Cox1 Fragments Research Articles

Seek, and you will find: Cryptic diversity of the cardiopulmonary nematode Angiostrongylus vasorum in the Americas

Angiostrongylus vasorum is a metastrongylid parasite infecting wild canids and domestic dogs. Its patchy distribution, high pathogenicity and taxonomical classification makes the evolutionary history of A. vasorum intriguing and important to study. First larval stages of A. vasorum were recovered from feces of two grey foxes, Urocyon cinereoargenteus, from Costa Rica. Sequencing and phylogenetic and haplotypic analyses of the ITS2, 18S and cytochrome oxidase subunit 1 (cox1) fragments were performed. Then p- and Nei´s genetic distance, nucleotide substitution rates and species delimitation analyses were conducted with cox1 data of the specimens collected herein and other Angiostrongylus spp. Cophylogenetic congruence and coevolutionary events of Angiostrongylus spp. and their hosts were evaluated using patristic and phenetic distances and maximum parsimony reconciliations. Specimens from Costa Rica clustered in a separate branch from European and Brazilian A. vasorum sequences in the phylogenetic and haplotype network analyses using the ITS2 and cox1 data. In addition, cox1 p-distance of the sequences derived from Costa Rica were up to 8.6 % different to the ones from Europe and Brazil, a finding mirrored in Nei´s genetic distance PCoA. Species delimitation analysis supported a separate group with the sequences from Costa Rica, suggesting that these worms may represent cryptic variants of A. vasorum, a new undescribed taxon or Angiocaulus raillieti, a synonym species of A. vasorum described in Brazil. Moreover, nucleotide substitution rates in A. vasorum were up to six times higher than in the congener Angiostrongylus cantonensis. This finding and the long time elapsed since the last common ancestor between both species may explain the larger diversity in A. vasorum. Finally, cophylogenetic congruence was observed between Angiostrongylus spp. and their hosts, with cospeciation events occurring at deeper taxonomic branching of host order. Altogether, our data suggest that the diversity of the genus Angiostrongylus is larger than expected, since additional species may be circulating in wild canids from the Americas.

Coccidia of Guinea fowls: Validity of recorded Eimeria spp. (Apicomplexa: Eimeriidae) and first molecular identification of Eimeria grenieri Yvoré & Aycardi, 1967

Guinea fowls, Numida meleagris (L., 1758), are galliform birds native to sub-Saharan Africa, but introduced in several countries around the world for domestic breeding and/or animal production. This species is considered more resistant to disease by Eimeria spp. than other domestic galliform birds. Here we review the Eimeria spp. known to infect species of Numididae and provide the first molecular identification of an Eimeria sp. from Guinea fowls. There are currently 3 named eimerians from Guinea fowls; Eimeria numidae Pellerdy, 1962; Eimeria grenieri Yvoré and Aycardi, 1967; and Eimeria gorakhpuri Bhatia & Pande, 1967. We reviewed each of these species descriptions and documented their taxonomic shortcomings. From that, we suggest that E. gorakhpuri is a junior synonym of E. numidae. In conclusion, we have morphologically redescribed in detail E. grenieri from N. meleagris from Rio de Janeiro and provided molecular supplementation through sequencing of three non-overlapping loci in cox1 and cox3 genes and fragments of small and large subunit mitochondrial rDNA.

Fasciola Infection Unexpectedly Found During Cholecystectomy: Review on How to Avoid Increasing Surgery Interventions in Non-human Endemic Areas

