Haplotype divergence supports long-term asexuality in the oribatid mite Oppiella nova.

Sex strongly impacts genome evolution via recombination and segregation. In the absence of these processes, haplotypes within lineages of diploid organisms are predicted to accumulate mutations independently of each other and diverge over time. This so-called "Meselson effect" is regarded as a strong indicator of the long-term evolution under obligate asexuality. Here, we present genomic and transcriptomic data of three populations of the asexual oribatid mite species Oppiella nova and its sexual relative Oppiella subpectinata We document strikingly different patterns of haplotype divergence between the two species, strongly supporting Meselson effect-like evolution and long-term asexuality in O. nova: I) variation within individuals exceeds variation between populations in O. nova but vice versa in O. subpectinata; II) two O. nova sublineages feature a high proportion of lineage-specific heterozygous single-nucleotide polymorphisms (SNPs), indicating that haplotypes continued to diverge after lineage separation; III) the deepest split in gene trees generally separates the two haplotypes in O. nova, but populations in O. subpectinata; and IV) the topologies of the two haplotype trees match each other. Our findings provide positive evidence for the absence of canonical sex over evolutionary time in O. nova and suggest that asexual oribatid mites can escape the dead-end fate usually associated with asexual lineages.

Molecular characterization and phylogenetic analysis of Argas persicus (Oken, 1818) (Acari: Argasidae) from domestic birds in eastern Algeria.

Argas persicus (the fowl tick) is a species of soft tick commonly associated with poultry farms. It has a wide geographic distribution and colonizes different climate regions. Morphological identification of A. persicus has been reported worldwide, but genetic data regarding its molecular characterization is limited. The present study provides data for morphological identification and genetic characterization of A. persicus collected from domestic birds in traditional farms from east Algeria (Setif region). Additionally, A. persicus samples originating from Gansu province in China were included for comparative molecular study. In total, 1518 ticks collected from 30 infested farms were examined and morphologically identified as A. persicus. Furthermore, the 14 tick samples obtained from China were morphologically identified as A. persicus. Molecular analysis of 30 ticks from Algeria (one tick from each infested farm) and the 14 Chinese samples based on PCR, sequencing, and phylogenetic analysis of three mitochondrial genetic markers (16S rRNA, 12S rRNA, and cox1) confirmed morphological results where all samples belonged to the A. persicus group. However, phylogenetic analysis showed that all Algerian samples and two Chinese samples belong to A. persicus sensu stricto (s.s.), while the remaining Chinese samples represented A. persicus sensu lato (s.l.) (divergent lineage). The present study confirms the occurrence of A. persicus s.s. both in Algeria and China, as well as provides novel molecular data for a distinct Chinese lineage of A. persicus.

Genetic Diversity of Hard Ticks (Acari: Ixodidae) in the South and East Regions of Kazakhstan and Northwestern China.

To date, there is no report on the genetic diversity of ticks in these regions. A total of 370 representative ticks from the south and east regions of Kazakhstan (SERK) and Xinjiang Uygur Autonomous Region (XUAR) were selected for molecular comparison. A fragment of the mitochondrial cytochrome c oxidase subunit I (cox1) gene, ranging from 631 bp to 889 bp, was used to analyze genetic diversity among these ticks. Phylogenetic analyses indicated 7 tick species including Hyalomma asiaticum, Hyalomma detritum, Hyalomma anatolicum, Dermacentor marginatus, Rhipicephalus sanguineus, Rhipicephalus turanicus and Haemaphysalis erinacei from the SERK clustered together with conspecific ticks from the XUAR. The network diagram of haplotypes showed that i) Hy. asiaticum from Almaty and Kyzylorda Oblasts together with that from Yuli County of XUAR constituted haplogroup H-2, and the lineage from Chimkent City of South Kazakhstan was newly evolved; and ii) the R. turanicus ticks sampled in Israel, Almaty, South Kazakhstan, Usu City, Ulugqat and Baicheng Counties of XUAR were derivated from an old lineage in Alataw City of XUAR. These findings indicate that: i) Hy. asiaticum, R. turanicus and Ha. erinacei shared genetic similarities between the SERK and XUAR; and ii) Hy. marginatum and D. reticulatus show differences in their evolution.

