a megadiverse insect order with high cryptic diversity. It attacks stored product pest beetles in human storage facilities. Recently, it has been shown to consist of two separate species. To further study its cryptic diversity, strains were collected to compare their relatedness using barcoding and nuclear genes. Nuclear genes identified two clusters which agree with the known two species, whereas the barcode fragment determined an additional third Clade. Total reproductive isolation (RI) according to the biological species concept (BSC) was investigated in crossing experiments within and between clusters using representative strains. Sexual isolation exists between all studied pairs, increasing from slight to strong with genetic distance. Postzygotic barriers mostly affected hybrid males, pointing to Haldane's rule. Hybrid females were only affected by unidirectional Spiroplasma-induced cytoplasmic incompatibility and behavioural sterility, each in one specific strain combination. RI was virtually absent between strains separated by up to 2.8% COI difference, but strong or complete in three pairs from one Clade each, separated by at least 7.2%. Apparently, each of these clusters represents one separate species according to the BSC, highlighting cryptic diversity in direct vicinity to humans. In addition, these results challenge the recent 'turbo-taxonomy' practice of using 2% COI differences to delimitate species, especially within parasitic Hymenoptera. The gradual increase in number and strength of reproductive barriers between strains with increasing genetic distance also sheds light on the emergence of barriers during the speciation process in L. distinguendus.

Highly transmissible cytoplasmic incompatibility by the extracellular insect symbiont Spiroplasma.

Cytoplasmic incompatibility (CI) is a form of reproductive manipulation caused by maternally inherited endosymbionts infecting arthropods, like Wolbachia, whereby matings between infected males and uninfected females produce few or no offspring. We report the discovery of a new CI symbiont, a strain of Spiroplasma causing CI in the parasitoid wasp Lariophagus distinguendus. Its extracellular occurrence enabled us to establish CI in uninfected adult insects by transferring Spiroplasma-infected hemolymph. We sequenced the CI-Spiroplasma genome and did not find any homologues of any of the cif genes discovered to cause CI in Wolbachia, suggesting independent evolution of CI. Instead, the genome contains other potential CI-causing candidate genes, such as homologues of high-mobility group (HMG) box proteins that are crucial in eukaryotic development but rare in bacterial genomes. Spiroplasma's extracellular nature and broad host range encompassing medically and agriculturally important arthropods make it a promising tool to study CI and its applications.

Comparative Proteomics of Coxiella like Endosymbionts (CLEs) in the Symbiotic Organs of Rhipicephalus sanguineus Ticks.

Maternally transmitted obligatory endosymbionts are found in the female gonads as well as in somatic tissue and are expected to provide missing metabolite to their hosts. These deficiencies are presumably complemented through specific symbiotic microorganisms such as Coxiella-like endosymbionts (CLEs) of Rhipicephalus ticks. CLEs are localized in specialized host tissue cells within the Malpighian tubules (Mt) and the ovaries (Ov) from which they are maternally transmitted to developing oocytes. These two organs differ in function and cell types, but the role of CLEs in these tissues is unknown. To probe possible functions of CLEs, comparative proteomics was performed between Mt and Ov of R. sanguineus ticks. Altogether, a total of 580 and 614 CLE proteins were identified in Mt and Ov, respectively. Of these, 276 CLE proteins were more abundant in Mt, of which 12 were significantly differentially abundant. In Ov, 290 CLE proteins were more abundant, of which 16 were significantly differentially abundant. Gene Ontology analysis revealed that most of the proteins enriched in Mt are related to cellular metabolic functions and stress responses, whereas in Ov, the majority were related to cell proliferation suggesting CLEs function differentially and interdependently with host requirements specific to each organ. The results suggest Mt CLEs provide essential nutrients to its host and Ov CLEs promote proliferation and vertical transmission to tick progeny. IMPORTANCE Here we compare the Coxiella-like endosymbionts (CLEs) proteomes from Malpighian tubule (Mt) and the ovaries (Ov) of the brown dog tick Rhipicephalus sanguineus. Our results support the hypothesis that CLEs function interdependently with host requirements in each of the organs. The different functional specificity of CLE in the same host suggest that metabolic capabilities evolved according to the constrains imposed by the specific organ function and requirements. Our findings provide specific CLE protein targets that can be useful for future studies of CLE biology with a focus on tick population control.

