Effects of Electromagnetic Radiation on Neuropeptide Transcript Levels in the Synganglion of Ixodes ricinus.

Anthropogenic electromagnetic radiation is an important environmental factor affecting the functionality of biological systems. Sensitivity to various frequencies of electromagnetic radiation has been detected in ixodid ticks in the past. However, the physiological aspects of radiation effects have not yet been studied in ticks. In the presented experiment, 360 Ixodes ricinus ticks, 180 males and 180 females, were divided into 16 irradiated and 8 control groups. The irradiated groups were exposed to two different intensities of electromagnetic radiation with a frequency of 900 MHz at different lengths of exposure time. RT-PCR was utilized to determine the changes in mRNA levels in tick synganglia after irradiation. Four randomly selected neuropeptide genes were tested-allatotropin (at), FGLa-related allatostatins (fgla/ast), kinin, and arginine-vasopressin-like peptide (avpl). A significant decrease in transcript levels in all female groups exposed to higher intensity radiofrequency radiation for 1 to 3 h was found. After one hour of radiofrequency exposure, a significant downregulation in allatotropin expression in males was detected. A consistent downregulation of the at gene was detected in males irradiated with at a higher intensity. Unfortunately, the specific functions of the studied neuropeptides in ticks are not known yet, so a more comprehensive study is necessary to describe the effects of EMF on observed neuropeptides. This study represents the first report on the effects of the abiotic environment on tick neurophysiology.

Interspecific differences in the behavioral response of ticks exposed to radiofrequency electromagnetic radiation.

Artificial electromagnetic radiation is a new environmental factor that affects animals. Experiments with the effect of radio frequency electromagnetic radiation were focused on both vertebrates and invertebrates. Ticks showed a significant affinity to radiation. Our study is a continuation of this research and its aim was to monitor the effect of radiation on the behavior of four tick species: Ixodes ricinus, Dermacentor reticulatus, Dermacentor marginatus and Haemaphysalis inermis. In total 1,200 ticks, 300 of each species, were tested in modules allowing the choice of an exposed or shielded area. During the test, the ticks were exposed to electro-magnetic radiation of 900 MHz for 24 h. The position of the individuals was recorded and we evaluated the obtained data statistically. We observed a significant preference to the exposed area in both sexes of I. ricinus. Males of D. reticulatus and D. marginatus also showed an affinity to radiation, but not females of both species, nor females and males of H. inermis. The results of the study support the assumption that ticks perceive the electromagnetic field and the observed differences in their response have the potential to help understand the mechanism of perception.

The Behavior of Rickettsia-Positive Dermacentor reticulatus Ticks under Laboratory Conditions.

Considering that tick-borne diseases are on the rise, a full understanding of how pathogen-tick-host interactions can lead to vector behavioral modifications is of high value. Successful transmission of pathogens to their hosts depends on vector mobility and their ability to quest for prey and attack hosts. In our research, the behavior of Dermacentor reticulatus ticks (n = 88) that were positive or negative for Rickettsia spp. (prevalence-36.36%) were analyzed using novel behavioral apparatuses. Tick locomotion and their preference for light or darkness were studied utilizing a multirod arena (MA) and a light/dark test (LDT) optimized for ticks. Behavioral tasks were evaluated using a Smart video-tracking system (Panlab, Spain). The majority of ticks (p < 0.0001) explored in the center of MA during the first 15 min. Despite that, most of them (p < 0.01) chose the periphery for questing or resting postures. They also preferred the elevated parts of the rods (p < 0.001) for this behavior. Ticks significantly (p < 0.0001) preferred the light part of the LDT. Rickettsia-infected ticks had higher locomotor activity, manifesting as longer trajectories (p = 0.0221). Our results revealed the possible impact of pathogens on some behavioral patterns of ticks as their vectors, which may significantly increase the probability of pathogen transmission.

Dermacentor reticulatus - a tick on its way from glacial refugia to a panmictic Eurasian population.

