Foci report on indigenous Dermacentor reticulatus populations in Belgium and a preliminary study of associated babesiosis pathogens

The occurrence of autochthonous clinical cases of canine and equine babesiosis in Belgium during the last two decades suggests that the vector of the pathogens responsible for these diseases, Dermacentor reticulatus (Ixodida: Ixodidae), may be present in this country. Consequently, evidence for the presence of this tick species in different locations within Belgium was investigated. Four different locations were monitored by flagging in 2010; these included the locations at which D. reticulatus was previously found on a dog in 2009 and on two red deer in 2007. Two different species of tick were identified, Ixodes ricinus (Ixodida: Ixodidae) and D. reticulatus. A total of 282 D. reticulatus adult ticks (98 males, 184 females) were collected from the four sites. Ticks were found mainly from early March until the end of May and a peak in activity was apparent in March. A Babesia spp. (Piroplasmida: Babesiidae) genus-specific polymerase chain reaction test based on the amplification of a fragment of the 18S rRNA gene was used to investigate the potential presence of Babesia spp. All DNA extracts isolated from the total tick samples yielded negative results. Additional studies to accurately determine the distribution and vectorial capacity of this important tick species in Belgium are warranted.     

Seasonal abundance and activity of the tick Dermacentor silvarum in northern China

Studies on the seasonal abundance and activity of larvae, nymphs and adults of Dermacentor silvarum (Ixodida: Ixodidae), both on and off the host, were carried out over a 2-year period from February 2008 to January 2010 in northern China. During the study period, feeding ticks were removed weekly from domestic sheep and free-living ticks were collected weekly by flag-dragging in four different habitat types. Rodents were captured and inspected for immature ticks from June to September 2008. The results of this study suggest that the ticks mainly reside in shrubs and complete only one generation per year with sequential seasonal distribution and little overlap between the activity of the larvae and nymphs. Adults were most prevalent from late February to late May and peaked in number in mid-April. Larvae were found from early June to early September and peaked in number in mid-July. Nymphs were mainly distributed from late June to late September and peaked in mid-August. Adult and nymphal D. silvarum were found primarily on the ears of sheep, but no larvae were found on sheep. Additionally, an overwintering male adult population was detected on sheep after October, but no free-living adults were found by dragging.     

Seasonal activity and spatial distribution of host-seeking adults of the tick Dermacentor variabilis

The seasonal temporal and spatial distribution and emergence of overwintering and summer cohorts of host-seeking American dog tick, Dermacentor variabilis (Say) (Acari: Ixodidae), adults were examined for 5 years in the Bluegrass region of Kentucky. Weekly drag cloth sampling was conducted from early March until late September 1992-1995 and early March until the end of May 1996. Overwintering adult cohorts became active in early to mid-April followed by bimodal host-seeking activity each year, with the duration and intensity of the first activity period being related to the previous year's summer cohort activity. The first activity peak extended from mid-April through late May and the second activity peak occurred in July. All host-seeking activity ceased by early August, except in 1995, when activity ended on 22 August. Laboratory survival of adults, collected weekly during 1993-1996, was used to estimate the emergence of summer adult cohorts. Summer adult cohorts became active in late May or early June and overlapped with overwintering adult host-seeking activity. The termination of host-seeking activity in overwintering adults could not be determined using the methods employed in this study and is most likely to have continued for the entire season. Host-seeking adults displayed contagious distribution patterns along trails bordering ecotone habitats, as determined by fitting a negative binomial frequency distribution to weekly sampling data. These patterns did not vary from year to year. In addition, the location of D. variabilis activity did not change from year to year, which suggests that microhabitats within apparently homogeneous environments enhance adult survival and lead to 'hot spots' of tick activity despite the ability of vertebrate hosts to disperse D. variabilis populations more widely. These data better define our understanding of D. variabilis adult seasonal and spatial host-seeking activity, estimate for the first time the intensity of summer cohort host-seeking activity, and relate summer cohort activity with spring activity the following year.     

Development and characterization of 12 polymorphic microsatellite loci in the American dog tick (Dermacentor variabilis)

Twelve polymorphic microsatellite loci were developed for the American dog tick (Dermacentor variabilis), an important vector of infectious diseases in humans and animals. Four multiplexed panels comprising the loci were developed and 45 ticks collected from two raccoons (Procyon lotor) were genotyped. The number of alleles per locus ranged from nine to 30, and single locus heterozygosities ranged from 0.18 to 0.93. Data generated using these markers will further our understanding of factors affecting gene flow in D. variabilis, thus helping to elucidate the transmission dynamics of diseases associated with this vector.     