Ecological Contacts and Host Specificity Promote Replacement of Nutritional Endosymbionts in Ticks.

Symbiosis with vitamin-provisioning microbes is essential for the nutrition of animals with some specialized feeding habits. While coevolution favors the interdependence between symbiotic partners, their associations are not necessarily stable: Recently acquired symbionts can replace ancestral symbionts. In this study, we demonstrate successful replacement by Francisella-like endosymbionts (-LE), a group of B-vitamin-provisioning endosymbionts, across tick communities driven by horizontal transfers. Using a broad collection of Francisella-LE-infected tick species, we determined the diversity of Francisella-LE haplotypes through a multi-locus strain typing approach and further characterized their phylogenetic relationships and their association with biological traits of their tick hosts. The patterns observed showed that Francisella-LE commonly transfer through similar ecological networks and geographic distributions shared among different tick species and, in certain cases, through preferential shuffling across congeneric tick species. Altogether, these findings reveal the importance of geographic, ecological, and phylogenetic proximity in shaping the replacement pattern in which new nutritional symbioses are initiated.

A dual endosymbiosis supports nutritional adaptation to hematophagy in the invasive tick Hyalomma marginatum.

Many animals are dependent on microbial partners that provide essential nutrients lacking from their diet. Ticks, whose diet consists exclusively on vertebrate blood, rely on maternally inherited bacterial symbionts to supply B vitamins. While previously studied tick species consistently harbor a single lineage of those nutritional symbionts, we evidence here that the invasive tick Hyalomma marginatum harbors a unique dual-partner nutritional system between an ancestral symbiont, Francisella, and a more recently acquired symbiont, Midichloria. Using metagenomics, we show that Francisella exhibits extensive genome erosion that endangers the nutritional symbiotic interactions. Its genome includes folate and riboflavin biosynthesis pathways but deprived functional biotin biosynthesis on account of massive pseudogenization. Co-symbiosis compensates this deficiency since the Midichloria genome encompasses an intact biotin operon, which was primarily acquired via lateral gene transfer from unrelated intracellular bacteria commonly infecting arthropods. Thus, in H. marginatum, a mosaic of co-evolved symbionts incorporating gene combinations of distant phylogenetic origins emerged to prevent the collapse of an ancestral nutritional symbiosis. Such dual endosymbiosis was never reported in other blood feeders but was recently documented in agricultural pests feeding on plant sap, suggesting that it may be a key mechanism for advanced adaptation of arthropods to specialized diets.

Serological and Molecular Prevalence of Babesia caballi in Apparently Healthy Horses in Israel.

Babesia caballi is a tick-borne hemoparasite of equines and one of the causative agents of equine piroplasmosis, which poses a great concern for the equine industry regarding animal welfare and international horse movement. The parasite is endemic in Israel; however, its seroprevalence in the area was never evaluated due to antigenic heterogenicity in the gene used in the commercially available kit. Blood samples were collected from 257 horses at 19 farms throughout the country and screened for the presence of anti-B. caballi antibodies via an indirect immunofluorescent antibody test (IFAT) and for the presence of parasite DNA by nested PCR. The seroprevalence of B. caballi was 69.6% and its molecular prevalence was 9.7%. The geographical area, horse's sex, breed, housing, exposure to ticks, and specifically to Hyalomma marginatum, and co-infection with Theileria equi were found to be significantly associated with serologic exposure in univariable analysis, while the geographical area and horses' sex remained significant in the multivariable analysis. The results of this study demonstrate a high level of exposure to B. caballi and identify important risk factors for infection. The difference between the serological and molecular prevalence, probably related to parasite clearance, is also highlighted.

Twenty Years of Equine Piroplasmosis Research: Global Distribution, Molecular Diagnosis, and Phylogeny.

Equine piroplasmosis (EP), caused by the hemoparasites Theileria equi, Theileria haneyi, and Babesia caballi, is an important tick-borne disease of equines that is prevalent in most parts of the world. Infection may affect animal welfare and has economic impacts related to limitations in horse transport between endemic and non-endemic regions, reduced performance of sport horses and treatment costs. Here, we analyzed the epidemiological, serological, and molecular diagnostic data published in the last 20 years, and all DNA sequences submitted to GenBank database, to describe the current global prevalence of these parasites. We demonstrate that EP is endemic in most parts of the world, and that it is spreading into more temperate climates. We emphasize the importance of using DNA sequencing and genotyping to monitor the spread of parasites, and point to the necessity of further studies to improve genotypic characterization of newly recognized parasite species and strains, and their linkage to virulence.