PurposeFascioliasis is caused by Fasciola hepatica of almost worldwide distribution and F. gigantica in wide regions of Asia and Africa. Their adult stage develops in the biliary canals and gallbladder. Infection follows an initial, 3–4 month long invasive, migratory or acute phase, and a several year-long biliary, chronic or obstructive phase.MethodsThe unexpected finding of a fasciolid inside the gallbladder during a cholecystectomy for obstructive lithiasis suspicion in a patient is reported from an area of Iran where human infection had been never reported before and studies on fascioliasis in livestock are absent.ResultsThe fluke obtained was phenotypically classified as F. hepatica by morphometry and genotypically as F. gigantica by mtDNA cox1 fragment sequencing, although with F. hepatica scattered mutations in species-differing nucleotide positions. The clinical, radiological, and biological signs observed at the acute and chronic phases often lead to some misdiagnosis. Serological methods may be useful in cases of negative coprology. Diagnostic techniques with insufficient resolution leading to unnecessary invasive interventions are analyzed. The way to avoid unnecessary surgery is described, including analyses to be made, diagnostic tools to be used, and aspects to be considered.ConclusionReaching a correct diagnosis in the confusing presentations avoids procedure delays and unnecessary surgery. A correct drug treatment may be sufficient. Except in extreme pathological presentations, lesions decrease in number and size and finally disappear or calcify after a successful treatment. Finally, the need to increase awareness of physicians about fascioliasis is highlighted, mainly in non-human endemic areas.

Elucidating Spirocerca lupi spread in the Americas by using phylogenetic and phylogeographic analyses

Spirocerca lupi is a parasitic nematode of domestic and wild canids of the world. This nematode induces esophageal spirocercosis and may eventually lead to carcinomas, aortic aneurisms, and death of the animal. Two genotypes of S. lupi have been described based on specimens from Europe, Asia, Africa, and Oceania, but no profound analysis has been conducted with S. lupi from the Americas. To study this, S. lupi specimens isolated from domestic dogs from Mexico, Costa Rica, and the United States, were molecularly characterized using 18S rDNA and cox1 fragments. Bayesian inference (BI) phylogenetic trees, Templeton-Crandall-Sing (TCS) haplotype networks and Principal coordinate analysis on nucleotide distances were constructed for each locus separately. In addition, a phylogeographic study using a fragment of the cox1 gene was used to infer the evolutionary history of the genus. BI cox1 trees grouped S. lupi from the Americas in genotype 1, together with Israeli specimens, and showed a high nucleotide identity with those worms. In the TCS network, American specimens clustered next to Israeli S. lupi. Furthermore, the 18S rDNA gene fragment separated Costa Rican worms from African, Asian, and European specimens and other species of the family Spiruridae. Interestingly, the phylogeographic analysis suggested that the origin of S. vulpis was in Europe, and it later diverged into S. lupi that spread first to Africa, then to Asia and finally to the Americas. Therefore, we suggest that the worms from the American continent might have originated from Asia by dispersion of infected intermediate, paratenic or definitive hosts.

Molecular diversity of Sarcocystis spp. in opossums (Didelphis spp.) from Southeastern and Midwestern Brazil.

South American opossums (Didelphis spp.) are definitive hosts of Sarcocystis neurona, Sarcocystis speeri, Sarcocystis lindsayi and Sarcocystis falcatula. In Brazil, diverse studies have demonstrated a high frequency of Sarcocystis falcatula-like in sporocysts derived from opossums, and high genetic diversity has been observed in surface antigen-encoding genes (SAGs). In this study, genetic diversity of Sarcocystis spp. derived from Didelphis albiventris and Didelphis aurita from the cities of Campo Grande and São Paulo, was accessed by sequencing SAG2, SAG3, SAG4, the first internal transcribed spacer (ITS-1) and cytochrome c oxidase subunit I (cox1). Molecular identification was performed for 16 DNA samples obtained from sporocyst or culture-derived merozoites. The ITS-1, cox1, and SAG3 fragments were cloned, whereas SAG2 and SAG4 were sequenced directly from PCR products. Four alleles variants were found for SAG2, 13 for SAG3 and seven for SAG4, from which four, 13 and four, respectively, were novel. Twenty-seven allele variants were found for ITS-1, all phylogenetically related to S. falcatula-like previously described in Brazil. Sarcocystis sp. phylogenetically related to Sarcocystis rileyi was evidenced by cox1 in three opossums. Further studies are needed to clarify the role of Didelphis spp. as definitive hosts of Sarcocystis spp. other than that previous described.