Molecular detection of Babesia canis in Dermacentor reticulatus ticks collected in Slovakia

Dermacentor reticulatus ticks are recognized as the most important vectors of Babesia canis, the aetiological agent of canine babesiosis occurring throughout Europe. Vector competence of D. reticulatus for B. canis is well described and experimentally determined; however, by using molecular analysis it was proven so by one recent study in Russia. Herein, the additional molecular evidence of B. canis infection in D. reticulatus ticks collected in Slovakia is provided. Using PCR followed by sequencing of distinctive amplicons we determined the presence of Babesia canis canis in one of 100 tested adult ticks. Two zoonotic pathogens, Francisella tularensis and Coxiella burnetii, were previously isolated from D. reticulatus ticks in Slovakia. In our samples, we detected only the presence of F. tularensis.     

Genomic evidence for host-associated differentiation in an animal parasite,Dermacentor variabilis,the American dog tick

The process responsible for the formation of genetically distinct populations associated with different host species is known as host-associated differentiation (HAD). Many insect parasites of plants have been shown to exhibit HAD but there have been fewer studies of HAD in parasites of vertebrate animals. Previous to this study, HAD has been documented in at least three species of ticks. The American dog tick, Dermacentor variabilis (Say) (Acari: Ixodidae) was chosen as the focal species for this study due to its importance as the vector of tularemia and Rocky Mountain spotted fever. Previous population genetic studies of this tick found the existence of various haplotypes but the tick’s host origins were unknown. In this study, ticks were collected from 15 vertebrate host species to test for HAD using single nuclear polymorphisms (SNPs). In total, 136 individual D. variabilis ticks were sequenced using ddRADseq. Genomic evidence was found to point to D. variabilis exhibiting HAD on eight different hosts. A STRUCTURE analysis showed that the highest posterior probability was obtained with a population size of eight and these populations correlated with host species. Pairwise F_ST values were as high as 0.622 and indicated a range of genetic distinction between host groups. In addition, ticks collected from the vegetation appeared as one homogenous distinct genotype suggesting the existence of nidicolous (nest dwelling) and non-nidicolous genotypes. The identification of host race formation occurring in this animal parasite has implications for the understanding of D. variabilis pathogen transmission and targeted control efforts because genetically distinct populations can differ in traits relevant to these applications.     

Attraction of immature stages of the American dog tick (Dermacentor variabilis) to 2,6-dichlorophenol

To determine whether 2,6-dichlorophenol is solely a sex pheromone, the response to it by the various stages of the American dog tick, Dermacentor variabilis, were compared. In contrast to adults, 2,6-dichlorophenol was attractive to unfed nymphs and to unfed larvae. Use of this chemical also prompted the expression of a novel type of feeding posture behavior in adults. The overlap in attraction to other substituted phenols plus the lack of functional value of this response for larvae and nymphs rules out the possibility that 2,6-dichlorophenol is a general attractant. However, 2,6-dichlorophenol likely plays a dual role as an attachment stimulant in the adult tick.Undergraduate Research Program in Biology     

Optimal sampling and spatial distribution of Ixodes pacificus, Dermacentor occidentalis and Dermacentor variabilis ticks (Acari: Ixodidae)

A common method for sampling tick populations is flagging, which is a method of dragging a white cloth over a plant substrate for a fixed distance along a transect. Flagging over rough physical surfaces or using long subtransect lengths could lead to the underestimation of tick densities. Using estimates of the drop-off rates of adult Ixodes pacificus in flag sampling, optimal sampling schemes (the length and number of subtransects) were examined using the relationships between the tick drop-off rates (c), the tick density and distribution patterns and the roughness of the sampled plant substrate. It was found that the optimal number of subtransects and Lmax, the longest subtransect length which did not significantly underestimate the tick density from c, were affected by the tick density, substrate roughness and tick distribution pattern. This study also showed that the density and distribution of I. pacificus and Dermacentor occidentalis varied greatly over time in the populations sampled, while the Dermacentor variabilis densities were low and showed no significant changes over time. Both I. pacificus and D. occidentalis had clumped distributions along trails and these clumps were aggregated. However, the clump sizes (or individual clump areas) changed significantly over time because of density fluctuation or the movement of ticks. Finally, a positive association between the number of I. pacificus and D. occidentalis adults was observed from 2 m subtransect flag collections in March 1995; no relationship was found in 1994. © Rapid Science Ltd. 1998     