Identification and Molecular Analysis of Ixodid Ticks (Acari: Ixodidae) Infesting Domestic Animals and Tick-Borne Pathogens at the Tarim Basin of Southern Xinjiang, China.

Livestock husbandry is vital to economy of the Tarim Basin, Xinjiang Autonomous Region, China. However, there have been few surveys of the distribution of ixodid ticks (Acari: Ixodidae) and tick-borne pathogens affecting domestic animals at these locations. In this study, 3,916 adult ixodid ticks infesting domestic animals were collected from 23 sampling sites during 2012-2016. Ticks were identified to species based on morphology, and the identification was confirmed based on mitochondrial 16S and 12S rRNA sequences. Ten tick species belonging to 4 genera were identified, including Rhipicephalus turanicus, Hyalomma anatolicum, Rh. bursa, H. asiaticum asiaticum, and Rh. sanguineus. DNA sequences of Rickettsia spp. (spotted fever group) and Anaplasma spp. were detected in these ticks. Phylogenetic analyses revealed possible existence of undescribed Babesia spp. and Borrelia spp. This study illustrates potential threat to domestic animals and humans from tick-borne pathogens.

Genomic insights into mite phylogeny, fitness, development, and reproduction.

BACKGROUND: Predatory mites (Acari: Phytoseiidae) are the most important beneficial arthropods used in augmentative biological pest control of protected crops around the world. However, the genomes of mites are far less well understood than those of insects and the evolutionary relationships among mite and other chelicerate orders are contested, with the enigmatic origin of mites at one of the centres in discussion of the evolution of Arachnida. RESULTS: We here report the 173 Mb nuclear genome (from 51.75 Gb pairs of Illumina reads) of the predatory mite, Neoseiulus cucumeris, a biocontrol agent against pests such as mites and thrips worldwide. We identified nearly 20.6 Mb (~ 11.93% of this genome) of repetitive sequences and annotated 18,735 protein-coding genes (a typical gene 2888 bp in size); the total length of protein-coding genes was about 50.55 Mb (29.2% of this assembly). About 37% (6981) of the genes are unique to N. cucumeris based on comparison with other arachnid genomes. Our phylogenomic analysis supported the monophyly of Acari, therefore rejecting the biphyletic origin of mites advocated by other studies based on limited gene fragments or few taxa in recent years. Our transcriptomic analyses of different life stages of N. cucumeris provide new insights into genes involved in its development. Putative genes involved in vitellogenesis, regulation of oviposition, sex determination, development of legs, signal perception, detoxification and stress-resistance, and innate immune systems are identified. CONCLUSIONS: Our genomics and developmental transcriptomics analyses of N. cucumeris provide invaluable resources for further research on the development, reproduction, and fitness of this economically important mite in particular and Arachnida in general.

Gene cloning and difference analysis of vitellogenin in Neoseiulus barkeri (Hughes).

Neoseiulus barkeri (HUGHES) is the natural enemy of spider mites, whiteflies and thrips. Screening for chemically-resistant predatory mites is a practical way to balance the contradiction between the pesticide using and biological control. In this study, the number of eggs laid by fenpropathrin-susceptible and resistant strains of N. barkeri was compared. Additionally, we cloned three N. barkeri vitellogenin (Vg) genes and used quantitative real-time polymerase chain reaction to quantify Vg expression in susceptible and resistant strains. The total number of eggs significantly increased in the fenpropathrin-resistant strain. The full-length cDNA cloning of three N. barkeri Vg genes (NbVg1, NbVg2 and NbVg3) revealed that the open reading frames of NbVg1, NbVg2 and NbVg3 were 5571, 5532 and 4728 bp, encoding 1856, 1843 and 1575 amino acids, respectively. The three N. barkeri Vg possessed the Vitellogenin-N domain (or lipoprotein N-terminal domain (LPD_N)), von Willebrand factor type D domain (VWD) and the domain with unknown function 1943 (DUF1943). The NbVg1 and NbVg2 expression levels were significantly higher in the resistant strain than in the susceptible strain, while the NbVg3 expression level was lower in the resistant strain. Thus, we speculate that the increased number of eggs laid by the fenpropathrin-resistant strain of N. barkeri may be a consequence of changes in Vg gene expression.