The ornate dog tick (Dermacentor reticulatus) shows a recently expanding geographic distribution. Knowledge on its intraspecific variability, population structure, rate of genetic diversity and divergence, including its evolution and geographic distribution, is crucial to understand its dispersal capacity. All such information would help to evaluate the potential risk of future spread of associated pathogens of medical and veterinary concern. A set of 865 D. reticulatus ticks was collected from 65 localities across 21 countries, from Portugal in the west to Kazakhstan and southern Russia in the east. Cluster analyses of 16 microsatellite loci were combined with nuclear (ITS2, 18S) and mitochondrial (12S, 16S, COI) sequence data to uncover the ticks' population structures and geographical patterns. Approximate Bayesian computation was applied to model evolutionary relationships among the found clusters. Low variability and a weak phylogenetic signal showing an east-west cline were detected both for mitochondrial and nuclear sequence markers. Microsatellite analyses revealed three genetic clusters, where the eastern and western cluster gradient was supplemented by a third, northern cluster. Alternative scenarios could explain such a tripartite population structure by independent formation of clusters in separate refugia, limited gene flow connected with isolation by distance causing a "bipolar pattern", and the northern cluster deriving from admixture between the eastern and western populations. The best supported demographic scenario of this tick species indicates that the northern cluster derived from admixture between the eastern and western populations 441 (median) to 224 (mode) generations ago, suggesting a possible link with the end of the Little Ice Age in Europe.

Behavioral Repertoire on a Vertical Rod-An Ethogram in Dermacentor reticulatus Ticks.

Ticks are important vectors of pathogens that endanger humans and animals. Study of their behavior under laboratory conditions is important for both predicting their behavior in natural conditions and understanding their involvement in transmission cycles of pathogens, which may lead to effective prevention of tick-borne disease transmission or establishment of effective preventive measures. The aim of our study was to describe the behavior of D. reticulatus ticks using laboratory assay. We focused on the description of individual behavioral units during their vertical movement. The assay consisted of glass beakers filled with sand and an embedded glass rod. We observed 10 different behavioral units, 4 of which have not yet been described: body posturing called "jogger", leg grooming, and body or leg jerking. The most frequent tick behavior observed was an upwards positioning of the two front legs while the body remained motionless (88.9%). Other common observations were both horizontal (63%) and vertical (58.0%) body posturing with all legs lowered, followed by questing behavior (51.9%). Ticks spent the most time questing (75.2%), crawling (54.7%), and grooming legs on the right side (23%). We did not observe any differences between males and females.

The first detection of relapsing fever spirochete Borrelia miyamotoi in Ixodes ricinus ticks from the northeast Czech Republic.

Borrelia miyamotoi, a relapsing fever spirochete, is considered a human pathogen. Knowledge of this borrelia is currently limited. Data about its potential impact on public health, circulation in nature, or its occurrence in natural environments are insufficient. For our study, a total of 505 questing Ixodes ricinus ticks (337 nymphs, 85 females and 83 males) from Hradec Králové Region in the Czech Republic were collected. Additionally, 160 winged Lipoptena deer keds from Hradec Králové Region, from Pardubice Region, Czech Republic, and from one location in western Slovakia were collected. The presence of B. miyamotoi in ticks and deer keds was determined using polymerase chain reaction (PCR) targeting a gene encoding glycerophosphodiester phosphodiesterase (glpQ), antigenic protein specific to the relapsing fever spirochetes. Borrelia miyamotoi was identified in six nymphs and four females of I. ricinus ticks. The overall prevalence was 2%. None of the examined Lipoptena specimens were found to be infected. Although no human case of infection with B. miyamotoi has been reported in the Czech Republic yet, this spirochete is widespread in ticks, and therefore the risk of human infection exists.

Locomotor Activity of Ixodes ricinus Females in 900 MHz Electromagnetic Field.

Mobile telecommunications technologies have become an indispensable part of people's lives of all ages around the world. They affect personal life and social interactions and are a work tool in the work routine. Network availability requirements and the quality of the Internet connection are constantly increasing, to which telecommunications providers are responding. Humans and wildlife live in the permanent presence of electromagnetic radiation with just a minor knowledge of the impact this radiation has. The aim of our study was to investigate the effect of a 900 MHz electromagnetic field (EMF) on the locomotor behavior of female Ixodes ricinus ticks under laboratory conditions. Experiments were performed in the radiation-shielded tube (RST) test and radiation-shielded circular open-field arena placed in an anechoic chamber. Altogether, 480 female I. ricinus ticks were tested. In the RST arena, no differences in preference for irradiated and shielded parts of experimental modules were observed; in the open-field arena, the time spent and the trajectory passed was significantly longer in the part exposed to the EMF.

