Genetic and morphological characterization of Ixodes apronophorus from Western Siberia,Russia

Genetic variability of I. apronophorus from Western Siberia, Russia was examined using the nuclear internal transcribed spacer 2 (ITS2) and mitochondrial 16S rRNA and cytochrome c oxidase subunit 1 (cox1) genes and compared to those of Ixodes persulcatus and Ixodes trianguliceps from the same site. The I. apronophorus sequences demonstrated the highest nucleotide and haplotype diversity for both mitochondrial genes, whereas I. persulcatus was more variable in the nuclear ITS2. Phylogenetic analysis of the molecular sequence data showed that I. apronophorus differed from other Ixodes species, including Romanian I. apronophorus. The level of identity between 16S rRNA gene sequences of Siberian and Romanian I. apronophorus was only 91%; these sequences did not form a monophyletic group, indicating that I. apronophorus from Siberia and Romania could be different tick species. The analysis of morphological features of the Siberian I. apronophorus confirmed their consistency with those for the previously described I. apronophorus species. Based on the 16S rRNA and ITS2 sequences, Siberian I. apronophorus clustered together with Ixodes kazakstani and Ixodes scapularis, which are the recognized members of the Ixodes ricinus-I. persulcatus species complex within the subgenus Ixodes, and can be assigned to this complex.     

Natural hybridization of the ticks Ixodes persulcatus and Ixodes pavlovskyi in their sympatric populations in Western Siberia

Hybridization of ticks of the genus Ixodes has been described for several species under laboratory conditions although no molecular genetics evidence confirming interspecific hybridization in nature is available. We have designed a real time PCR targeted on nuclear (ITS2) and mitochondrial (cox1) markers to accurately identify tick species and to detect interspecific hybrids of Ixodes persulcatus and Ixodes pavlovskyi in their sympatric populations in Western Siberia. A survey of 783 individual ticks from a suburb of Tomsk showed that 44.2% of ticks belong to I. pavlovskyi species and 55.8% to I. persulcatus, based on the mtDNA data. Results obtained with the nuclear marker were not consistent, indicating that approximately 10% of the ticks were hybrids and about 5% revealed mtDNA introgression. Both hybridization and introgression have been shown to occur bidirectionally but more efficiently in the mating pair female I. pavlovskyi × male I. persulcatus than vice versa. The existence of the first generation hybrids and backcrosses challenges the existing view about effective reproductive barriers between I. pavlovskyi and I. persulcatus. While using only mitochondrial markers can lead to errors in determining tick species, we propose to use nuclear or both markers instead. The results obtained in the present paper and published earlier suggest that hybridization between closely related tick species in their sympatric zones is common rather than exceptional. The role of hybrid populations of vectors in the evolution of transmitted pathogens is also discussed.     

DNA条形码技术用于进口货物中肩突硬蜱的鉴定简

目的利用DNA条形码技术对入境货物中截获未知种类的蜱进行种属鉴定。方法抽取蜱DNA,用特异性扩增线粒体细胞色素C氧化酶第Ⅰ亚基（COⅠ）基因片段,并测序。利用NCBI进行BLAST比对。结果蜱样本COⅠ基因序列与肩突硬蜱（Ixodes scapularisSay,1821）COⅠ基因匹配度达100%。形态学上,该蜱符合肩突硬蜱的形态学特征。该蜱种在我国尚无检出报道。结论该蜱为肩突硬蜱。这是国内首次报道从进口货物中截获肩突硬蜱。     

Description of a new species of Ixodes (Acari: Ixodidae) and first report of Ixodes lasallei and Ixodes bocatorensis in Brazil

In a recent study, we relegated the taxon Ixodes aragaoi Fonseca, 1935 to a junior synonym of Ixodes fuscipes Koch, 1844, and reinstated Ixodes spinosus Neumann, 1899 (a former synonym of I. fuscipes) to a valid species. We examined all lots of ticks formerly identified as I. fuscipes or I. aragaoi in three tick collections of Brazil. Through morphological analysis, some of the examined specimens could not be assigned to either I. fuscipes or I. spinosus based on the examination of the type specimens of these two species. Herein, we report these ticks to represent three different species: Ixodes catarinensis n. sp. Onofrio & Labruna, Ixodes lasallei Méndez Arocha and Ortiz, 1958, and Ixodes bocatorensis Apanaskevich and Bermúdez, 2017. The latter two species are reported for the first time in Brazil. Phylogenetic analysis inferred from the tick 16S rRNA gene partial sequences corroborated our morphological analysis, indicating that I. spinosus, I. lasallei, I. bocatorensis, and I. catarinensis n. sp. form a natural group of neotropical ticks. With the present study, the number of Ixodes species in Brazil increases from 9 to 12. We propose a new identification key for females and males of Ixodes species currently recognized in Brazil.     