A genomic approach to identify and monitor a novel pyrethroid resistance mutation in the redlegged earth mite, Halotydeus destructor.

Resistance mechanisms are typically uncovered by identifying sequence variation in known candidate genes, however this strategy can be problematic for species with no reference data in known relatives. Here we take a genomic approach to identify resistance to pyrethroids in the redlegged earth mite, Halotydeus destructor, a member of the Penthalidae family of mites that are virtually uncharacterized genetically. Based on shallow genome sequencing followed by a genome assembly, we first identified contigs of the H. destructor parasodium channel gene. By linking variation in this gene to known resistant phenotypes, we located a single nucleotide polymorphism in resistant mites. This polymorphism results in a leucine (L) to phenylalanine (F) amino acid substitution in the II6 region (predicted) of the gene (L1024F). This novel mutation has not previously been linked to pyrethroid resistance, although other polymorphisms have been identified in the two-spotted spider mite, Tetranychus urticae at the same locus (L1024V). The sequencing approach was successful in generating a candidate polymorphism that was then validated using laboratory bioassays and field surveys. A high throughput Illumina-based sequencing diagnostic was developed to rapidly assess resistance allele frequencies in pools of mites sourced from hundreds of populations across Australia. Resistance was confirmed to be widespread in the southern wheatbelt region of Western Australia. Two different resistance mutations were identified in field populations, both resulting in the same amino acid substitution. The frequency and distribution of resistance amplicon haplotypes suggests at least two, and probably more independent origins of resistance.

Suppression of Plant Defenses by Herbivorous Mites Is Not Associated with Adaptation to Host Plants.

Some herbivores suppress plant defenses, which may be viewed as a result of the coevolutionary arms race between plants and herbivores. However, this ability is usually studied in a one-herbivore-one-plant system, which hampers comparative studies that could corroborate this hypothesis. Here, we extend this paradigm and ask whether the herbivorous spider-mite Tetranychus evansi, which suppresses the jasmonic-acid pathway in tomato plants, is also able to suppress defenses in other host plants at different phylogenetic distances from tomatoes. We test this using different plants from the Solanales order, namely tomato, jimsonweed, tobacco, and morning glory (three Solanaceae and one Convolvulaceae), and bean plants (Fabales). First, we compare the performance of T. evansi to that of the other two most-commonly found species of the same genus, T. urticae and T. ludeni, on several plants. We found that the performance of T. evansi is higher than that of the other species only on tomato plants. We then showed, by measuring trypsin inhibitor activity and life history traits of conspecific mites on either clean or pre-infested plants, that T. evansi can suppress plant defenses on all plants except tobacco. This study suggests that the suppression of plant defenses may occur on host plants other than those to which herbivores are adapted.

Secondary structure of expansion segment D1 in LSU rDNA from Arachnida and its phylogenetic application in Eriophyoid mites and in Acari.

An increasing number of researchers have applied secondary-structure based multiple alignments of rDNA genes in phylogeny. These studies mostly depended on a few valuable divergent domains in LSU and SSU rDNA. Yet other divergent domains, e.g. D1, were poorly investigated and rarely used. However, these domains might contain additional evolutionary data and play a vital role in DNA-based phylogenetic study. Here, we investigated all available D1 sequences of Arachnida taxa and predicted corresponding secondary structures to help identify homologous positions in the D1 region. Long insertions were found exclusive to Eriophyoidea and folded into three newly proposed helices. Non-Acari taxa were all GC rich. In Acari, most Trombidiformes and all Mesostigmata (Parasitiformes) taxa were AT rich and Ixodida (Parasitiformes) GC rich; however there was no consistent base bias in Sarcoptiformes sequences. For Eriophyoid mites, genera Cecidophyopsis and Aceria were both well supported in MP, NJ, ME and ML tress based on D1 sequences, and clusters of Cecidophyopsis species were identical with former study. This demonstrated that the D1 region could act as a valuable molecular marker in phylogenetic reconstruction of Eriophyoidea. Additionally, D1 has been proven suitable in phylogenetic analysis at the family and genus level in Acari, but not in Opiliones.