Tick infection rates with Borrelia Ixodes ricinus versus Haemaphysalis concinna and Dermacentor reticulatus in two locations in eastern Germany

Unfed nymphal Ixodes ricinus, Haemaphysalis concinna, and adult Dermacentor reticulatus were collected in two locations of Saxony in July and September 1991 by flagging. In July, the abundance of nymphal I. ricinus was about 2-3 times higher than that of nymphal H. concinna, a time of the year when nymphs of both species are reported to have a seasonal peak of activity. No D. reticulatus were flagged concurrently. In September, host-seeking activity of nymphal I. ricinus was again quite high as was that of adult D. reticulatus but only low numbers of nymphal H. concinna were collected. The flagged ticks were individually examined for Borrelia by an indirect immunofluorescence assay (I. ricinus: n = 414; H. concinna: n = 96; D. reticulatus: n = 116). The prevalence of Borrelia (probably B. burgdorferi) in I. ricinus varied from 12.1% to 21.0%. No borreliae were found in H. concinna. Of the examined D. reticulatus from one site (n = 97) 11.3% contained either B. burgdorferi or a related Borrelia. This may be the first finding of Borrelia in an Eurasian Dermacentor species.     

Evidence for the reproductive isolation of Dermacentor marginatus and Dermacentor reticulatus (Acari: Ixodidae) ticks based on cross-breeding, morphology and molecular studies

The species status of Dermacentor marginatus and Dermacentor reticulatus was evaluated by scanning electron microscope (SEM) examination of adult ticks, cross-breeding experiments and molecular biological analysis of eggs derived from transspecific pairings. The SEM investigations including the morphometric quantification of phenotypic features resulted in an unequivocal differentiation of adult D. marginatus and D. reticulatus ticks. The cross-breeding experiments demonstrated that irrespective of whether female ticks of both species were applied with con- or transspecific male ticks or without males to sheep, they engorged and laid eggs. The larvae, however, developed only in eggs which originated from conspecific matings. A nested polymerase chain reaction (PCR) of the second internal transcribed spacer (ITS2) using the DNA of eggs from transspecific pairings and sequencing of the PCR products revealed two different genotypes. The genotypes of eggs originating from D. marginatus and D. reticulatus females of these pairings differed. However, the eggs deposited by D. marginatus always possessed the same two genotypes as did the eggs produced by D. reticulatus. These results argue for a strict reproductive isolation of D. marginatus and D. reticulatus and, therefore, for a separate species status.     

Spirochete-like cells in a Dominican amber Ambylomma tick (Arachnida: Ixodidae)

Amber preserves microscopic, soft-bodies organisms and is a good medium in which to trace the evolution of pathogen–vector associations. Spirochetes-like cells (Spirochaetales: Spirochaetaceae) in the hemocoel and lumen of the alimentary tract of a larva tick (Amblyomma sp. Arachnida: Ixodidae) in Dominican amber are described in the collective fossil genus and species, Palaeoborrelia dominicanan. gen., n. sp. The size and shape of the fossil spirochetes closely resemble those of present-day Borrelia species. This discovery represents the first record of spirochete-like cells associated with fossil ticks.     

Frequent isolation of Francisella tularensis from Dermacentor reticulatus ticks in an enzootic focus of tularaemia

Abstract. A total of 924 questing Dermacentor reticulatus (Fabricius), 504 Ixodes ricinus (L.), sixty Haemaphysalis concinna Koch and 718 mosquitoes (Aedes spp.) were examined in a floodplain forest ecosystem during the 1994-95 outbreak of tularaemia in South Moravia, Czech Republic. Francisella tularensis was not isolated from H.concinna ticks or Aedes spp. mosquitoes, whereas twenty-one isolates were recovered from the other haematophagous arthropods. Dermacentor reticulatus revealed a significantly higher infection rate (2.6%) than I.ricinus (0.2%). This tick species acts as principal vector for tularaemia in the enzootic focus. Monitoring of D.reticulatus for F.tularensis thus seems to be a very efficient approach in the surveillance of tularaemia in the flood-plain forest ecosystems of Europe.     

