Partial ITS Research Articles

A conventional multiplex PCR for the detection of four common soil-transmitted nematodes in human feces: development and validation.

Soil-transmitted helminth (STH) infections, mainly caused by Ascaris lumbricoides, Trichuris trichiura, and hookworms, are among the most common intestinal parasites that infect humans. The infections are widely distributed throughout tropical and subtropical countries, including Malaysia, particularly in underprivileged communities. Microscopic and culture techniques have been used as a gold standard for diagnostic techniques. However, these methods yield low sensitivity and specificity, laborious and time-consuming. Therefore, simple, rapid, and accurate alternative methods are needed for the simultaneous detection of STH infections. Although advanced technologies such as real-time multiplex PCR have been established, the use of this technique as a routine diagnostic is limited due to the high cost of the instrument. Therefore, a single-round multiplex conventional PCR assay for rapid detection of four STH species in the fecal sample was developed in this study. To perform the single-round multiplex PCR, each pair of species-specific primers was selected from target genes, including Ancylostoma duodenale (Internal Transcribed Spacer 2; accession No. AJ001594; 156 base pair), Necator americanus (ITS 2; accession No. AJ001599; 225 base pair), Ascaris lumbricoides (Internal Transcribed Spacer 1; accession No. AJ000895; 334 base pair) and Trichuris triciura (partial ITS 1, 5.8s rRNA and partial ITS 2; accession No. AM992981; 518 base pair). The results showed that the newly designed primers could detect the DNA of STH at low concentrations (0.001 ng/ μl) with no cross-amplification with other species. This assay enables the differentiation of single infections as well as mixed infections. It could be used as an alternative and is a convenient method for the detection of STHs, especially for the differentiation of N. americanus and A. duodenale.

Morphological and molecular characterization of adult Dermacentor pavlovskyi Olenev, 1927 (Acari: Ixodidae)

Dermacentor pavlovskyi Olenev, 1927 is the type tick species of subgenus Asiacentor Filippova & Panova, 1974 in the genus Dermacentor, which is only found in Xinjiang of China and Central Asia. In the present study, the males and females of D. pavlovskyi from Xinjiang were firstly characterized in detail morphologically by scanning electron microscopy and molecularly by partial 16S rDNA, COI and ITS2 sequences. The results of phylogenetic analysis indicated that D. pavlovskyi and Palaearctic subgenus Dermacentor (s. str.) species clustered to form a phylogenetic lineage, and their genetic distances were smaller than those between Palaearctic and non-Palaearctic subgenus Dermacentor (s. str.) species. Among Palaearctic subgenus Dermacentor (s. str.), D. pavlovskyi seems to be much closer to D. raskemensis, D. marginatus and D. niveus than to D. silvarum, D. nuttalli, D. everestianus and D. reticulatus. We also suggested that D. montanus, as the closest relative species of D. pavlovskyi, should be further investigated.

Investigations on Sarcocystis species in the leg muscles of the bird family Corvidae in Lithuania.

Although three species of Sarcocystis, S. cornixi, S. corvusi and S. kutkienae, have been described in corvids, molecular studies of sarcocysts isolated from these birds are incomplete. Leg muscles of 83 corvids, 35 hooded crows (Corvus cornix), 21 western jackdaws (Coloeus monedula), 11 rooks (Corvus frugilegus), 9 common ravens (Corvus corax), 4 common magpies (Pica pica) and 3 Eurasian jays (Garrulus glandarius), from Lithuania were examined for the presence of Sarcocystis spp. in the present study. In methylene blue-stained squashed samples, sarcocysts were detected in 26 birds (31.0%). Under a light microscope, two morphological types of sarcocysts were distinguished (type A and type B). Sarcocysts of type A had a smooth and thin (about 1μm) cyst wall, while cysts of type B were characterised by a thicker (1.4-2.5μm) cyst wall. Based on ITS1 sequence comparison, sarcocysts of type A were identified as S. halieti and Sarcocystis sp. ex Corvus corax, whereas cysts of type B belonged to S. kutkienae and S. cornixi. Furthermore, it was demonstrated that a single bird could host two different Sarcocystis spp. Sarcocystis halieti was detected in corvids for the first time in the common raven and the hooded crow. Also, this study presents the first evidence of S. kutkienae in the hooded crow and the common magpie, and S. cornixi in the western jackdaw. Sarcocystis sp. ex Corvus corax was genetically characterised using almost complete 18S rDNA, partial 28S rDNA and complete ITS1 sequences. Sarcocystis sp. ex Corvus corax clustered together with S. columbae, S. corvusi and S. halieti in phylogenetic trees reconstructed using 28S rDNA and ITS1 sequences.