Characterization of Five New Earthworm Mitogenomes (Oligochaeta: Lumbricidae): Mitochondrial Phylogeny of Lumbricidae

Identification based on conventional morphological characteristics is typically difficult and time-consuming. The development of molecular techniques provides a novel strategy that relies on specific mitochondrial gene fragments to conduct authentication. For this study, five newly sequenced partial mitogenomes of earthworms (Bimastos parvus, Dendrobaena octaedra, Eisenia andrei, Eisenia nordenskioldi, and Octolasion tyrtaeum) with lengths ranging from 14,977 to 15,715 were presented. Each mitogenome possessed a putative control region that resided between tRNA-Arg and tRNA-His. All of the PCGs were under negative selection according to the value of Ka/Ks. The phylogenetic trees supported the classification of Eisenia and Lumbricus; however, the trees based on cox1 did not. Through various comparisons, it was determined that cox1 fragments might be more suitable for molecular identification. These results lay the foundation for further phylogenetic studies on Lumbricidae.

Echinococcus granulosus sensu stricto G1 is the predominant genotype in human and livestock isolates from Turkey and Iran, based on mitochondrial nad5 gene differentiation

BackgroundDifferent genotypes of Echinococcus granulosus sensu stricto (s.s.) isolated from livestock and humans have been identified based on cox1 and nad1 genomic fragments. The present study was performed to differentiate the G1/G3 genotypes of Echinococcus granulosus (s.s.) isolated from humans and livestock (sheep and cattle) from Azerbaijan in northwestern Iran, Fars Province in southern Iran, and Van province in Eastern Turkey, using the nad5 gene fragment as a suitable marker to distinguish these two genotypes.MethodsA total of 60 pathologically confirmed human hydatid cysts and 90 hydatid cyst samples from livestock were collected from Turkey and Iran. PCR was performed on all of the samples, targeting the nad5 gene. Based on PCR product quality, host type, and the geographical area where the samples were obtained, 36 of the samples were sequenced and were used in the phylogenetic analysis.ResultsOut of 36 evaluated samples, 26 (72.2%) samples belonged to G1, and 10 (27.8%) samples belonged to the G3 genotype. Out of 21 samples from Turkey, 16 (76.2%) were G1 and 5 (23.8%) were G3, while out of 15 samples from Iran, 10 (66.7%) were G1 and 5 (33.3%) were the G3 genotype. None of the samples isolated from humans in Iran or from sheep in Turkey were G3. Overall, between the two countries, 18.18% of E. granulosus isolates in cattle, 41.66% of isolates in sheep, and 23.07% of human samples were identified as G3, and the others as the G1 genotype. The G3 genotype was not detected in human samples from Iran or sheep samples from Turkey.ConclusionThe findings of the study revealed that the G1 genotype of E. granulosus s.s. is the predominant genotype in humans and livestock, both in Turkey and Iran. The ratio of the E. granulosus s.s. G1 to G3 genotype was 3.2 in Turkey and 2 in Iran. The study also further confirmed that the nad5 gene properly differentiated the G1/G3 isolates of E. granulosus from both humans and livestock.Graphical

Invasive American mink (Neovison vison) as potential definitive host of Sarcocystis elongata, S. entzerothi, S. japonica, S. truncata and S. silva using different cervid species as intermediate hosts.