Modus operandi of oviposition in Dermacentor reticulatus (Acari: Ixodidae)

The process of oviposition in D. reticulatus was observed and found to be a sequence of exactly coordinated, interlocking events independent of the phase of oviposition. The average period of oviposition in the investigated ticks was 31.6 days at 20 °C and 95% relative humidity. The number of eggs deposited on each day increased until reaching a maximum on the fifth day of oviposition and then decreased continuously. As a result, most of the eggs were deposited during the initial phase of oviposition. The total number of eggs was proportional to the ticks' weight replenishment. Egg-laying commenced with the lowering of the capitulum and the simultaneous spread of the pedipalps which were lowered to the body wall embracing the genital aperture on both sides. Immediately afterwards the cuticular sac of Gene's organ was pushed out and retracted several times. At the cuticular sac's maximum extension, the vestibulum vaginae prolapsed, forming the ovipositor as an extended tube which handed over an egg to the two horns of the cuticular sac after a brief, but intensive, contact with the cuticular sac. Then the vestibulum vaginae invaginated, the pedipalps closed, and the cuticular sac was retracted. Finally, the egg was transported onto the dorsal area of the tick by means of a vigorous rising of the capitulum. During the course of oviposition most of the events, especially the period of egg embracement by the cuticular sac, were prolonged, as was the total time for laying one egg. Similarly, the intervals between successive egg-laying processes increased continuously.The number of eggs deposited was not dependent on the functional ability of Gene's organ, as shown by similar numbers of deposited eggs from ticks with and without mechanical blocking of the cuticular sac. But the participation of the organ in the process of oviposition proved to be a prerequisite for the viability of the eggs. Larvae developed and hatched only from those eggs which were deposited from ticks with an undisturbed Gene's organ. In comparison, eggs without contact to the cuticular sac of Gene's organ dried up and shrivelled immediately after being deposited and did not hatch. Consequently, it strongly suggests, together with the results from other studies, that Gene's organ covers the eggs with a secretion that prevents the loss of water.     

The effect of female body size on male mounting behaviour in Dermacentor variabilis and D. andersoni

Abstract. This study was undertaken to determine if body size of female ticks was an important factor in eliciting the mating behaviour of Dermacentor variabilis and D. andersoni males. Dummy female ticks (DFTs) representing varying sizes of feeding females were prepared from plastic beads. Size of DFT was shown to be important in determining both the time spent by males in contact with DFTs and the extent of male mating response released. Dermacentor varlabilis males preferred the smaller-sized DFT, whereas D. andersoni males preferred the larger sizes. Males of both species were able to discriminate between size of DFT only when mounting sex pheromone (MSP) was present.
Size of the DFT was more important than the composition of the MSP extract in determining the time males spent in contact with DFTs.
Males of both species were sensitive to variations in concentration of extract, and spent 2–6 times longer in contact with DFTs when the concentration was optimal than when it was not. No significant difference in the male's mating response was seen in response to variation in concentration of heterospecific extract.
Male mating response, i.e. the behaviour of the male as it progresses through the initial contact, climbing onto the dorsal surface and turning onto the venter, was shown not to be dependent upon how long the male spent in contact with the DFT. Male D. andersoni had a much lower mating response to DFTs than D. variabilis males, suggesting that some further stimulus may be required.     

The role of direct chilling injury and inoculative freezing in cold tolerance of Amblyomma americanum, Dermacentor variabilis and Ixodes scapularis

Abstract. Supercooling points and chill tolerance were compared among nymphs and adults of the ixodid ticks Dermacentor variabilis, Amblyomma americanum and Ixodes scapularis (Acari: Ixodidae).Supercooling points in the range of <-22 to -18°C were observed for nymphs, and -22 to -8°C for adults.The lower lethal temperatures observed under dry conditions, -14 to -10°C, were warmer than the supercooling points, but still much colder than -4.8°C, the lowest temperature recorded from a likely tick habitat in southwestern Ohio.Based on our experiments, spontaneous freezing and direct chilling injury are not significant mortality factors in these species in the field.Mortality was observed between -5 and -3°C for A.americanum and D.variabilis nymphs chilled for 2 h while in direct contact with ice.This mortality is probably due to inoculative freezing.Given the requirement for a rather humid microhabitat for off-host survival, these findings suggest that inoculative freezing is an important cause of overwintering mortality in these medically important species.     