Colletotrichum fructicola associated with fruit anthracnose of persimmon

AbstractAnthracnose, a major problem for persimmon exporters worldwide, reduces fruit yield and quality. In this study, a pathogen causing anthracnose of an imported persimmon fruit from an unknown country of origin was identified using a polyphasic approach. The pathogenicity of two fungal isolates, MBPER001A and MBPER002, was assessed by wound inoculation on healthy persimmon fruits. Anthracnose symptoms were observed 14 days after inoculation. Koch's postulates were fulfilled after the same fungus was re‐isolated from the inoculated fruits. Morphologies of both MBPER001A and MBPER002 isolates, original and re‐isolated, showed characteristics similar to Colletotrichum species in the gloeosporioides species complex. Multi‐locus analysis of the partial ITS, ACT, GAPDH and TUB2 gene regions showed MBPER001A and MBPER002 grouped within the Colletotrichum fructicola clade. Results from this study demonstrate the ability of C. fructicola to cause anthracnose in wounded persimmon fruits. Preventing wounding and damaging that expose the fruit during postharvest handling is therefore important to reduce infection. This study is the first report of C. fructicola in persimmon in the Philippines.

Two new species of Diaporthe (Diaporthaceae, Diaporthales) associated with tree cankers in the Netherlands.

Diaporthe (Diaporthaceae, Diaporthales) is a common fungal genus inhabiting plant tissues as endophytes, pathogens and saprobes. Some species are reported from tree branches associated with canker diseases. In the present study, Diaporthe samples were collected from Alnusglutinosa, Fraxinusexcelsior and Quercusrobur in Utrecht, the Netherlands. They were identified to species based on a polyphasic approach including morphology, pure culture characters, and phylogenetic analyses of a combined matrix of partial ITS, cal, his3, tef1 and tub2 gene regions. As a result, four species (viz. Diaporthepseudoalnea sp. nov. from Alnusglutinosa, Diaporthesilvicola sp. nov. from Fraxinusexcelsior, D.foeniculacea and D.rudis from Quercusrobur) were revealed from tree branches in the Netherlands. Diaporthepseudoalnea differs from D.eres (syn. D.alnea) by its longer conidiophores. Diaporthesilvicola is distinguished from D.fraxinicola and D.fraxini-angustifoliae by larger alpha conidia.

Molecular identification of the rodent-borne pathogen Rodentolepis nana using the genetic markers of ITS-1, 18S, and 28S rDNA.

Rodentolepis nana (syn. Hymenolepis nana), the most common cyclophyllid tapeworm infecting rodents, is a well-studied gastrointestinal parasite in mice and belongs to the family Hymenolepididae. The present study focuses on the molecular analysis for the nuclear genes (ITS-1, 18S, and 28S rDNA) used for the accurate recognition of the recovered Rodentolepis species. The annotated partial ITS-1, 18S, and 28S rDNA gene regions were deposited in GenBank (gbǀ MW310394.1, gbǀ MW327585.1, and gbǀ MW324479.1, respectively) and further used in the maximum likelihood method (ML) to clarify their genetic relationships at the species level. The interrogation sequence of R. nana was aligned and belonged to the family Hymenolepididae, in the same group as all Hymenolepis species, which were distinct from Cyclophyllidea cestodes, especially species belonging to Anoplocephalidae and Taeniidae. Sequence data support the paraphyly of Hymenolepis species. The phylogeny supports the availability of the ITS-1, 18S, and 28S rDNA genes as reliable genetic markers for evolutionary relationships.