Infection dynamics of Theileria equi in carrier horses is associated with management and tick exposure.

The tick-borne equine hemoparasite, Theileria equi, is endemic in many parts of the world where prevalence may be high, and most infected horses are apparently healthy but serve as life-long carriers. To determine the factors that affect T. equi dynamics, we followed parasitic loads in apparently healthy horses at four time points during one year. A total of 1094 blood samples were collected from 395 horses, along with ticks and demographic and clinical data. Infection and load of T. equi were tested by PCR and qPCR, and for the spring dataset, infection was also tested serologically by IFAT (n = 268). Theileria equi was molecularly detected in 64.8 % of the horses. The agreement between molecular and serological results was 79.8 % (K > 0.674) and positively correlated with parasitic load. Infection was associated with pale mucus membranes, lower packed cell volume and higher total solids (all P < 0.001), although these changes had only minor clinical importance. While parasitic loads in qPCR-positive samples (n = 561) were generally low (mean = 7.9-10-4% parasitized erythrocytes), younger horses showed higher loads, possibly suggesting development of immunity. Infection and parasitic load were associated with housing management and tick exposure, illustrating different patterns of exposure. Endemic stability is suggested in pasture farms with constant exposure to ticks, where parasite prevalence was high (96 %) and associated with T. equi 18S rRNA genotype D, low parasitemia and high antibody titers. Endemic instability can be suggested in case were horses are kept in paddocks (prevalence = 49 %) with intermittent exposure to ticks, where infection was associated with high parasitemia when ticks were present. A steady state is suggested in stabled horses (prevalence = 46 %), with no exposure to ticks; where infection was associated with genotype A, low parasitemia and low antibody titers. The ability to identify different risk groups within endemic areas may improve the administration of suitable treatment and control practices in an effort to reduce the risk of clinical disease.

Cellular Localization of Two Rickettsia Symbionts in the Digestive System and within the Ovaries of the Mirid Bug, Macrolophous pygmaeus.

Bacterial symbionts in arthropods are common, vary in their effects, and can dramatically influence the outcome of biological control efforts. Macrolophus pygmaeus (Heteroptera: Miridae), a key component of biological control programs, is mainly predaceous but may also display phytophagy. M. pygmaeus hosts symbiotic Wolbachia, which induce cytoplasmic incompatibility, and two Rickettsia species, R. bellii and R. limoniae, which are found in all individuals tested. To test possible involvement of the two Rickettsia species in the feeding habits of M. pygmaeus, we first showed that the microbiome of the insect is dominated by these three symbionts, and later described the distribution pattern of the two Rickettsia species in its digestive system. Although both Rickettsia species were located in certain gut bacteriocyes, in caeca and in Malpighian tubules of both sexes, each species has a unique cellular occupancy pattern and specific distribution along digestive system compartments. Infrequently, both species were found in a cell. In females, both Rickettsia species were detected in the germarium, the apical end of the ovarioles within the ovaries, but not in oocytes. Although the cause for these Rickettsia distribution patterns is yet unknown, it is likely linked to host nutrition while feeding on prey or plants.

Convergence of Nutritional Symbioses in Obligate Blood Feeders.

Symbiosis with intracellular or gut bacteria is essential for the nutrition of animals with an obligate blood-feeding habit. Divergent bacterial lineages have independently evolved functional interactions with obligate blood feeders, but all converge to an analogous biochemical feature: the provisioning of B vitamins. Although symbionts and blood feeders coevolved interdependently for millions of years we stress that their associations are not necessarily stable. Ancestral symbionts can be replaced by recently acquired bacteria with similar biochemical features, a dynamic that emerges through a combination of phylogenetic and ecological constraints. Specifically, we highlight the lateral transfer of a streamlined biotin (B7 vitamin) operon, and conjecture that its extensive spread across bacterial lineages may drive the emergence of novel nutritional symbioses with blood feeders.

Coxiella-Like Endosymbiont of Rhipicephalus sanguineus Is Required for Physiological Processes During Ontogeny.