The meadow tick Dermacentor reticulatus in natural foci of tick-borne encephalitis in Udmurtia

In southern Udmurtia, where occur both Dermacentor reticulatus and Ixodes persulcatus, in the years of high abundance of small mammals feeding periods of larvae and nymphs of these ticks coincide completely. The duration of simultaneous parasitism of preimago of the above ixodid species on animals is of epidemiological significance as far as within this period the infection of young stages of D. reticulatus with the virus of tick-borne encephalitis occurs. This is confirmed by the isolation of I strain of the virus from D. reticulatus collected at the state of engorged nymphs on hosts common with I. persulcatus and moulted in the laboratory.     

Risk factors associated with ixodid tick species distributions in the Basque region in Spain

Ixodid tick abundance was investigated in the Basque region in Spain in two 1-year longitudinal studies, in 1992-1993 and 2003-2004. Forty zones were visited monthly and 162 672 ticks (87% larvae, 12% nymphs and 1% adults) were collected by blanket dragging. Eleven tick species belonging to the genera Ixodes, Haemaphysalis, Rhipicephalus and Dermacentor were identified including Haemaphysalis concinna Koch, which had not previously been reported in Spain. Tick species abundance differed between zones, studies and seasons. In 1992-1993, Haemaphysalis punctata Canestrini & Fanzago was the predominant species and distinct spring-summer and autumn-early winter peaks of activity were observed. In 2003-2004, Ixodes ricinus (Linneaus) was the most common species and was active throughout the winter. Larval and nymph seasonal activity patterns coincided in both 1993 and 2003 and this could facilitate co-feeding transmission of pathogens. Higher tick abundance was associated with increased livestock abundance in 1992-1993 and milder winter temperatures in 2003-2004. Tick collection rates in areas with moderate and high tick density were positively associated with the interaction between ambient temperature at sampling and rainfall 7 days prior to sampling. Collection rates were also significantly higher at medium rather than higher altitude, in forested areas than in open grasslands and lower in recreational areas frequented by people and with wet vegetation at sampling.     

The status of the barbel, Barbus barbus (L.) (Teleostei, Cyprinidae), in the United Kingdom

The barbel was originally a fish with a relatively restricted range in the British Isles. However, despite the general environmental degradation of many British rivers, and in contrast with some native freshwater fishes, the barbel is now more widely distributed than it was a century ago. It may even be more abundant numerically.     

Immunocytochemical mapping of FMRFamide-like peptides in the argasid tick Ornithodoros parkeri and the ixodid tick Dermacentor variabilis

FMRFamide-like immunoreactivity was studied in the argasid tick Ornithodoros parkeri and the ixodid tick Dermacentor variabilis using immunocytochemistry based on the peroxidase-antigeroxidase method. FMRFamide-like immunoreactive cells are widely distributed in various regions of the tick synganglion including protocerebral, cheliceral, stomodeal, palpal, pedal I–IV, and opisthosomal regions in both species. However, there is one layer of immunoreactive cells located on the dorsal surface of the postoesophageal part of the synganglion that is found only in D. variabilis. Besides the immunoreactivity within the cell body and its axons, the neuropile and the neural lamella (the extracellular sheath of the synganglion) are rich in immunoreactive materials. Some coxal muscles are innervated by the FMRFamide-like immunoreactive processes of the nerve from the pedal ganglion.     

Ultrastructural localization of acetylcholinesterase in the synganglion of the tick,Dermacentor variabilis (Say)

Summary Light- and electron-microscopic enzyme cytochemistry was used to localize acetylcholinesterase (AChE) activity in the synganglion (brain) of the tick Dermacentor variabilis. High AChE activity was observed throughout the neuropil as well as adjacent to most neuronal perikarya. Intracellular activity was not observed by light microscopy. By electron microscopy, reaction product was localized at the plasma membrane of glia and neurons. Enzyme activity was not associated with the olfactory globuli neurons. In other types of neurons, small amounts of reaction product were observed in the Golgi apparatus and nuclear envelope. Large neurosecretory neurons contained activity that appeared to be associated with deep invaginations of the plasma membrane as well as intracellular membranes. AChE activity was also associated with processes of both neurons and glia. In most peripheral nerves AChE activity was associated with virtually all axons. Clearly then, AChE is associated with glia and non-cholinergic neurons as well as with presumed cholinergic neurons. The widespread localization and large amounts of AChE in the tick brain exceeds that reported for other invertebrates and vertebrates. As has been suggested for other animals, AChE in the tick brain may have functions in addition to its known role in cholinergic neurotransmission.