Characterization of Diaporthe species on Camelliaoleifera in Hunan Province, with descriptions of two new species.

Tea-oil tree (Camelliaoleifera Abel.) is an important edible oil woody plant with a planting area over 3,800,000 hectares in southern China. Species of Diaporthe inhabit a wide range of plant hosts as plant pathogens, endophytes and saprobes. At present, relatively little is known about the taxonomy and genetic diversity of Diaporthe on C.oleifera. Here, we conducted an extensive field survey in Hunan Province in China to identify and characterise Diaporthe species associated with tea-oil leaf spots. As a result, eleven isolates of Diaporthe were obtained from symptomatic C.oleifera leaves. These isolates were studied by applying a polyphasic approach including morphological and phylogenetic analyses of partial ITS, cal, his3, tef1 and tub2 gene regions. Two new Diaporthe species (D.camelliae-oleiferae and D.hunanensis) were proposed and described herein, and C.oleifera was revealed to be new host records of D.hubeiensis and D.sojae. This study indicated there is a potential of more undiscovered Diaporthe species from C.oleifera in China.

A fresh outlook on the smooth-spored species of Inocybe: type studies and 18 new species

On the basis of detailed morphological and molecular investigation, eighteen new species of Inocybe (I. alberichiana, I. beatifica, I. bellidiana, I. clandestina, I. drenthensis, I. dryadiana, I. gaiana, I. ghibliana, I. grusiana, I. knautiana, I. lampetiana, I. oetziana, I. orionis, I. plurabellae, I. rivierana, I. scolopacis, I. sitibunda and I. tiburtina) are described. All of them are smooth-spored, and most of them are pruinose only in the apical part of the stipe. The new species are compared to 40 type specimens, all of which are described here and for several of which (partial) ITS sequences have been generated. For eight species, epi-, lecto- or neotypes were selected, among these are I. geophylla, I. glabripes and I. tigrina. Based on these studies, we suggest twelve synonymies, i.e. that I. clarkii is synonymous with I. sindonia, I. conformata with I. cincinnata, I. elegans with I. griseolilacina, I. fuscidula with I. glabripes, I. griseotarda with I. psammobrunnea, I. obscurella with I. obscuroides, I. obscuromellea with I. semifulva, I. patibilis and I. tigrinella with I. tigrina, I. petroselinolens with I. tenuicystidiata and I. rubidofracta with I. pseudorubens and I. subporospora is synonymized with I. tjallingiorum. All of the new species are supported by phylogenetic analyses. Among the previously described species accepted here, sixteen are represented by types in the phylogenetic analyses and ten by own collections morphologically corresponding to the type. In summary, we here verify or provide morphological concepts associated with molecular data for 44 smooth-spored species of Inocybe.

Seasonal Shifts in Cold Tolerance and the Composition of the Gut Microbiome of Dendroctonus valens LeConte Occur Concurrently

Dendroctonus valens LeConte, an invasive bark beetle, has caused severe damage in pine forests and has the potential to disperse into new geographic ranges in China. Although the gut microbiota of D. valens and its fundamental role in host fitness have been investigated widely, little is known about the relationship between the seasonal shifts of both cold tolerance and the gut microbiome of D. valens during overwintering, which occurs at the larval stage. In this study, to examine seasonal variations in the composition of the microbiome, we collected D. valens larvae in September (autumn), January (winter), and May (spring), and then analyzed the bacterial and fungal communities of the gut via sequencing of partial 16S rRNA and ITS genes. In addition, changes in the supercooling capacity and antioxidant enzyme activities of D. valens larvae collected in the different seasons were evaluated. Overwintering resulted in changes to microbial communities. In particular, the abundances of Enterobacter, Serratia, Erwinia, and Klebsiella decreased during overwintering. Concurrent with these changes, the cold tolerance of D. valens larvae was enhanced during overwintering, and the activities of the antioxidant enzymes catalase and peroxidase were reduced. We hypothesize that seasonal shifts in the gut microbiome may be connected to changes in cold tolerance and antioxidant enzyme activity in D. valens. It will be worthwhile to confirm whether seasonal changes in the microbiome contribute to the success of host overwintering.