Canids and scavenger birds were shown to act as definitive hosts of numerous Sarcocystis species using members of the Cervidae family as an intermediate host, whereas definitive hosts spreading closely related S. elongata, S. entzerothi, S. japonica, S. matsuoae, S. rangiferi, S. truncata, S. silva and S. tarandi remain unknown. In the current study, the intestine samples of 40 American minks (Neovison vison) were molecularly tested for the presence of the above-mentioned Sarcocystis spp. Species-specific PCR of cytochrome c oxidase subunit I (cox1) fragments and subsequent sequencing revealed the presence of sporocysts/oocysts of five species, S. elongata (n=2), S. entzerothi (n=10), S. japonica (n=4), S. silva (n=13) and S. truncata (n=21) in the analysed samples. Sarcocystis infection was confirmed in 32/40 (80%) examined samples. In addition, half of the infected animals (50%) were infected with multiple Sarcocystis species suggesting that American minks had access to meat of different deer species, such as roe deer, red deer and sika deer. This causes concern about compliance of hunters and game processing companies with game waste management rules. Further research on the involvement of mustelids in the transmission of various Sarcocystis spp. from different geographical locations is needed.

Detection of DNA of filariae closely related to Mansonella perstans in faecal samples from wild non-human primates from Cameroon and Gabon

BackgroundThe Onchocercidae is a family of filarial nematodes with several species of medical or veterinary importance. Microfilariae are found in the blood and/or the dermis and are usually diagnosed in humans by microscopy examination of a blood sample or skin biopsy. The main objectives of this study were to evaluate whether filariae DNA can be detected in faecal samples of wild non-human primates (NHPs), whether the detected parasites were closely related to those infecting humans and whether filarial DNA detection in faeces is associated with co-infections with nematodes (Oesophagostumum sp. and Necator sp.) known to cause blood loss while feeding on the host intestinal mucosa.MethodsA total of 315 faecal samples from 6 species of NHPs from Cameroon and Gabon were analysed. PCRs targeted DNA fragments of cox1 and 12S rDNA genes, to detect the presence of filariae, and the internal transcribed spacer 2 (ITS2), to detect the presence of Oesophagostomum sp. and Necator sp. infections.ResultsAmong the 315 samples analysed, 121 produced sequences with > 90% homology with Onchocercidae reference sequences. However, 63% of the 12S rDNA and 78% of the cox1 gene sequences were exploitable for phylogenetic analyses and the amplification of the 12S rDNA gene showed less discriminating power than the amplification of the cox1 fragment. Phylogenetic analyses showed that the cox1 sequences obtained from five chimpanzee DNA faecal samples from Gabon and two from Cameroon cluster together with Mansonella perstans with high bootstrap support. Most of the remaining sequences clustered together within the genus Mansonella, but the species could not be resolved. Among the NHP species investigated, a significant association between filarial DNA detection and Oesophagostomum sp. and Necator sp. infection was observed only in gorillas.ConclusionsTo our knowledge, this is the first study reporting DNA from Mansonella spp. in faecal samples. Our results raise questions about the diversity and abundance of these parasites in wildlife, their role as sylvatic reservoirs and their potential for zoonotic transmission. Future studies should focus on detecting variants circulating in both human and NHPs, and improve the molecular information to resolve or support taxonomy classification based on morphological descriptions.

Comparative Genotyping of Echinococcus granulosus Infecting Livestock in Turkey and Iran

Echinococcus granulosus contains a complex of different strains that represent diversity in the pattern of the life cycle and also their host types. So far 10 genotypes of this parasite have been identified, using molecular methods. The current study aimed to evaluate and compare the genotypic diversity of E. granulosus metacestodes from livestock of Turkey and Iran. A total of 90 livestock liver and lung organs infected with hydatid cyst from industrial slaughterhouses of Bonab Province in the East Azerbaijan Province in Iran (60 samples, including 30 sheep and 30 cattle) and Van Province in Turkey (30 samples, including 15 sheep and 15 cattle) were collected. DNA was extracted from the protoscolices or germinal layers and polymerase chain reaction (PCR) were utilized, targeting the partial mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1) genes. PCR products were isolated from the electrophoresis gels and sequenced. The sequences were compared with each other, as well as with those related available sequences in the GenBank, using the BioEdit software and the BLAST algorithm. Finally, the phylogenetic trees were constructed by comparing sequences of cox1 and nad1 fragments, using the MEGA7 software and the maximum likelihood method. All samples sequenced from Iran corresponded to the genotype G1 (100%). Among the samples from Turkey, 15 samples (78.9%) were identified as G1 while only one sample (5.3%) corresponded to the genotype G3 and 3 isolates (15.8%) were defined as genotypes G1/G3. Five distinct haplotypes were determined within the examined isolates from sheep and cattle in both countries and all isolates clustered in one group. Phylogenetic analysis revealed that the intra-species genetic variations were 0.0-0.6% and 0.0-1.4% for cox1 and nad1, respectively. The dominant genotype of E. granulosus sensu stricto of livestock in both countries was the G1 (sheep strain) genotype. Our findings indicate that the sheep-dog cycle is the leading cycle of E. granulosus in these two areas. Hence, adopting regional common policies and bilateral cooperation helps to control the disease in livestock as well as in human in these two regions. Further study is required to compare the genetic diversity of human isolates of E. granulosus in these two countries.