Assessment of intraspecific mtDNA variability of European Ixodes ricinus sensu stricto (Acari: Ixodidae).

The Ixodes ricinus complex is composed of 14 species distributed worldwide. Some members of this complex are involved in the transmission of a number of diseases to animals and humans, in particular Lyme borreliosis, tick-borne encephalitis, ehrlichiosis and babesiosis. While the phylogenetic relationships between species of the I. ricinus complex have been investigated in the past, still little is known about the genetic structure within the species I. ricinus sensu stricto. We have investigated the intraspecific variability among 26 I. ricinus s.s. ticks collected in various European countries, including Switzerland, Italy, Austria, Denmark, Sweden, and Finland by using five mitochondrial gene fragments corresponding to the control region, 12S rDNA, cytb, COI, and COII. The five genes considered here showed a low genetic variability (1.6-5%). Our results based on both statistical parsimony (applied to the COI + COII + cytb + 12S + CR data set, for a total of 3423 bp) and maximum parsimony (applied to the COI + COII + cytb + 12S data set, for a total of 2980 bp) did not provide any evidence for a correlation between the identified haplotypes and their geographic origin. Thus, the European I. ricinus s.s. ticks do not seem to show any phylogeography structure.     

Phylogeographical structure of the tick Ixodes persulcatus: A novel view

The tick Ixodes persulcatus Schulze, 1930, has a wide distribution from the Baltic to the Far East and is a vector of a number of human pathogens. Thus, the study of the genetic structure and evolution of this species is of great epidemiological importance. rRNA genes were used as genetic markers to identify the phylogeographical structure of the ticks. The sequences of gene fragments of 28S (expansion segment D3) and mitochondrial 12S rRNA for 25 and 76 ticks, respectively, that had been collected in various regions of Russia in 2007–2011, were obtained. The sequences of the 28S rRNA D3 segment were identical for all ticks within the studied area. Analysis of the sequences of the mitochondrial 12S rRNA fragment revealed 4 haplotypes with one occurring at a frequency of 0.96. It is shown that the ‘deep’ population structure of I. persulcatus (McLain et al., 2001) was erroneous because of the inclusion of contaminating fungi sequences of 28S rRNA in the phylogenetic analysis. This was, possibly, due to the use of universal PCR primers that amplify the DNA of a wide range of eukaryotes, particularly of fungi which are common in samples of ticks. The influence of PCR conditions on the preferential amplification of the DNA of different organisms is also demonstrated.     

Isolation of Arsenophonus nasoniae from Ixodes ricinus ticks in Slovakia

The tick Ixodes ricinus is the most prevalent and widely distributed tick species in Central Europe, commonly found in woodlands, heaths, and forests and particularly abundant in the Alpine region. This tick readily bites humans and transmits a number of bacterial and viral pathogens. We collected 10 live nymphs of I. ricinus ticks from vegetation in the Rovinka forest, Slovakia, and isolated a strain of Arsenophonus nasoniae from one tick using the BME/CTVM2 cell line. A new isolate was then subcultured on axenic media (Columbia agar supplemented with 5% sheep blood). To the best of our knowledge, this bacterium was never previously isolated from hard ticks or identified in ticks in Europe. We amplified and sequenced the 16S rRNA, rpoB, and ftsY genes. Limited genetic characterization showed that the isolated strain is almost identical to a strain from the parasitic wasp Nasonia vitripennis. Electron microscopy revealed a typical morphology of a Gram-negative bacterium, without pili or flagellae. Its role in human and animal pathology remains to be evaluated.     