Morphological and molecular characterization of Gambierdiscus caribaeus (Dinophyceae), with a confirmation of its occurrence in the Colombian Caribbean Tayrona National Natural Park

Abstract Dinoflagellates in the genus Gambierdiscus are known to produce potent neurotoxins known as ciguatoxins. These toxins vector through the food web and can reach sufficiently high levels to cause ciguatera fish poisoning (CFP). In many regions of the Caribbean, little is known about the occurrence of CFP-causing species or the incidence of CFP, despite its apparent regional increase over the last few decades. Given the intraspecific variability in content and potencies of these toxins among Gambierdiscus species, the precise identification of local species of this genus is crucial. In this study we confirm the presence of the epibenthic dinoflagellate Gambierdiscus caribaeus in the Colombian continental Caribbean. Cells were collected in seagrass beds of Thalassia testudinum in Bonito Gordo, a site located on the western side of Bahía Concha, Tayrona National Natural Park, near the city of Santa Marta, Colombia. Subsequently, a single strain of Gambierdiscus could be established for further identification. Morphological (light and scanning electron microscopy), and molecular results (partial LSU and ITS rDNA sequencing) were consistent with the original description of G. caribaeus, as well as with the morphological characteristics shown by other authors in organisms obtained near the sampling area.

Bursaphelenchus parayongensis n. sp. (Tylenchina: Aphelenchoididae) found in packaging wood from China

Summary Bursaphelenchus parayongensis n. sp. was isolated from packaging wood of a consignment from China exported to Germany. The new species is characterised by the lateral field with three lines, 9-13 μm long stylet with small basal swellings, and the excretory pore located near the posterior part of metacorpus. The male spicules are mitten-shaped, the lamina smoothly tapers towards the distal tip. The condylus is broadly rounded, with a small dorsally bent hook, rostrum triangular with sharply pointed tip, and without cucullus. One precloacal papilla, one adcloacal pair and two postcloacal pairs of male papillae present, with the posteriormost pair forming ‘gland papillae’. Bursal flap long, spade-like with posterior margin truncate or irregular. Females have a small vulval flap of 2-3 μm long, and a post-vulval uterine sac extending for 55-77% of the vulva to anus distance. Tail cylindrical, mostly (about 70%) with a robust mucron, very short to less than 3 μm, but occasionally tail terminus bluntly rounded, without mucron. It is similar to species in subgroup 3 of the eggersi group including B. carpini, B. clavicauda, B. cryphali and B. yongensis, but morphologically different. Detailed phylogenetic analysis based on partial 18S, ITS and D2-D3 region of 28S sequences has confirmed the status of this nematode as a new species.

Leeches as the intermediate host for strigeid trematodes: genetic diversity and taxonomy of the genera Australapatemon Sudarikov, 1959 and Cotylurus Szidat, 1928