Prevalence of relapsing fever spirochete Borrelia miyamotoi in Ixodes ricinus ticks from eastern Slovakia.

Borrelia miyamotoi, recurrent fever borrelia, pathogenic to humans, has been found recently in Ixodes ricinus ticks. There is still little information about its circulation in nature and potential local impact on human health as well as the occurrence in natural localities. In our study, a total of 1609 ticks (745 nymphs, 358 females and 506 males) collected from different localities in eastern Slovakia were analysed using a polymerase chain reaction targeting a gene encoding glycerophosphodiester phosphodiesterase (glpQ), which is specific to the Borrelia species in the relapsing fever group. B. miyamotoi was found in 3 nymphs, 6 females and 9 males of quested I. ricinus ticks. Although no human case of infection with B. miyamotoi has been reported in Slovakia yet, its presence needs to be studied to obtain knowledge regarding the prevalence and the distribution of this human pathogen in questing ticks and to increase medical healthcare awareness.

Morphometric analysis - effect of the radiofrequency interface of electromagnetic field on the size of hatched Dermacentor reticulatus larvae.

INTRODUCTION AND OBJECTIVE: Electromagnetic radiation interactions with living systems have been one of determining factors in biological evolution. This study investigates the effect of 900 MHz radiofrequency (RF) electromagnetics field (EMF) exposure of eggs on the development of Dermacentor reticulatus larvae. The basic objective was to determine whether the 900 MHz RF-EMF has the potential to influence the size of the body of the hatched larvae of D. reticulatus ticks. MATERIAL AND METHODS: To this aim, eggs from 3 fully engorged females of D. reticulatus were included in the test procedure. Altogether four groups of eggs were designated which included eggs from each female. We used RF-EMF frequency of 900 MHz. Eggs were exposed to EMF for different time periods (30, 60 and 90 minutes) in dark, electromagnetically shielded anechoic chamber. After the irradiation eggs were allowed to hatch in climatic chamber. Randomly selected 200 larval individuals were measured to get basic morphological records. Four body traits including the total body length (TBL), length of gnathosoma with scutum (GSL), the total body width (TBW), and the width of basis capituli (BCW) were measured. RESULTS: The D. reticulatus larvae hatched from eggs exposed for 60 minutes, had demonstrably larger dimensions of all measured body traits not only as a control unexposed group but also as other experimental groups. CONCLUSIONS: The study shows, particularly, that artificial EMF that is used in smartphone technology impacts seriously D. reticulatus larvae development.

Lack of evidence of vertical transmission of Karyolysus blood parasites in Iberian green lizards (Lacerta schreiberi).

To understand the spread of parasite and the persistence of infection in an ecological environment, it is essential to investigate their transmission possibilities. Vertical transmission of pathogens from mother to offspring is a fundamental opportunity, notwithstanding a relatively under-researched topic, especially in wildlife animals. We studied whether there is vertical transmission of a haemogregarinid blood parasite of Iberian green lizard (Lacerta schreiberi). To study infection of mothers, embryos and freshly hatched juveniles, their blood smears and molecular analyses of their tail tissue were used. Examining blood smears, seventy-one percent of females were found to be infected, but molecular analyses of all mothers showed positive results for the blood parasite. Based on molecular data the parasite was identified as a Karyolysus species. In contrast, no parasite was found in the blood smears of the freshly hatched juveniles, and we could not detect Karyolysus in the embryos or tail tissue of offspring using molecular methods either. Based on our results, vertical transmission of Karyolysus blood parasites is unlikely in the Iberian green lizard.

First report on parasites of European beavers in the Slovak Republic.