Obligatory hematophagous arthropods such as lice, bugs, flies, and ticks harbor bacterial endosymbionts that are expected to complement missing essential nutrients in their diet. Genomic and some experimental evidence support this expectation. Hard ticks (Acari: Ixodidae) are associated with several lineages of bacterial symbionts, and very few were experimentally shown to be essential to some aspects of tick's fitness. In order to pinpoint the nature of interactions between hard ticks and their symbionts, we tested the effect of massive elimination of Coxiella-like endosymbionts (CLE) by antibiotics on the development and fitness of the brown dog tick (Rhipicephalus sanguineus). Administration of ofloxacin to engorged (blood fed) nymphs resulted in significant and acute reduction of their CLE loads - an effect that also persisted in subsequent life stages (aposymbiotic ticks). As a result, the post-feeding development of aposymbiotic female (but not male) nymphs was delayed. Additionally, aposymbiotic adult females needed a significantly prolonged feeding period in order to replete (detach from host), and had reduced engorgement weight and a lower capacity to produce eggs. Consequently, their fecundity and fertility were significantly reduced. Eggs produced by aposymbiotic females were free of CLE, and the resulting aposymbiotic larvae were unable to feed successfully. Our findings demonstrate that the observed fitness effects are due to CLE reduction and not due to antibiotic administration. Additionally, we suggest that the contribution of CLE is not mandatory for oocyte development and embryogenesis, but is required during feeding in females, when blood meal processing and tissue buildup are taking place. Presumably, under these extreme physiological demands, CLE contribute to R. sanguineus through supplementing essential micro- and macronutrients. Further nutrient complementary studies are required to support this hypothesis.

Parasite load and genotype are associated with clinical outcome of piroplasm-infected equines in Israel.

BACKGROUND: Equine piroplasmosis is a highly endemic protozoan disease of horses worldwide, caused by Theileria equi and Babesia caballi. While most horses in endemic areas are subclinically infected, the mechanisms leading to clinical outcome are vastly unknown. Moreover, since clinical signs of disease are not specific, and the prevalence in endemic areas is high, it is difficult to determine if equine piroplasmosis is the cause of disease. To identify possible mechanisms leading to the clinical outcome in an endemic area, we compared parasite loads and genotypes in clinically and subclinically infected horses. METHODS: Blood was collected from horses with clinical signs consistent with equine piroplasmosis, and from apparently healthy horses in Israel. Packed cell volume and total solids were measured. Quantitative and diagnostic polymerase chain reaction were used to identify, quantify and classify equine piroplasmosis infection. Phylogenetic analyses were used to determine the genotype of both parasites. RESULTS: For both parasites, clinical cases were associated with low mean packed cell volume and high mean parasite load (P < 0.001), enabling the determination of a cut-off value to distinguish between clinically and subclinically infected horses. Samples of Theileria equi from subclinical horses were classified into three different 18S rRNA genotypes, D (n = 23), A (n = 12) and C (n = 5), while samples from all clinical cases (n = 6) were classified as genotype A. The sequences of T. equi equi merozoite antigens 1 (ema-1, n = 9) and 2 (ema-2, n = 11) genes were fairly conserved and did not differ between clinical and subclinical cases. Babesia caballi rhoptry associated protein-1 (rap-1) was classified into sub-genotypes A1 (n = 14) and A2 (n = 5) with no association to clinical outcome. Classification of the 18S rRNA gene (sub-genotypes B1 and B2) agreed with the rap-1 classification. CONCLUSIONS: The results of this study suggest that quantification of parasite loads of infected horses may be used to distinguish between infections resulting in disease and subclinical cases. Although number of clinical cases is limited, we identified T. equi 18S rRNA genotype A to be associated with clinical disease. This finding emphasizes the importance of in-depth genetic characterization of T. equi genotypes to identify possible markers for virulence.

Transplacental Transmission of Theileria equi Is Not a Common Cause of Abortions and Infection of Foals in Israel.

Although the main route of transmission of Theileria equi is through tick feeding, transplacental transmission is also possible and may lead to abortion, or to the birth of a sick or carrier foal. The aim of this study was to evaluate the role of T. equi as a cause of abortions in Israel and the risk of foals being infected at a young age. Eight aborting mares were serologically evaluated for exposure to T. equi via the immunofluorescence antibody test (IFAT) and their aborted fetuses were evaluated using PCR and qPCR. In addition, five mares and their foals (aged 4-6 months) from a highly endemic farm were tested for T. equi infection using IFAT, PCR and qPCR. Five of the eight aborting mares were seropositive for T. equi; however, none of the aborted fetuses was infected. All five mares from the endemic farm were subclinically infected with T. equi. Of their five foals, one was infected, with relatively high parasitemia and different parasite genotype than its dam's, suggesting another source of infection. The results of this study suggest that transplacental transmission of T. equi is not common and does not appear to be a prominent cause of abortion in chronically infected mares.