Cryptic speciation in the Acari: a function of species lifestyles or our ability to separate species?

There are approximately 55,000 described Acari species, accounting for almost half of all known Arachnida species, but total estimated Acari diversity is reckoned to be far greater. One important source of currently hidden Acari diversity is cryptic speciation, which poses challenges to taxonomists documenting biodiversity assessment as well as to researchers in medicine and agriculture. In this review, we revisit the subject of biodiversity in the Acari and investigate what is currently known about cryptic species within this group. Based on a thorough literature search, we show that the probability of occurrence of cryptic species is mainly related to the number of attempts made to detect them. The use of, both, DNA tools and bioassays significantly increased the probability of cryptic species detection. We did not confirm the generally-accepted idea that species lifestyle (i.e. free-living vs. symbiotic) affects the number of cryptic species. To increase detection of cryptic lineages and to understand the processes leading to cryptic speciation in Acari, integrative approaches including multivariate morphometrics, molecular tools, crossing, ecological assays, intensive sampling, and experimental evolution are recommended. We conclude that there is a demonstrable need for future investigations focusing on potentially hidden mite and tick species and addressing evolutionary mechanisms behind cryptic speciation within Acari.

Phylogeny and species delineation in European species of the genus Steganacarus (Acari, Oribatida) using mitochondrial and nuclear markers.

Species of the genus Steganacarus are soil-living oribatid mites (Acari, Phthiracaridae) with a ptychoid body. The phylogeny and species status of the species of Steganacarus are not resolved, some authors group all ten German species of Steganacarus within the genus Steganacarus whereas others split them into three subgenera, Steganacarus, Tropacarus and Atropacarus. Additionally, two species, S. magnus and T. carinatus, comprise morphotypes of questionable species status. We investigated the phylogeny and species status of ten European Steganacarus species, i.e. S. applicatus, S. herculeanus, S. magnus forma magna, S. magnus forma anomala, S. spinosus, Tropacarus brevipilus, T. carinatus forma carinata, T. carinatus forma pulcherrima, Atropacarus striculus and Rhacaplacarus ortizi. We used two molecular markers, a 251 bp fragment of the nuclear gene 28S rDNA (D3) and a 477 bp fragment of the mitochondrial COI region. The phylogeny based on a combined analysis of D3 and COI separated four subgenera (Steganacarus, Tropacarus and Atropacarus, Rhacaplacarus) indicating that they form monophyletic groups. The COI region separated all ten species of the genus Steganacarus and showed variation within some species often correlating with the geographic origin of the species. Resolution of the more conserved D3 region was limited, indicating that radiation events are rather recent. Overall, our results indicate that both genes alone cannot be used for phylogeny and barcoding since variation is too low in D3 and too high in COI. However, when used in combination these genes provide reliable insight into the phylogeny, radiation and species status of taxa of the genus Steganacarus.

Cloning and differential expression of five heat shock protein genes associated with thermal stress and development in the polyphagous predatory mite Neoseiulus cucumeris (Acari: Phytoseiidae).

In order to explore the role of heat shock proteins (Hsps) during thermal stress and development in the predatory mite Neoseiulus cucumeris (Oudemans), we cloned and characterized five full-length Hsp genes. We investigated the expression levels of these genes by quantitative real-time PCR. The five genes characterized here were NcHsp90, NcHsp75, NcHsp70, NcHsp60, and NcHsp40. These Hsps showed high sequence conservation and had greatest identity with heat shock proteins of Metaseiulus occidentalis and other mite and insect species. All five NcHsp genes showed changes in their levels of expression during development. Higher levels of expression were observed in adult females than in adult males, but there were no significant changes between pre-oviposition and post-oviposition stages in the females. NcHsp90, NcHsp75, and NcHsp70 expression levels were up-regulated after a heat shock, and the increases in NcHsp75 and NcHsp70 expression levels were maintained for at least 3 h. Up-regulation of NcHsp60 and NcHsp40 was not detected after 1 h at a high temperature (35-45 °C); however, a significant down-regulation was observed after 3 h heat exposure at 35 °C and 3 h recovery at 25 °C. Cold shock treatment (-5 to 15 °C) for 1 h did not acute elicit changes in the expression levels of any of the genes. At 5 °C, the expression levels of NcHsp90 significantly increased after 6 or 24 h exposure compared to the levels after 1 h exposure. Thus, expression of Hsp genes in N. cucumeris reflected developmental changes, sexual difference, and variable induced response to thermal stress. Increased expression of Hsps might protect N. cucumeris individuals under extreme temperature conditions. Therefore, it may be possible to enhance the thermal tolerance of commercially available N. cucumeris using temperature acclimation. Treatment at 35 °C should be suitable for such acclimation.