European beaver (Castor fiber L. 1758) is the biggest rodent living in Europe. It is a semi-aquatic animal known for building dams and burrows. European beaver is a potential host for a wide range of parasites and other infectious diseases. In Slovakia, there is an increasing number of beavers but the data about their parasitic fauna are missing. Our work is the first documentation about the beaver's parasitofauna in Slovakia. In a 1-year study, we collected and examined 19 beaver fecal samples from the vicinity of beaver burrows inhabiting three particular localities at the Danube, Topla, and Laborec rivers in Slovakia. In these fecal samples, 4 different species of intestinal endoparasites were detected as follows: oocysts of Cryptosporidium, cysts of Giardia, eggs of Stichorchis subtriquetrus, and eggs and larvae of Travassosius rufus. Parasites were confirmed only in samples collected at river Topla. Based on our results, we can conclude that European beaver can be an important source of parasitic contamination of surface waters especially in the localities shared by people.

Infected Ixodes ricinus ticks are attracted by electromagnetic radiation of 900 MHz.

The electromagnetic field (EMF) is known to influence functions of the nervous, cardiovascular and reproductive systems of many animals, including ticks. The aim of this study was to test the behavior of ticks in the presence of radio-frequency EMF. For testing, 160 adult male and 140 adult female unfed Ixodes ricinus ticks were used. Individuals were exposed to 900 MHz EMF in the Radiation-Shielded Tube (RST). Ticks were attracted to the irradiated area. This effect was significantly stronger for ticks infected with Rickettsia spp., suggesting that pathogens can alter the ticks' response to environmental stimuli. These results lead to the question of whether man-made EMF may have an impact on I. ricinus activity and, as such, be a contributing factor to the ongoing changes in the distribution of the tick and its pathogens currently observed in Europe and elsewhere.

Candidatus Cryptoplasma Associated with Green Lizards and Ixodes ricinus Ticks, Slovakia, 2004-2011.

During 2004-2011, we collected green lizards and Ixodes ricinus ticks in Slovak Karst National Park in Slovakia; 90% (36/40) of lizards and 37% of ticks removed from lizards were infected with family Anaplasmataceae bacteria. Only Candidatus Cryptoplasma sp. REP (reptile) was identified in these samples. Green lizards transmit this bacterium.

Tick distribution along animal tracks: implication for preventative medicine.

INTRODUCTION: Tick abundance and the prevalence of the pathogens they carry have been increasing worldwide in the last decades, and is projected to increase even further. Despite the fact that problem is global, there still remain many gaps in the diagnosis and treatment of tick-borne diseases. The best protection from tick-borne pathogens, therefore, is prevention and avoidance of bites. Ticks mobility is limited so that their spatial distribution is strongly correlated with the presence of, especially with large mammals. In this study, the hypothesis was tested that tick abundance is higher on animal tracks in the forests than in adjacent habitats. This is an important issue because there are still several human habits and practices that can decrease the zoonoses risk. For example, during recreation in forest, people should always walk on the paths (including narrow animal's tracks) instead of wading through bushes. MATERIAL AND METHODS: Flagging of animal trails and near control transects were performed simultaneously. Next, collected ticks were counted, sexed and aged. RESULTS: The abundance of ticks was almost 5-fold (Ixodes ricinus) and 3-fold (Dermacentor spp.) higher on animal trails than on adjacent control transects. CONCLUSIONS: The results obtained support the hypothesis that ticks are more abundant on pathways than in adjacent habitats. Most likely, the pattern emerges because large mammals, like deer, which are the most important ticks hosts, use forest paths to move across the landscape and frequently move along the same routes. This research sends an important public message that these forest trails are hotspots of disease risk and should be avoided.

Electromagnetic radiation and behavioural response of ticks: an experimental test.