Genetic Identification of Spirometra erinaceieuropaei Spargana in Liaoning and Hubei Provinces, PR China.

Spargana were collected from human and frogs in Liaoning and Hubei Provinces, China. PCR amplification and direct sequencing of A cox1 fragment was PCR-amplified from genomic DNA extracted from 7 specimens (5 from humans and 2 from frogs). The cox1 fragment (390 bp) showed 97–100% similarity to the reference sequence of S. erinaceieuropaei and 88–89% to the reference sequence of S. decipiens. There were 1–12 bases different between these worms, but no obvious genetic variation (0–3.3%) to the references. There was little difference of cox1 gene between sparganum samples of humans and frogs (1–3%). This study is the first report on S. erinaceieuropaei spargana from humans in Liaoning and Hubei Provinces.

First mitochondrial and nuclear DNA sequences of Lamanema chavezi (Nematoda: Molineidae): Novel findings to improve its identification in feces from South American camelids

Lamanema chavezi (Family Molineidae) is a parasitic nematode of South American camelids (SACs). A few studies have reported this parasite in SACs, mainly in domestic camelid species (llama and alpaca). Parasite identification by means of copro-parasitological methods is non-invasive and allows performing epidemiological studies. However, egg misidentification and difficulty to culture third-stage larvae do not allow identifying nematodes to species level. In contrast, molecular tools allow identifying eggs of gastrointestinal nematodes more accurately. However, the little genomic information available in databases for some species prevents an accurate diagnosis. In the present work, L. chavezi females present in feces of llamas from northwestern Argentina were molecularly characterized to obtain genomic information and improve parasitological diagnosis of L. chavezi-like eggs present in guanaco feces from southeastern Argentina. An 833-bp fragment of nuclear ribosomal DNA (rDNA) and a 434-bp fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene from both L. chavezi females and eggs were amplified and sequenced. Comparison between sequences from females and eggs showed 99–99.6% identity to rDNA and 99.5–96.1% to the cox1 gene fragments, confirming egg morphological assignment. A higher divergence between sequences was observed in the cox1 fragment, with a maximum variation of 3.9%. The examination of eggs found in guanaco feces from southeastern Argentina and their specific molecular identification represent the first record for this host in Argentine Patagonia and contribute to improving the diagnosis of gastrointestinal nematodes in SACs, mainly in wild camelids.

Barcoding hybrids: heterogeneous distribution of Schistosoma haematobium × Schistosoma bovis hybrids across the Senegal River Basin.

Hybridization events between Schistosoma species (Digenea, Platyhelminthes) are reported with increasing frequency, largely due to improved access to molecular tools. Nevertheless, little is known about the distribution and frequency of hybrid schistosomes in nature. Screening for hybrids on a large scale is complicated by the need for nuclear and mitochondrial sequence information, precluding a 'simple' barcoding approach. Here we aimed to determine and understand the spatiotemporal distribution of Schistosoma haematobium × Schistosoma bovis hybrids in the Senegal River Basin. From ten villages, distributed over the four main water basins, we genotyped a total of 1236 schistosome larvae collected from human urine samples using a partial mitochondrial cox1 fragment; a subset of 268 parasites was also genotyped using ITS rDNA. Hybrid schistosomes were unevenly distributed, with substantially higher numbers in villages bordering Lac de Guiers than in villages from the Lampsar River and the Middle Valley of the Senegal River. The frequency of hybrids per village was not linked with the prevalence of urinary schistosomiasis in that village. However, we did find a significant positive association between the frequency of hybrids per village and the prevalence of Schistosoma mansoni. We discuss the potential consequences of adopting a barcoding approach when studying hybrids in nature.