Homoplastic evolution and host association of Eriophyoidea (Acari, Prostigmata) conflict with the morphological-based taxonomic system.

The superfamily Eriophyoidea is exceptionally diverse and its members are highly host-specific. Currently, the taxonomy of this group is based on morphology only. However, phylogenetic relationships in this group could be incorrect if the diagnostic morphological characters are homoplastic. Therefore, the phylogeny of 112 representative taxa of Eriophyoidea from China was determined using 18S, 28S D2-5 and D9-10 rRNA. Phylogenetic relationships were inferred through Bayesian, maximum likelihood and maximum parsimony methods, and then a number of clades or major clades were defined according to robust phylogenetic topologies combined with morphological comparison. Tests of monophyly showed that two of three families of Eriophyoidea as well as one subfamily and four tribes were not monophyletic. Ancestral character state reconstruction (ACSR) showed that five diagnostic morphological characters evolved several times, confounding the current taxonomy. Additionally, reconstruction of the history of host plant colonization suggested host switching occurred in a limited range of host plants. The host association data made it possible to determine taxonomic relationships more accurately. These results show that by integrating morphological and molecular information and host plant choice, it is possible to obtain a more accurate taxonomy and a deeper phylogenetic understanding of Eriophyoidea.

Multivariate discrimination among cryptic mites of the genus Androlaelaps (Acari: Mesostigmata: Laelapidae) parasitic of sympatric akodontine rodents (Cricetidae: Sigmodontinae) in northeastern Argentina: possible evidence of host switch followed by speciation, with the description of two new species.

Laelapids are among the most common ectoparasites of rodents. Currently, it is under discussion whether there is a single polixenous species that parasites a variety of hosts, or whether there are cryptic species highly host specific. Herein, multivariate morphometric analyses of cryptic sympatric laelapids of the genus Androlaelaps allowed us to identify different species. These species are specific of their akodontine hosts, Akodon montensis and Thaptomys nigrita, in localities situated in northeastern Argentina. In addition, we analyzed similar laelapids associated with the akodontines Deltamys kempi and Akodon cursor. Using principle component analyses we differentiated four laelapid species, each one host specific, independent of sympatry of the hosts, and without geographical variation. From these four species, we described two new species (Androlaelaps navonae n. sp. and Androlaelaps wingei n. sp.). We determined the four species based on a range of variations in several characters, mainly size. These four laelapid species belong to the Androlaelaps rotundus species group, specific to akodontines. These species are very similar among them but differ from the remainder species of the group by their small size, distance between j6 setae similar to the distance between the z5 setae, strong ventral setae, opisthogaster with 13 pairs of strong setae (one close to the distal margin of epigynal shield), and anal shield wider than long. Further studies will elucidate whether they constitute a new laelapid genus. Phylogenetic and ecological factors influencing host-specificity are discussed, and we propose that host colonization could have taken place by host switching of a single laelapid species among rodent species, followed by speciation.

Genetic and morphological diversity of Trisetacus species (Eriophyoidea: Phytoptidae) associated with coniferous trees in Poland: phylogeny, barcoding, host and habitat specialization.