BackgroundLeeches (Hirudinida) play a significant role as intermediate hosts in the circulation of trematodes in the aquatic environment. However, species richness and the molecular diversity and phylogeny of larval stages of strigeid trematodes (tetracotyle) occurring in this group of aquatic invertebrates remain poorly understood. Here, we report our use of recently obtained sequences of several molecular markers to analyse some aspects of the ecology, taxonomy and phylogeny of the genera Australapatemon and Cotylurus, which utilise leeches as intermediate hosts.MethodsFrom April 2017 to September 2018, 153 leeches were collected from several sampling stations in small rivers with slow-flowing waters and related drainage canals located in three regions of Poland. The distinctive forms of tetracotyle metacercariae collected from leeches supplemented with adult Strigeidae specimens sampled from a wide range of water birds were analysed using the 28S rDNA partial gene, the second internal transcribed spacer region (ITS2) region and the cytochrome c oxidase (COI) fragment.ResultsAmong investigated leeches, metacercariae of the tetracotyle type were detected in the parenchyma and musculature of 62 specimens (prevalence 40.5%) with a mean intensity reaching 19.9 individuals. The taxonomic generic affiliation of metacercariae derived from the leeches revealed the occurrence of two strigeid genera: Australapatemon Sudarikov, 1959 and Cotylurus Szidat, 1928. Phylogenetic reconstructions based on the partial 28S rRNA gene, ITS2 region and partial COI gene confirmed the separation of the Australapatemon and Cotylurus clades. Taking currently available molecular data and our results into consideration, recently sequenced tetracotyle of Australapatemon represents most probably Au. minor; however, unclear phylogenetic relationships between Au. burti and Au. minor reduce the reliability of this conclusion. On the other hand, on the basis of the obtained sequences, supplemented with previously published data, the metacercariae of Cotylurus detected in leeches were identified as two species: C. strigeoides Dubois, 1958 and C. syrius Dubois, 1934. This is the first record of C. syrius from the intermediate host.ConclusionsThe results of this study suggest the separation of ecological niches and life cycles between C. cornutus (Rudolphi, 1808) and C. strigeoides/C. syrius, with potential serious evolutionary consequences for a wide range of host–parasite relationships. Moreover, phylogenetic analyses corroborated the polyphyletic character of C. syrius, the unclear status of C. cornutus and the separate position of Cotylurus raabei Bezubik, 1958 within Cotylurus. The data demonstrate the inconsistent taxonomic status of the sequenced tetracotyle of Australapatemon, resulting, in our opinion, from the limited availability of fully reliable, comparative sequences of related taxa in GenBank.Graphical

A new species of Neoheterobothrium Price, 1943 (Monogenea, Diclidophoridae) from Syacium papillosum (Linnaeus) (Pleuronectiformes, Paralichthyidae) in the Yucatan Shelf, with notes on the validity of the subfamilies in the Diclidophoridae

Neoheterobothrium papillosum n. sp. (Monogenea: Diclidophoridae) is described based on specimens collected from the gills of the dusky flounder Syacium papillosum (Linnaeus) (Paralichthyidae) in the Yucatan Shelf. The new species can be differentiated from other congeneric species by the following characteristics: (1) number of hooks in the genital atrium (8 hooks), (2) number of testes (20-34), (3) pharynx size, (4) peduncle length, and (5) egg size. Neoheterobothrium papillosum n. sp. resembles Neoheterobothrium syacii; however, detailed morphological analysis allowed for their separation into two different species. Sequences of the nuclear ribosomal DNA partial 28S and ITS1 were obtained and compared with available sequences of other species and genera of the family Diclidophoridae from GenBank. A phylogenetic analysis was conducted, including 33 sequences, 18 of which represented 17 species and 11 genera of the family Diclidophoridae. The phylogenetic tree showed that Diclidophoridae is a monophyletic family and species of Diclidophora were grouped as the sister group of Neoheterobothrium papillosum n. sp. Our phylogenetic analyses were consistent with Mamaev's hypothesis, who divided Diclidophoridae into Diclidophorinae and Choricotylinae and concluded that Neoheterobothrium was a genus belonging to Diclidophorinae.

The Bacterial Community of the Foliose Macro-lichen Peltigera frigida Is More than a Mere Extension of the Microbiota of the Subjacent Substrate