Factors associated with the increased usage of electronic devices, wireless technologies and mobile phones nowadays are present in increasing amounts in our environment. All living organisms are constantly affected by electromagnetic radiation which causes serious environmental pollution. The distribution and density of ticks in natural habitats is influenced by a complex of abiotic and biotic factors. Exposure to radio-frequency electromagnetic field (RF-EMF) constitutes a potential cause altering the presence and distribution of ticks in the environment. Our main objective was to determine the affinity of Dermacentor reticulatus ticks towards RF-EMF exposure. Originally designed and constructed radiation-shielded tube (RST) test was used to test the affinity of ticks under controlled laboratory conditions. All test were performed in an electromagnetic compatibility laboratory in an anechoic chamber. Ticks were irradiated using a Double-Ridged Waveguide Horn Antenna to RF-EMF at 900 and 5000 MHz, 0 MHz was used as control. The RF-EMF exposure to 900 MHz induced a higher concentration of ticks on irradiated arm of RST as opposed to the RF-EMF at 5000 MHz, which caused an escape of ticks to the shielded arm. This study represents the first experimental evidence of RF-EMF preference in D. reticulatus. The projection of obtained results to the natural environment could help assess the risk of tick borne diseases and could be a tool of preventive medicine.

Patterns in the distribution and directional asymmetry of fleas living on the northern white-breasted hedgehog Erinaceus roumanicus.

Fleas infecting northern white-breasted hedgehogs, Erinaceus roumanicus (Barrett-Hamilton), collected from 2009-2011 in Budapest (Hungary) were studied. A total of 305 white-breasted hedgehogs were captured and 1,251 fleas were collected. The flea community comprised two species, the hedgehog flea Archaeopsylla erinacei (Bouche, 1835) and the dog flea Ctenocephalides canis (Curtis, 1826), although the latter was only found on three hedgehogs. Fleas were found on half of the host specimens (51%; n = 156) where their distribution was strongly aggregated. The sex ratio of A. erinacei was biased towards females and was correlated with host size. Interestingly, the sex ratio of fleas became more equal on heavier hosts. It had been expected that, under high competition, the sex ratio would be female biased because it is known that female ectoparasites dominate on poorer hosts. The body size of a random sample of 200 fleas (100 female and 100 male) was measured under a microscope. The analyses showed directional asymmetry in two features - the distance between the top of the head and the eye, and head length. In this two body traits the left side was significantly greater than right side in both sexes of A. erinacei. Our data shed light on the complex nature of the flea population infecting northern white-breasted hedgehogs in an urban area.

Identification of Hepatozoon erhardovae Krampitz, 1964 from bank voles (Myodes glareolus) and fleas in Southern Hungary.

In order to investigate the prevalence and life cycle of apicomplexan parasites, small mammals were live-trapped with modified Sherman traps in Southern Hungary between 2010 and 2012. Altogether, 528 rodents (Apodemus flavicollis Melchior, 1834, Apodemus agrarius Pallas, 1771, Myodes glareolus Schreber, 1780, Microtus agrestis Linnaeus, 1761, Mus musculus Linnaeus, 1758 and Micromys minutus Pallas, 1771) were collected and four shrews (Sorex spp.) were by-catched. Captured animals belonging to non-protected species were euthanized, and spleen samples were preserved for histological and molecular analyses. During the examination of spleen smears, Hepatozoon parasites were observed in eight out of 48 bank voles (M. glareolus). DNA was isolated from altogether 221 spleen samples, and 18S rDNA was amplified using two different PCR protocols. The eight bank vole samples were positive with PCR, but none of the other M. glareolus spleen samples or any of the tissue samples from other species were found to be infected. Sequenced amplicons were very similar to Hepatozoon spp. detected in M. glareolus in Spain and Poland. Ectoparasites were collected from the small mammal carcasses and from the vegetation. Hepatozoon DNA was not found in the 181 ticks removed from the small mammals or in the 162 ticks collected with flagging, but was detected in all three flea species (4/43 Megabothris turbidus Rothschild, 1909, 3/10 Ctenophthalmus assimilis Taschenberg, 1880 and 7/78 Ctenophthalmus agyrtes Heller, 1896). Based on gamont morphology, vertebrate and arthropod host species and DNA sequences, the parasites in our study can be identified as Hepatozoon erhardovae.

Morphological and molecular characterization of Karyolysus--a neglected but common parasite infecting some European lizards.