Eriophyoid species belonging to the genus Trisetacus are economically important as pests of conifers. A narrow host specialization to conifers and some unique morphological characteristics have made these mites interesting subjects for scientific inquiry. In this study, we assessed morphological and genetic variation of seven Trisetacus species originating from six coniferous hosts in Poland by morphometric analysis and molecular sequencing of the mitochondrial cytochrome oxidase subunit I gene and the nuclear D2 region of 28S rDNA. The results confirmed the monophyly of the genus Trisetacus as well as the monophyly of five of the seven species studied. Both DNA sequences were effective in discriminating between six of the seven species tested. Host-dependent genetic and morphological variation in T. silvestris and T. relocatus, and habitat-dependent genetic and morphological variation in T. juniperinus were detected, suggesting the existence of races or even distinct species within these Trisetacus taxa. This is the first molecular phylogenetic analysis of the Trisetacus species. The findings presented here will stimulate further investigations on the evolutionary relationships of Trisetacus as well as the entire Phytoptidae family.

Hidden in the mangrove forest: the cryptic intertidal mite Carinozetes mangrovi sp. nov. (Acari, Oribatida, Selenoribatidae).

The small archipelago of Bermuda is a geologically young landmass in the Western Atlantic Ocean and recently turned out to be inhabited by a number of intertidal oribatid mites. One newly described species, Carinozetes bermudensis, showed an unusual vast range of habitats like sandy beaches, rocky substrate and mangroves. In the present study, 13 Bermudian populations of C. bermudensis were analysed to verify species integrity of specimens from different microhabitats. A morphometric analysis of 17 continuous variables as well as a molecular genetic investigation of the mitochondrial cytochrome oxidase subunit I revealed the existence of a new species Carinozetes mangrovi sp. nov., inhabiting exclusively intertidal algae growing on mangrove roots. Although both species are morphologically nearly identical, the configuration of the genus-specific ventral carinae represents a clear diagnostic character. The high genetic divergence of approximately 12 % of the cytochrome oxidase subunit I gene sequence between C. bermudensis and C. mangrovi sp. nov. suggests that these two species diverged before the emergence of the Bermuda islands. Accordingly, both of them are older than the geologically young archipelago of Bermuda.

New records of soft ticks (Acari: Argasidae) from caves in Brazil, with a morphological study of Ornithodoros fonsecai and an analysis of the taxonomic status of Antricola inexpectata.

In this study, we report soft ticks from bat-inhabiting caves in different areas of Brazil. From 2010 to 2019, we collected 807 tick specimens from nine caves located in four Brazilian states among two biomes. Ticks were morphologically identified as Antricola guglielmonei (282 specimens), Ornithodoros cavernicolous (260 specimens), and Ornithodoros fonsecai (265 specimens). Whereas A. guglielmonei was collected on bat guano in hot caves, O. cavernicolous and O. fonsecai were collected in cracks and crevices on the walls of cold caves, sometimes in the same chamber. Morphological identifications were corroborated by molecular and phylogenetic analyses inferred from tick mitochondrial 16S rRNA gene partial sequences. The sequences of A. guglielmonei, O. cavernicolous and O. fonsecai collected in this study clustered with conspecific GenBank sequences from different localities of Brazil. Remarkably, a clade containing 12 sequences of O. fonsecai was clearly bifurcated, denoting a degree of genetic divergence (up to 5 %) of specimens from Cerrado/Atlantic Forest biomes with the specimens from the Caatinga biome. To further evaluate this divergence, we performed morphometric analysis of the larval stage of different O. fonsencai populations by principal component analysis, which indicated that the larvae from Caatinga populations were generally smaller than the larvae from other biomes. Some of the present A. guglielmonei specimens were collected from the type locality of Antricola inexpectata. Comparisons of these specimens with the type specimens of A. inexpectata and A. guglielmonei indicated that they could not be separated by their external morphology. Hence, we are relegating A. inexpectata to a synonym of A. guglielmonei. This proposal is corroborated by our phylogenetic analysis.

Molecular detection of Borrelia sp. in Ornithodoros cavernicolous (Acari: Argasidae) in midwestern Brazil.