Stress conditions do not affect Theileria equi parasitemia levels in sub-clinically infected horses.

Stress has been suggested as a risk factor for Theileria equi peracute disease and may lead to relapse in clinical signs in chronically infected horses. The aim of this study was to assess the effect of stress on T. equi parasitemia in sub-clinically infected horses in two settings: horses hospitalized at a veterinary teaching hospital and horses from an endurance farm. Blood samples were collected from the hospitalized horses (n = 32) upon admission (T0) and at discharge (T1) from the hospital, and results were compared between horses that underwent surgery (stress) and other hospitalized horses (control). Blood samples were collected from an endurance farm (n = 20) six weeks before (T0) and two days after (T1) participation in an 80-km endurance event, and results were compared between horses that participated (stress) or did not participate (control) in the event. Theileria equi parasite load was determined using qPCR, and T1/T0 ratio was calculated for each horse. Mean parasite load at both time points did not differ statistically between the stress group and the controls in both settings. Theileria equi genotype was determined based on the 18S rRNA gene, when possible. Parasite genotypes were similar to strains previously characterized in the region and classified as genotypes A and D. The results of this study contradict the common assumption that stress may lead to increased parasite load in horses with a subclinical T. equi infection.

Genetic characteristics of Theileria equi in zebras, wild and domestic donkeys in Israel and the Palestinian Authority.

Equine piroplasmosis (EP) is an important tick-borne disease of equids, caused by Theileria equi and Babesia caballi. It is endemic in most parts of the world, including Israel, and has clinical and economic consequences. This study was set to evaluate the presence of EP parasites in domestic donkeys and in wild equids in Israel and the Palestinian Authority (PA). To assess subclinical EP infection in 98 domestic donkeys (Equus africanus asinus), 9 Asiatic wild donkeys (Equus hemionus), 8 zebras (Equus quagga), 7 African wild donkeys (Equus africanus) and 5 mules, were tested using PCR and qPCR. Positive samples were characterized by amplification and sequencing of a 1600 bp fragment of the 18S rRNA gene. Babesia caballi was not detected in any of the animals. Theileria equi was detected in 32% of the donkeys, 89% of Asiatic wild donkeys, 57% of African wild donkeys, 62% of zebras and none of the mules. Parasitemia was low in all of the positive samples. Risk factors associated with infection in donkeys included one farm (Kiryat Gat) and animal sex (male). The sequences of the 18S rRNA gene from domestic donkeys were all similar, and belonged to the T. equi genotype D, similar to the genotype sequenced from horses in the same area, while sequences from wild donkeys were unique and belonged to the T. equi genotype A. Verification of the T. equi genotype in zebras could not be concluded, suggesting major genetic variation.

Reproductive isolation due to prezygotic isolation and postzygotic cytoplasmic incompatibility in parasitoid wasps.

The reproductive barriers that prevent gene flow between closely related species are a major topic in evolutionary research. Insect clades with parasitoid lifestyle are among the most species-rich insects and new species are constantly described, indicating that speciation occurs frequently in this group. However, there are only very few studies on speciation in parasitoids. We studied reproductive barriers in two lineages of Lariophagus distinguendus (Chalcidoidea: Hymenoptera), a parasitoid wasp of pest beetle larvae that occur in human environments. One of the two lineages occurs in households preferably attacking larvae of the drugstore beetle Stegobium paniceum ("DB-lineage"), the other in grain stores with larvae of the granary weevil Sitophilus granarius as main host ("GW-lineage"). Between two populations of the DB-lineage, we identified slight sexual isolation as intraspecific barrier. Between populations from both lineages, we found almost complete sexual isolation caused by female mate choice, and postzygotic isolation, which is partially caused by cytoplasmic incompatibility induced by so far undescribed endosymbionts which are not Wolbachia or Cardinium. Because separation between the two lineages is almost complete, they should be considered as separate species according to the biological species concept. This demonstrates that cryptic species within parasitoid Hymenoptera also occur in Central Europe in close contact to humans.