Genetic variation of Thelazia callipaeda among isolates collected from patients in Zunyi City, Guizhou Province

To identify the genetic variation and possible sources of Thelazia callipaeda isolates collected from patients in Zunyi City, Guizhou Province. Seven cases of T. callipaeda infection in Zunyi City, 2016 were verified, and DNA (s) were extracted from the T. callipaeda's body collected from the thelaziasis patients. A mitochondrial COX1 fragment was amplified and sequenced. The sequence alignment and phylogenetical analysis were performed to compare the genetic variation of the gene sequence with the homologous sequences downloaded from Genebank. COX1 genes of T. callipaeda were differed among the samples from the seven cases, which had low variation. Zunyi City is a new area with endemic of thelaziasis. The isolates from Zunyi City include either Asian origin or European origin of T. callipaeda. Moreover, at least four haplotypes are identified among the seven isolates.

Molecular phylogeny of the tribe Erotini with description of a new genus from China (Coleoptera: Lycidae)

AbstractErotini is a small tribe of net‐winged beetles known from the northern temperate zone. We investigated relationships and limits of tribes and genera using a molecular phylogeny inferred from rrnL, cox1 and nad5 mtDNA fragments. Lopheros Leconte, 1881 and Pseudaplatopterus Kleine, 1940 were inferred in current molecular analyses as terminal lineages within Erotini. Therefore, we consider Lopherotini Kazantsev, 2012 and Pseudaplatopterina Kazantsev, 2012 as junior synonyms of Erotini Leconte, 1881. The Platycis genus group comprises 19 species representing several genus‐group taxa previously placed in Platycis s. l. Konoplatycis Nakane, 1969 was recovered as a deeply rooted lineage of Erotini and we inferred another independent lineage for which we propose a new genus Sinoplatycis gen. nov. Additionally, we studied the diversity of Platycis s. l. in Asia, found their highest diversity in the temperate forest habitats of Eastern Asia and inferred the paraphyletic character of the subgenus Erotides s. str. Five new species are described from China: Sinoplatycis cardinalis sp. nov., S. zhani sp. nov. (type species of Sinoplatycis gen. nov.), Erotides brunnescens sp. nov., E. pusillus sp. nov. and E. slipinskii sp. nov. The molecular phylogeny shows deep splits of Japanese species with respect to the continental Eurasian fauna and the role of shallow seas and mountain systems in the diversification of the Platycis genus group.

Involvement of two genetic lineages of Sarcoptes scabiei mites in a local mange epizootic of wild mammals in Japan.