Lichens host highly diverse microbial communities, with bacteria being one of the most explored groups in terms of their diversity and functioning. These bacteria could partly originate from symbiotic propagules developed by many lichens and, perhaps more commonly and depending on environmental conditions, from different sources of the surroundings. Using the narrowly distributed species Peltigera frigida as an object of study, we propose that bacterial communities in these lichens are different from those in their subjacent substrates, even if some taxa might be shared. Ten terricolous P. frigida lichens and their substrates were sampled from forested sites in the Coyhaique National Reserve, located in an understudied region in Chile. The mycobiont identity was confirmed using partial 28S and ITS sequences. Besides, 16S fragments revealed that mycobionts were associated with the same cyanobacterial haplotype. From both lichens and substrates, Illumina 16S amplicon sequencing was performed using primers that exclude cyanobacteria. In lichens, Proteobacteria was the most abundant phylum (37%), whereas soil substrates were dominated by Acidobacteriota (39%). At lower taxonomic levels, several bacterial groups differed in relative abundance among P. frigida lichens and their substrates, some of them being highly abundant in lichens but almost absent in substrates, like Sphingomonas (8% vs 0.2%), and others enriched in lichens, as an unassigned genus of Chitinophagaceae (10% vs 2%). These results reinforce the idea that lichens would carry some components of their microbiome when propagating, but they also could acquire part of their bacterial community from the substrates.

Morphological and Molecular Characterization of Trichoderma Species Prevailing in The Major Pulse Growing Areas of India

The present study was aimed to characterize the Trichoderma species prevailing in the major pulse growing areas of India. The Trichoderma isolates (54) were collected from the rhizospheric soils of chickpea, pigeonpea and rice-pulse cropping system in Madhya Pradesh, Uttar Pradesh and Maharashtra states of India. The isolates were characterized using their culture-morphological and molecular characteristics. Compact mycelial growth of Trichoderma spp. was seen in PDA medium, less dense growth in malt extract agar and powdery growth in oatmeal agar. Colony colour in media was generally green to dark green and occasionally yellowish green to dark green with white tinge; and the pigmentation in the reverse side varied from white, yellowish or faint yellow. Phialides were ampulliform to sub-globose or lageniform divergent or crowded whorls of 2-5, the middle of phialides were markedly swollen, conidia were subglobose to ovoid, the conidiophores highly branched having intercalary or terminal chlamydospores of sub-globose to ellipsoid or pyriform shape. Based on PCR amplification of ITS region sequences 44 isolates were characterized at molecular level. The partial ITS sequences were analyzed using MegaBLAST with NCBI GenBank database to confirm their identity. The study revealed the presence of 5 species of Trichoderma viz., T. viride, T. asperelloides, T. koningiopsis, T. asperellum and T. harzianum.

Morphological and Molecular Characterization of Quinisulcius curvus from China.

A stunt nematode species, Quinisulcius curvus, recovered from the rhizosphere of sea randa (Guettarda speciosa), is described and characterized herein based on integrative taxonomy. Morphometrics and distribution of all reported populations of Q. curvus are also discussed. The Chinese population of Q. curvus displayed slight variation in stylet length; however, the rest of the characters matches well with the original description. This is the first record of Q. curvus from Hainan, China, and the first molecular characterization for this species. Phylogenetic analysis based on partial 18S, 28S and ITS sequences placed Q. curvus with related stunt nematodes species, but clearly separated from them. The present study expanded the geographic record and provided molecular data on Q. curvus from China.

Integrative taxonomy, distribution, and host associations of Geocenamus brevidens and Quinisulcius capitatus from southern Alberta, Canada.

Two stunt nematode species, Geocenamus brevidens and Quinisulcius capitatus, were recovered from the potato growing regions of southern Alberta, described and characterized based on integrative taxonomy. Morphometrics, distribution, and host associations of both species are discussed. The Canadian populations of both species displayed minor variations in morphometrical characteristics (viz., slightly longer bodies and tails) from the original descriptions. The populations of G. brevidens and Q. capitatus species examined in this study are proposed as standard and reference populations for each respective species until topotype specimens become available and molecularly characterized. Phylogenetic analyses, based on partial 18S, 28S, and ITS sequences, placed both species with related stunt nematode species. The present study updates the taxonomic records of G. brevidens and Q. capitatus from a new location, southern Alberta, Canada, and will aid in the decision whether these stunt nematodes should be included in nematode management programs.

Development of a recombinase polymerase amplification (RPA-EXO) and lateral flow assay (RPA-LFA) based on the ITS1 gene for the detection of Angiostrongylus cantonensis in gastropod intermediate hosts.