Ticks are obligate hematophagous parasites that can transmit to vertebrate hosts several pathogens, including viruses, bacteria, protozoa and helminths. Among these agents, some Borrelia species some Borrelia species cause disease in humans and other vertebrate hosts; therefore, they have medical and veterinary health importance. To gather additional information on Borrelia species in Brazil, the current study aimed to detect the presence of these species in Ornithodoros cavernicolous ticks collected in September 2019 from cement pipes that are used by bats as shelter in a farm located in the midwestern region of Brazil. DNA samples obtained from 18 specimens of O. cavernicolous were subjected of two polymerase chain reactions, targeting a segment of the Borrelia fla B gene. Of the samples tested, only one (6 %, 1/18) showed amplification. The nucleotide sequence of the amplified DNA showed more than 97 % (293/300) identity with a sequence of a Borrelia sp. detected in blood collected from a bat from Macaregua Cave, Colombia, and more than 97 % (292/300) detected in lungs from vampire bats from northeastern Brazil. The deduced amino acid sequences were identical to each other. Phylogenetic analysis indicated that these sequences formed a group of Borrelia species (putatively associated with bats) that is closely related to sequences of Borrelia species of the Lyme borreliosis group. Further investigations should be carried out in order to determine whether the sequence of the Borrelia sp. we found belongs to a new taxon. It will also be of great importance to determine which vertebrate hosts, besides bats, O. cavernicolous ticks can parasitize in order to investigate whether the Borrelia sp. we found may be transmitted and cause disease to the other vertebrate hosts.

Secondary structure prediction for complete rDNA sequences (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, and comparison of divergent domains structures across Acari.

According to base pairing, the rRNA folds into corresponding secondary structures, which contain additional phylogenetic information. On the basis of sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2 and 28S rDNA) of Demodex, we predicted the secondary structure of the complete rDNA sequence (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, which was in concordance with that of the main arthropod lineages in past studies. And together with the sequence data from GenBank, we also predicted the secondary structures of divergent domains in SSU rRNA of 51 species and in LSU rRNA of 43 species from four superfamilies in Acari (Cheyletoidea, Tetranychoidea, Analgoidea and Ixodoidea). The multiple alignment among the four superfamilies in Acari showed that, insertions from Tetranychoidea SSU rRNA formed two newly proposed helixes, and helix c3-2b of LSU rRNA was absent in Demodex (Cheyletoidea) taxa. Generally speaking, LSU rRNA presented more remarkable differences than SSU rRNA did, mainly in D2, D3, D5, D7a, D7b, D8 and D10.

Molecular identification of four phenotypes of human Demodex mites (Acari: Demodicidae) based on mitochondrial 16S rDNA.

Classification of Demodex mites has long depended on hosts and morphological characteristics. However, the fact that two species coexist in the same host and phenotype is easily influenced by environment causes difficulty and indeterminacy in traditional classification. Genotype, which directly reflects the molecular structure characteristics, is relatively stable. In this study, species identification of four phenotypes of human Demodex mites was conducted. Mites were morphologically classified into four phenotypes: long- and short-bodied Demodex folliculorum with finger-like terminus and Demodex brevis with finger- or cone-like terminus. The mitochondrial 16S ribosomal DNA (rDNA) fragment of individual mite was amplified, cloned, sequenced, and aligned. Sequence divergences, genetic distances, transition/transversion rates, and phylogenetic trees were analyzed. The results demonstrated that the 16S rDNA sequence of three phenotypes with finger-like terminus was 337 bp, and that of phenotype with cone-like terminus was 342 bp. The divergences, genetic distances, and transition/transversion rates among the three phenotypes with finger-like terminus were 0.0-2.7%, 0.000-0.029, and 5.0-7/0 (5/1-7/0), respectively, indicating an intraspecific variation. Yet, those between these three phenotypes and the one with cone-like terminus were 21.6-22.8%, 2.510-2.589, and 0.47-0.59 (22/47-27/46), respectively, suggesting an interspecific variation. The five phylogenetic trees showed that the three phenotypes with finger-like terminus clustered into one branch, while the phenotype with cone-like terminus clustered into another. In conclusion, terminus is a major morphological characteristic for the identification of human Demodex species. The three phenotypes with finger-like terminus belong to D. folliculorum, while the phenotype with cone-like terminus belongs to D. brevis. Molecular identification can verify and replenish morphological identification.