BACKGROUND: Blood parasites of the genus Karyolysus Labbé, 1894 (Apicomplexa: Adeleida: Karyolysidae) represent the protozoan haemogregarines found in various genera of lizards, including Lacerta, Podarcis, Darevskia (Lacertidae) and Mabouia (Scincidae). The vectors of parasites are gamasid mites from the genus Ophionyssus. METHODS: A total of 557 individuals of lacertid lizards were captured in four different localities in Europe (Hungary, Poland, Romania and Slovakia) and blood was collected. Samples were examined using both microscopic and molecular methods, and phylogenetic relationships of all isolates of Karyolysus sp. were assessed for the first time. Karyolysus sp. 18S rRNA isolates were evaluated using Bayesian and Maximum Likelihood analyses. RESULTS: A total of 520 blood smears were examined microscopically and unicellular protozoan parasites were found in 116 samples (22.3% prevalence). The presence of two Karyolysus species, K. latus and K. lacazei was identified. In total, of 210 samples tested by polymerase chain reaction (PCR), the presence of parasites was observed in 64 individuals (prevalence 30.5%). Results of phylogenetic analyses revealed the existence of four haplotypes, all part of the same lineage, with other parasites identified as belonging to the genus Hepatozoon. CONCLUSIONS: Classification of these parasites using current taxonomy is complex - they were identified in both mites and ticks that typically are considered to host Karyolysus and Hepatozoon respectively. Furthermore although distortions to the intermediate host erythrocyte nuclei were observed, the defining characteristic of Karyolysus, the haplotypes were nearly identical to those reported from lizards in the Iberian Peninsula, where such distortions were not reported and which were thus identified as Hepatozoon. Based on the phylogenetic analyses, neither vertebrate host, nor geographical patterns of the studied blood parasites could be established.

Anaplasmataceae and Borrelia burgdorferi sensu lato in the sand lizard Lacerta agilis and co-infection of these bacteria in hosted Ixodes ricinus ticks.

BACKGROUND: Anaplasmataceae and Borrelia burgdorferi s.l. are important tick-borne bacteria maintained in nature by transmission between ticks and vertebrate hosts. However, the potential role of lizards as hosts has not been sufficiently studied. RESULTS: The current study showed that 23 of 171 examined sand lizards Lacerta agilis were PCR positive for Anaplasmataceae. The nucleotide sequences of the several selected PCR products showed 100% homology with Anaplasma spp. found in Ixodes ricinus collected in Tunisia and Morocco (AY672415 - AY672420). 1.2% of lizard collar scale samples were PCR positive for B. lusitaniae. In addition, 12 of 290 examined I. ricinus were PCR positive for B. burgdorferi s.l. and 82 were PCR positive for Anaplasmatacea. The number of ticks per lizard and the number of ticks PCR positive for both microorganisms per lizard were strongly correlated. Moreover, we found a significant correlation between numbers of ticks infected with Anaplasmataceae and with B. burgdorferi s.l. living on the same lizard. However, there was no significant correlation between detection of both bacteria in the same tick. CONCLUSIONS: To the best of our knowledge, this is the first report of Anaplasmataceae DNA and additionally the second report of B. burgdorferi s.l DNA detection in the sand lizard.

Polymerase chain reaction confirmation of Babesia canis canis and Anaplasma phagocytophilum in dogs suspected of babesiosis in Slovakia.

Canine babesiosis was considered an imported tick transmitted disease until the first case of autochthonous canine babesiosis in Slovakia was described in 2002. Since then, the number of cases kept increasing every year. The causative agent of babesiosis in dogs is not yet characterized; therefore, the aim of our study was to determine the agent and the rate of infection in the vector tick D. reticulatus in Slovakia. Babesia canis canis was detected in 80 out of 87 blood samples from dogs with clinical manifestations of babesiosis. Six dogs suspected of babesiosis tested positive for presence of Anaplasma phagocytophilum, and one mixed infection of B. c. canis and A. phagocytophilum was detected. B. c. canis was detected in 35.6% questing adults of D. reticulatus. The obtained sequences from blood samples showed 99.7% and from D. reticulatus, 99.4% similarity with the B. c. canis (AY072926) from dogs infected in Croatia. In our study, we characterized the agent of canine babesiosis from blood samples of naturally infected dogs and D. reticulatus, the vector tick. Further, the presence of A. phagocytophilum, bacterium responsible for the canine granulocytic anaplasmosis, was recorded in dogs for the first time in Slovakia.