Similar to wild mammals on the continents, mange caused by the mange mite, Sarcoptes scabiei (Acari: Sarcoptidae) is spreading in wild mammals in most of Japan. We collected crusted or alopetic skin from 120 raccoon dogs (Nyctereutes procyonoides viverrinus), three raccoons (Procyon lotor), six Japanese badgers (Meles anakuma), one Japanese marten (Martes melampus), one stray dog (Canis lupus familiaris), four wild boars (Sus scrofa leucomystax), and one Japanese serow (Capricornis crispus), mainly in an area where mangy wild animals have been increasingly noted in the past 4 yr. The second internal transcribed spacer (ITS2) region of the ribosomal RNA gene and the partial 16S and cytochrome c oxidase subunit I (cox-1) genes of mitochondrial DNA (mtDNA) were characterized in these skin samples. The ITS2 sequencing (404 base pairs [bp]) identified the causative mite for mangy skin lesions of 128 animals as S. scabiei, regardless of host origin. The cat mite (Notoedres cati) was the cause in one raccoon dog and one raccoon. Most mites had almost identical ITS2 nucleotide sequences to those recorded in a variety of mammals worldwide. Partial 16S and cox-1 fragments of mtDNA amplified and sequenced successfully (331 bp and 410 bp, respectively) showed an identical nucleotide sequence except for one site (C vs. T) for the former and four sites (G, C, C, C vs. A, T, T, T, respectively) for the latter fragment. These substitutions were always synchronized, with the two mitochondrial DNA haplotypes (i.e., C/GCCC and T/ATTT) appearing to separately colonize in geographic units. The T/ATTT haplotype fell into a clade where animal-derived mites worldwide dominated, whereas the C/GCCC haplotype formed a geographic branch unique to Japanese isolates. These results suggest that heterologous populations of monospecific S. scabiei are expanding their populations and distributions regardless of host species in an apparently local mange epizootic of wild mammals in Japan.

Population identification of Sarcoptes hominis and Sarcoptes canis in China using DNA sequences.

There has been no consistent conclusion on whether Sarcoptes mites parasitizing in humans and animals are the same species. To identify Sarcoptes (S.) hominis and S. canis in China, gDNA was extracted from individual mites (five from patients with scabies and five from dogs with mange) for amplification of rDNA ITS2, mtDNA 16S, and cox1 fragment sequences. Then, the sequences obtained were aligned with those from different hosts and geographical locations retrieved from GenBank and sequence analyses were conducted. Phylogenetic trees based on 317-bp mtDNA cox1 showed five distinctive branches (species) of Sarcoptes mites, four for S. hominis (S. hominis Chinese, S. nr. hominis Chinese, S. hominis Australian, and S. hominis Panamanian) and one for S. animal (S. animal). S. animal included mites from nine animal species, with S. canis China, S. canis Australia, and S. canis USA clustering as a subbranch. Further sequence divergence analysis revealed no overlap between intraspecific (≤ 2.6 %) and interspecific (2.6-10.5 %) divergences in 317-bp mtDNA cox1. However, overlap was detected between intra- and interspecific divergences in 311-bp rDNA ITS2 or 275-bp mtDNA 16S when the divergences exceeded 1.0 %, which resulted in failure in identification of Sarcoptes. The results showed that the 317-bp mtDNA cox1 could be used as a DNA barcode for molecular identification of Sarcoptes mites. In addition, geographical isolation was observed between S. hominis Chinese, S. hominis Australian, and S. hominis Panamanian, but not between all S. canis. S. canis and the other S. animal belonged to the same species.

Molecular identification and phylogenetic study of Demodex caprae.

The DNA barcode has been widely used in species identification and phylogenetic analysis since 2003, but there have been no reports in Demodex. In this study, to obtain an appropriate DNA barcode for Demodex, molecular identification of Demodex caprae based on mitochondrial cox1 was conducted. Firstly, individual adults and eggs of D. caprae were obtained for genomic DNA (gDNA) extraction; Secondly, mitochondrial cox1 fragment was amplified, cloned, and sequenced; Thirdly, cox1 fragments of D. caprae were aligned with those of other Demodex retrieved from GenBank; Finally, the intra- and inter-specific divergences were computed and the phylogenetic trees were reconstructed to analyze phylogenetic relationship in Demodex. Results obtained from seven 429-bp fragments of D. caprae showed that sequence identities were above 99.1% among three adults and four eggs. The intraspecific divergences in D. caprae, Demodex folliculorum, Demodex brevis, and Demodex canis were 0.0-0.9, 0.5-0.9, 0.0-0.2, and 0.0-0.5%, respectively, while the interspecific divergences between D. caprae and D. folliculorum, D. canis, and D. brevis were 20.3-20.9, 21.8-23.0, and 25.0-25.3, respectively. The interspecific divergences were 10 times higher than intraspecific ones, indicating considerable barcoding gap. Furthermore, the phylogenetic trees showed that four Demodex species gathered separately, representing independent species; and Demodex folliculorum gathered with canine Demodex, D. caprae, and D. brevis in sequence. In conclusion, the selected 429-bp mitochondrial cox1 gene is an appropriate DNA barcode for molecular classification, identification, and phylogenetic analysis of Demodex. D. caprae is an independent species and D. folliculorum is closer to D. canis than to D. caprae or D. brevis.