Description of Amblyomma monteiroae n. sp. (Acari: Ixodidae), a parasite of the great horned owl (Strigiformes: Strigidae) in southern Brazil

In 2020, adult hard ticks (males and females) were collected from great horned owls [Bubo virginianus (Gmelin, 1788)] in the coastal region in southern Brazil. The engorged females were allowed to oviposit in the laboratory and hatched larvae could be obtained. Analyses of the external morphology of the adult ticks revealed that they represent a new species, which was named Amblyomma monteiroae n. sp. Partial sequences of the mitochondrial 16S rRNA gene and the nuclear second internal transcribed spacer (ITS2) were generated from a male and a female. Their 16S rRNA haplotypes were identical to each other and closest (96% identity) to corresponding sequences of Amblyomma parvitarsum Neumann, 1901, and 90% identical to Amblyomma neumanni Ribaga, 1902. Their ITS2 haplotypes were 95.8 to 96.0 identical to the single ITS-2 partial sequence of A. parvitarsum available in GenBank. In the phylogenetic trees inferred by both 16S rRNA and ITS2 partial sequences, A. monteiroae n. sp. formed a clade with A. parvitarsum, with A. neumanni branching sister to this clade. Amblyomma monteiroae n. sp. is genetically and morphologically related to A. parvitarsum. Both tick species are unique in combining the following morphological characters: scutum extensively ornate; eyes rounded and bulging; coxa I with two moderate pointed spurs, the external longer than the internal; a single triangular short spur on coxae II-III; presence of two spines on the tibia of legs II-IV; hypostomal dentition 3/3, trochanters without spurs. However, the males of the two species can be separated by specific features in palps and festoons, whereas the females differ in specific features of the coxal spurs. The larva of A. monteiroae n. sp. can be morphologically distinguished from A. parvitarsum only by morphometry, with the former species being slightly smaller. Currently, A. monteiroae n. sp. is restricted to southern Brazil, and the only known host is B. virginianus (Strigiformes: Strigidae). The present study increases the Amblyomma Brazilian fauna to 34 species.     

A survey of hard ticks associated with cave dwelling mammals in Turkey

Members of the subgenus Eschatocephalus Frauenfeld, 1853 are highly specialized cave-dwelling ectoparasites of bats. We conducted a comprehensive field-based survey on bat ticks in Turkey and provided information about the phylogenetic placement of collected species. Sampling was carried out at 26 caves from 18 provinces around Turkey between 2019 and 2021. Eighty-one tick specimens collected from the cave environment or on various cave roosting bats resulted in five species: Ixodes vespertilionis, Ixodes simplex, Ixodes ariadnae, Ixodes kaiseri, and Haemaphysalis erinacei. While I. simplex was the most frequently collected species with a rate of 56.2% mainly from bats (Miniopterus schreibersii), I. vespertilionis was the most prevalent species (65.4%) and found mainly on cave walls. The first record of I. ariadnae was provided for Turkey. Phylogenetic trees were built using mt 16S rDNA and COI markers. Our results demonstrated the presence of two distinct lineages of I. vespertilionis in Turkey; one lineage grouped with European isolates, whereas three sequences clustered separately. The phylogenetic pattern of I. simplex was consistent with previous results; this clade was clustered distantly to other bat tick species. The significance of the surprising records of H. erinacei and I. kaiseri in caves is also discussed.     

A seven-legged tick: Report of a morphological anomaly in Ixodes scapularis (Acari: Ixodidae) biting a human host from the Northeastern United States

Cases of morphological anomalies in the blacklegged tick, Ixodes scapularis (Acari: Ixodidae), have recently been reported from the Northeastern and upper Midwestern United States, potentially complicating identification of this important vector of human disease-causing pathogens. We hereby report a case of a morphological anomaly in I. scapularis, biting a human host residing in Norwich, Connecticut. Using a dichotomous morphological key, high-resolution and scanning electron microscopy images, as well as DNA sequencing, the tick was identified as an adult female I. scapularis with three legs on the left side of the abdomen versus four on the right side, which we believe is the first case of ectromely in an adult I. scapularis. Using diagnostic genes in polymerase chain reaction, the specimen tested positive for Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum, the causative agents for Lyme disease and anaplasmosis, respectively, and also showed evidence of a rickettsial endosymbiont. Here we discuss recent reports of morphological anomalies in I. scapularis, and emphasize the significance of additional studies of teratology in this important tick species and its potential implications.     