Linking morphological and molecular taxonomy for the identification of poultry house, soil, and nest dwelling mites in the Western Palearctic.

Because of its ability to expedite specimen identification and species delineation, the barcode index number (BIN) system presents a powerful tool to characterize hyperdiverse invertebrate groups such as the Acari (mites). However, the congruence between BINs and morphologically recognized species has seen limited testing in this taxon. We therefore apply this method towards the development of a barcode reference library for soil, poultry litter, and nest dwelling mites in the Western Palearctic. Through analysis of over 600 specimens, we provide DNA barcode coverage for 35 described species and 70 molecular taxonomic units (BINs). Nearly 80% of the species were accurately identified through this method, but just 60% perfectly matched (1:1) with BINs. High intraspecific divergences were found in 34% of the species examined and likely reflect cryptic diversity, highlighting the need for revision in these taxa. These findings provide a valuable resource for integrative pest management, but also highlight the importance of integrating morphological and molecular methods for fine-scale taxonomic resolution in poorly-known invertebrate lineages.

Activity of native and commercial strains of Metarhizium spp. against the poultry red mite Dermanyssus gallinae under different environmental conditions.

The poultry red mite (PRM), Dermanyssus gallinae, is a major pest of laying hens with extremely limited control means. To evaluate the potential of natural and commercial entomopathogenic fungi (EPF) for use against D. gallinae, we tested four wild populations of D. gallinae from Israeli farms. The genus Aspergillus was identified as the most abundant isolates from the mites. Additionally, eight new isolates of Metarhizium belonging to the specie M. brunneum were identified. At all sites from which mites were collected in both seasons, the abundance of fungi on D. gallinae was greater during the winter season than during the summer season. Through indirect inoculations of adult D. gallinae, we examined the virulence of the native Metarhizium species, the commercial strain Ma-43 and a previously described acaropathogenic strain (Ma-7). All of the Metarhizium strains caused 56-95% mortality of adult mites by seven days after inoculation at a concentration of 5 × 105 conidia per cm2. The efficacies of Ma-43, Ma-7 and the most promising native strain were tested under optimal abiotic conditions (28°C; 85-100% RH) and abiotic conditions similar to those typically found in a poultry house (30 °C; 60% RH). Under optimal conditions, the efficacy of all three stains ranged between 85 and 92%. In contrast, under poultry-house conditions, the efficacy of control ranged between 30 and 40%. The incidence of mycoses on mite cadavers was significantly decreased under poultry-house conditions. These results demonstrate the potential of native and commercial Metarhizium strains for use as biopesticides. Future research should address suitable delivery methods and formulations for the effective control of D. gallinae under poultry-house conditions.

Species distribution and seasonal dynamics of equine tick infestation in two Mediterranean climate niches in Israel.

BACKGROUND: Ticks are important ectoparasites of horses that can affect animal welfare and vector several infectious, including zoonotic, diseases. In order to investigate the species distribution, epidemiology and seasonal dynamics of ticks infesting horses in Israel, 3267 ticks were collected from 396 horses in 24 farms across the country from July 2014 to June 2015. RESULTS: Ticks were found on 50% of the farms and on 25% of the horses, with Hyalomma being the most prevalent genus (70% of ticks). Pasture was the most prominent risk factor for tick infestation (99% of ticks, P < 0.001), and is represented here by two areas with a Mediterranean climate that differ in their environmental characteristics: the Golan Heights (GH, 74% of ticks); and the Carmel mountain ridge (CMR, 24%). Although these two sites are less than 100 km apart, the composition of the tick populations infesting horses differed significantly between them. In GH the most abundant tick species was Hyalomma excavatum (P < 0.001), while in CMR it was Hyalomma marginatum (P < 0.001). The GH also hosted a more diverse tick fauna than the CMR, including Haemaphysalis parva (peaking in the autumn, P < 0.001) and Rhipicephalus turanicus (peaking in the spring, P < 0.001), which were not found at the other sites. A few Rhipicephalus bursa, Hyalomma rufipes and Hyalomma turanicum were also found on horses. CONCLUSIONS: The current findings can be used in epidemiological studies assessing the risk of tick-borne equine diseases in the area. Further analysis is needed to determine the specific distribution and habitat preferences of each tick species.