Dermacentor everestianus Hirst, 1926 (Acari: Ixodidae): phylogenetic status inferred from molecular characteristics.

Dermacentor everestianus Hirst, 1926, is only reported in Northwestern China and Nepal. Few researches about this species have been involved, especially for molecular characteristics. The taxonomy studies of D.everestianus are mainly based on morphological features, and its taxonomic status is an ongoing controversy. To clarify the molecular characteristics and phylogenetic status of D.everestianus and other related species, the sequences of mitochondrial 16S ribosomal DNA (rDNA) and cox1 fragments were analyzed in the present study. Analysis of 16S rDNA and cox1 sequences showed 99.3-100% identity within D.everestianus individuals, with the genetic divergence among them was 0-0.0086. The interspecific distance of 16S rDNA and cox1 between D.everestianus and some other Palaearctic species including D. silvarum, D. nuttalli, and D. marginatus was much smaller than that between D.everestianus and Nearctic Dermacentor ticks (D.albipictus, D.nitens, and D.variabilis). Such relationships of these ticks were also verified in the phylogenetic analysis. Two major clades were recovered within Dermacentor spp. with more than 90% bootstrap support in the phylogenetic trees. D.everestianus together with D.silvarum, D.nuttalli, and D.marginatus were included in the clade I (Eurasia lineage). Other analyzed tick species including D.variabilis, D.nitens, and D.albipictus formed clade II, which are distributed in Nearctic realm. These indicated that the genus Dermacentor was at least composed of two lineages. Thus, further researches including additionally molecular markers on all Dermacentor species globally should be taken to precisely resolve relationships within Dermacentor.

Population structure and distribution patterns of the sibling mosquito species Culex pipiens and Culex torrentium (Diptera: Culicidae) reveal different evolutionary paths.

Nowadays a number of endemic mosquito species are known to possess vector abilities for various diseases, as e.g. the sibling species Culex pipiens and Culex torrentium. Due to their morphological similarity, ecology, distribution and vector abilities, knowledge about these species' population structure is essential. Culicidae from 25 different sampling sites were collected from March till October 2012. All analyses were performed with aligned cox1 sequences with a total length of 658 bp. Population structure as well as distribution patterns of both species were analysed using molecular methods and different statistical tests like distance based redundancy analysis (dbDRA), analysis of molecular variances (AMOVA) or McDonald & Kreitman test and Tajima's D. Within both species, we could show a genetic variability among the cox1 fragment. The construction of haplotype networks revealed one dominating haplotype for Cx. pipiens, widely distributed within Germany and a more homogeneous pattern for Cx. torrentium. The low genetic differences within Cx. pipiens could be a result of an infection with Wolbachia which can induce a sweep through populations by passively taking the also maternally inherited mtDNA through the population, thereby reducing the mitochondrial diversity as an outcome of reproductive incompatibility. Pairwise population genetic differentiation (FST) ranged significantly from moderate to very great between populations of Cx. pipiens and Cx. torrentium. Analyses of molecular variances revealed for both species that the main genetic variability exists within the populations (Cx. pipiens [88.38%]; Cx. torrentium [66.54%]). Based on a distance based redundancy analysis geographical origin explained a small but significant part of the species' genetic variation. Overall, the results confirm that Cx. pipiens and Cx. torrentium underlie different factors regarding their mitochondrial differentiation, which could be a result of endosymbiosis, dispersal between nearly located populations or human introduction.