Ticks (Acari: Ixodidae,Argasidae) associated with wild birds in Argentina

The aim of this study was to report tick infestations on wild birds from four Phytogeographic Provinces of Argentina. A total of 1085 birds was captured (124 species, 97 genera, 29 families and 13 orders), and ticks were collected from 265 birds (48 species, 40 genera and five orders). A total of 1469 ticks (1102 larvae, 363 nymphs and 4 females) belonging to 15 tick species (Amblyomma calcaratum, Amblyomma dubitatum, Amblyomma nodosum, Amblyomma ovale, Amblyomma parvum, Amblyomma sculptum, Amblyomma tigrinum, Amblyomma triste, Haemaphysalis juxtakochi, Haemaphysalis leporispalustris, Ixodes auritulus sensu lato, Ixodes pararicinus, Ixodes silvanus, Ixodes sp. cf. I. affinis and Ornithodoros sp. cf. O. mimon). Eighty-one new associations between bird species and stages of tick species are detected. The families Thamnophilidae, Turdidae, Thraupidae, Passerellidae, Furnariidae and Troglodytidae were the most prevalent. According to the Phytogeographic Provinces involved in this study, the prevalence of infection for each of them in birds was: (1) Chaco: 28.2% (11 tick species); (2) Yungas: 22.0% (8 tick species); (3) Espinal: 11.1% (2 tick species); and (4) Pampa: 3.9% (1 tick species). This study provided information on the diversity of tick species that parasitize wild birds, the variability of the specific tick-bird associations between the different Phytogeographic Provinces and the relevance of some families of birds as hosts of different tick species.     

Phylogenetic analysis of Babesia species in China based on cytochrome b (COB) gene

In this study, a mitochondrial marker consisting of an approximately 550-bp region of the Cytochrome b genes (COB) was amplified by polymerase chain reaction (PCR) and sequenced from individual Babesia species. Sequence variation between Babesia species from China was 1.6–30.8%. The constructed phylogenetic tree based on the three unlinked gene sequences (partial COB gene, 18S rDNA and ITS) that evolve at different rates by the method of Neighbor-joining revealed the phylogenetic relationship of Babesia species in China compared with other published corresponding sequences from Babesia species. These data indicate that the 18S rDNA more reliably distinguish the deeper branches among some Babesia species than the partial COB gene and ITS, however, the partial COB gene sequence is better for recognizing close lineages among some Babesia species than the 18S rDNA and ITS sequences. So the combined phylogenetic analysis based on the multiple unlinked loci with different evolving rates can facilitate to establish the more reliable phylogenetic relationship of the Babesia genus. The data could be applicable for the survey of parasite dynamics, epidemiological studies as well as prevention and control of the disease.     

Pathogens in Ixodes persulcatus and Ixodes ricinus ticks (Acari,Ixodidae) in Karelia (Russia)

Ixodid ticks (Acarina, Ixodidae) are vectors of dangerous human infections. The main tick species that determine the epidemiological situation for tick-borne diseases in northern Europe are Ixodes ricinus and Ixodes persulcatus. In recent years, significant changes in the number and distribution of these species have been observed, accompanied by an expansion of the sympatric range. This work summarizes the data of long-term studies carried out in Karelia since 2007 on the infection of I. persulcatus and I. ricinus ticks with various pathogens, including new viruses with unclear pathogenic potential. As a result, tick-borne encephalitis virus (TBEV, Siberian genotype), Alongshan virus, several representatives of the family Phenuiviridae, Borrelia afzelii, Borrelia garinii, Ehrlichia muris, Candidatus Rickettsia tarasevichiae and Candidatus Lariskella arthropodarum were identified. Data were obtained on the geographical and temporal variability of tick infection rates with these main pathogens. The average infection rates of I. persulcatus with TBEV and Borrelia burgdorferi sensu lato were 4.4% and 23.4% and those of I. ricinus were 1.1% and 11.9%, respectively. We did not find a correlation between the infection rate of ticks with TBEV, B. burgdorferi s.l. and Ehrlichia muris/chaffeensis with the sex of the vector. In general, the peculiarities of the epidemiological situation in Karelia are determined by the wide distribution and high abundance of I. persulcatus ticks and by their relatively high infection rate with TBEV and B. burgdorferi s.l. in most of the territory, including the periphery of the range.     

Ixodes persulcatus,a major vector of Alphaproteobacteria in Russia

Ixodes persulcatus Schulze, 1930 (I. persulcatus) is distributed from the western to the eastern borders of Russia where it is found in the taiga and in mixed forests of the European type. This tick is a known vector of viral (tick-borne encephalitis virus), spirochetal (Borrelia spp.), and protozoan (Babesia spp.) agents. In this work, we analyzed the phylogenetic position of I. persulcatus within the Prostriata based on its 18S rRNA gene. Furthermore, we review the relationship of this tick with 9 Alphaproteobacteria from the genera Rickettsia, Ehrlichia, and Anaplasma which are found in I. persulcatus in the territory of Russia. These observations suggest that I. persulcatus may be one of the more important vectors and reservoirs of Alphaproteobacteria and other microorganisms in Russia.     

Anomalous morphologies in Ixodes scapularis feeding on human hosts

Cases of anomalous morphologies in the blacklegged tick, Ixodes scapularis, have been reported in both field-collected and human-biting specimen in the Northeastern and Midwestern United States, complicating the identification of this medically important tick species. We herein describe four cases of morphological anomalies in I. scapularis females exhibiting nanism and abnormally small genital apertures. We also report a female I. scapularis displaying slight asymmetry in the lower abdomen oriented toward the right side and an abnormal anal groove completely enclosing the anus. The identity of each specimen was confirmed using taxonomic keys, high resolution light and scanning electron microscopy imaging, and DNA sequencing of the 18S rRNA gene. All specimens described in this study were found parasitizing human hosts and were submitted to the Connecticut Agricultural Experiment Station-Tick Testing Laboratory in 2021 for species identification and pathogen screening. Here, we also discuss recent reports of teratological abnormalities in I. scapularis as well as likely causes for such deformities and potential implications.     

Synonymy of Ixodes aragaoi with Ixodes fuscipes,and reinstatement of Ixodes spinosus (Acari: Ixodidae)

Ixodes fuscipes Koch, 1844, described from Brazil, has been considered a valid species with records from Brazil and Peru. Ixodes spinosus Neumann, 1899, also described from Brazil, has been considered a synonym of I. fuscipes. In 2014, Ixodes aragaoi Fonseca, 1935 was redescribed as a valid species for Brazil and Uruguay. Until the present study, one major difference between the females of I. fuscipes and I. aragaoi was the horn-like auriculae in the former versus the ridge-like auriculae in the later, but this morphological difference was not supported after examination of the holotype of I. fuscipes and a syntype female of I. spinosus. Surprisingly, we found the type of I. fuscipes to have ridge-like auriculae, in contrast to the horn-like auriculae of I. spinosus. Comparisons of the I. fuscipes holotype with the syntypes of I. aragaoi revealed that they correspond to the same species. Therefore, we redescribe I. fuscipes, relegate I. aragaoi to a junior synonym of I. fuscipes, and reinstate I. spinosus as a valid species. After examining all lots of I. fuscipes in four tick collections, no specimen was recognized as I. fuscipes when compared with the type specimen of this taxon. On the other hand, specimens previously identified as I. aragaoi are now confirmed as I. fuscipes, with bona fide records for Brazil and Uruguay. Some of the specimens previously reported as I. fuscipes are now confirmed as I. spinosus, with records in two Brazilian biomes, Amazon and Atlantic rainforest. We present lists of the ticks examined in this study, with their current taxonomic status.     

Reconstruction of mitochondrial genomes from raw sequencing data provides insights on the phylogeny of Ixodes ticks and cautions for species misidentification

High-throughput sequencing (HTS) technology has profoundly been involved in sequencing whole genomes of several organisms in a fast and cost-effective manner. Although HTS provides an alternative biomonitoring method to the time-consuming and taxonomy-expertise dependent morphological approach, still we cannot rule out the possibility of the impediment and misidentification biases. In this article we aim to retrieve whole mitochondrial genome (mitogenome) sequences from publicly available raw sequencing data for phylogenetic comparison of Ixodes persulcatus. For this comparison, we sequenced whole mitogenomes of four I. persulcatus ticks from Japan and constructed mitogenomes from raw sequencing data of 74 I. persulcatus ticks from China. Bayesian phylogenetic trees were inferred by the concatenated fifteen mitochondrial genes. We further tested our results by the phylogenetic analysis of cytochrome c oxidase subunit 1 (cox1) gene and internal transcribed spacer 2 (ITS2) sequences. Our findings showed that 70 constructed mitogenomes from China were clustered with the sequenced four mitogenomes of I. persulcatus from Japan. We also revealed that mitogenome sequences retrieved from two data sets CRR142297 and CRR142298 were clustered with Ixodes nipponensis. Moreover, other two mitogenome sequences from CRR142310 and CRR142311 formed a clade with Ixodes pavlovskyi. The phylogenetic analysis of cox1 gene and ITS2 sequences confirmed the identification errors of these four samples. The overall phylogenetics in our study concluded that accurate morphological identification is necessary before implementing HTS to avoid any misidentification biases.     

First records of Ixodes brunneus (Acari: Ixodidae) in Delaware

Eight adult specimens of Ixodes brunneus (Acari: Ixodidae) were collected in Delaware via a combination of methods. The first was an engorged female recovered from a deceased Northern Mockingbird (Mimus polyglottos), a window strike victim, in Kent County. The other seven were four males and three females, all apparently unfed, collected by flagging and dragging vegetation in Kent, New Castle, and Sussex Counties during routine tick surveillance. These collections represent a new host record as well as new state and county records, and indicate a statewide presence of this species.     

Identification of novel conserved Ixodes vaccine candidates; a promising role for non-secreted salivary gland proteins

Ixodes ricinus and Ixodes scapularis are the main vectors for the causative agents of Lyme borreliosis and a wide range of other pathogens. Repeated tick-bites are known to lead to tick rejection; a phenomenon designated as tick immunity. Tick immunity is mainly directed against tick salivary gland proteins (TSGPs) and has been shown to partially protect against experimental Lyme borreliosis. TSGPs recognized by antibodies from tick immune animals could therefore be interesting candidates for an anti-tick vaccine, which might also block pathogen transmission. To identify conserved Ixodes TSGPs that could serve as a universal anti-tick vaccine in both Europe and the US, a Yeast Surface Display containing salivary gland genes of nymphal I. ricinus expressed at 24, 48 and 72 h into tick feeding was probed with either sera from rabbits repeatedly exposed for 24 h to I. ricinus nymphal ticks and/or sera from rabbits immune to I. scapularis. Thus, we identified thirteen TSGP vaccine candidates, of which ten were secreted. For vaccination studies in rabbits, we selected six secreted TSGPs, five full length and one conserved peptide. None of these proteins hampered tick feeding. In contrast, vaccination of guinea pigs with four non-secreted TSGPs – two from the current and two from a previous human immunoscreening - did significantly reduce tick attachment and feeding. Therefore, non-secreted TSGPs appear to be involved in the development of tick immunity and are interesting candidates for an anti-tick vaccine.     

Identification and functional analysis of ferritin 2 from the Taiga tick Ixodes persulcatus Schulze

Ferritin 2 (FER2) is an iron storage protein, which has been shown to be critical for iron homeostasis during blood feeding and reproduction in ticks and is therefore suitable as a component for anti-tick vaccines. In this study, we identified the FER2 of Ixodes persulcatus, a major vector for zoonotic diseases such as Lyme borreliosis and tick-borne relapsing fever in Japan, and investigated its functions. Ixodes persulcatus-derived ferritin 2 (Ip-FER2) showed concentration-dependent iron-binding ability and high amino acid conservation, consistent with FER2s of other tick species. Vaccines containing the recombinant Ip-FER2 elicited a significant reduction of the engorgement weight of adult I. persulcatus. Interestingly, the reduction of engorgement weight was also observed in Ixodes ovatus, a sympatric species of I. persulcatus. In silico analyses of FER2 sequences of I. persulcatus and other ticks showed a greater similarity with I. scapularis and I. ricinus and lesser similarity with Hyalomma anatolicum, Haemaphysalis longicornis, Rhipicephalus microplus, and R. appendiculatus. Moreover, it was observed that the tick FER2 sequences possess conserved regions within the primary structures, and in silico epitope mapping analysis revealed that antigenic regions were also conserved, particularly among Ixodes spp ticks. In conclusion, the data support further protective tick vaccination applications using the Ip-FER2 antigens identified herein.