Angiostrongylus cantonensis is a parasitic nematode known to infect humans through the ingestion of third stage larvae which can cause inflammation and damage to the central nervous system. Currently, polymerase chain reaction (PCR) is one of the most reliable diagnostic methods for detecting A. cantonensis in humans as well as in gastropod hosts, but requires expensive and specialized equipment. Here, we compare the sensitivity and accuracy of a recombinase polymerase amplification Exo (RPA-EXO) assay, and a recombinase polymerase amplification lateral flow assay (RPA-LFA) with a traditional quantitative PCR (qPCR) assay currently available. The three assays were used to test 35 slugs from Hawai'i for the presence of A. cantonensis DNA. Consistent results among the three tests were shown in 23/35 samples (65.7%), while 7/35 (20%) were discordant in low infection level samples (<0.01 larvae per mg tissue), and 5/35 (14.3%) were equivocal. To evaluate sensitivity, a partial ITS1 gene was cloned, and serial plasmid dilutions were created ranging from 100 copies μL-1 to ~1 copy μL-1. All three assays consistently detected 50-100 copies μL-1 in triplicate and qPCR was able to detect ~13 copies μL-1 in triplicate. RPA-EXO was able to detect 25 copies μL-1 in triplicate and RPA-LFA was not able to amplify consistently below 50 copies μL-1. Thus, our RPA-EXO and RPA-LFA assays do not appear as sensitive as the current qPCR assay at low DNA concentrations; however, these tests have numerous advantages that may make them useful alternatives to qPCR.

Genetic data of the ITS1-5.8S rDNA sequences of small liver flukes (Opisthorchiidae) from cats in Northern Vietnam

Domestic cats and humans are favorite final hosts for some hepatic trematodes of Opisthorchiidae. The present study examined the sequence variation in the nuclear ribosomal DNA (rDNA) region, including the internal transcribed spacer (ITS1) and the 5.8S gene, of two human opisthorchiid species Clonorchis sinensis (Cobbold, 1875) Looss, 1907 and Opisthorchis viverrini (Poirier, 1886) Stiles &amp; Hassal, 1896, in cats sampled in Northern Vietnam. The length of 30 partial ITS1‒5.8S rDNA sequences of C. sinensis from different hosts and localities varied from 761 to 766 bp. The results showed completely identity for all investigated 5.8S rDNA sequences of C. sinensis. In contrast, the variations were revealed in the complete ITS1 sequences of C. sinensis. Three polymorphic sites, indel and new sites with intragenomic polymorphism were detected. The length of the complete ITS1 rDNA region ranged from 656 to 661 bp. There are no substitutions downstream of the 5 bp insertion, and the different ratio of nucleotide in polymorphic sites with presence/absence of insertion leads to the existence of 6 genotypes in the investigated samples. The major genotype was obtained in 70% of sequences, other genotypes were presented by 1, 2 or 3 sequences. Nucleotide diversity in whole sample of C. sinensis was 0.00122 ± 0.00073. Partial sequences of ITS1‒5.8S rDNA of O. viverrini were 745 bp in length, and no difference was observed. Phylogenetic analyses by neighbor joining phylogram inferred from ITS1‒5.8S rDNA of 3 sequences of O. viverrini and 30 sequences of C. sinensis were formed two separately monophyletic clades.

Comparative analysis of mitochondrial DNA datasets indicates that Cylicostephanus minutus represents a species complex

Cyathostomins are one kind of the most important parasites in equids. Cylicostephanus minutus is a member of the subfamily Cyathostominae. In the present study, we determined the complete mitochondrial (mt) genomes from four Cs. minutus isolates and reconstructed the phylogenetic relationship of Strongylidae to test the hypothesis that Cs. minutus represents a species complex. The complete mt genome sequences of Cs. minutus were 13,772–13,822 bp in length, and contained 36 genes (12 protein coding genes, 22 tRNA genes, two rRNA genes), and two non-coding regions (NCRs). The intraspecific identity of nucleotide sequences and amino acid sequences in Cs. minutus (1–4) were 89.3–97.9% and 97.0–98.8%, respectively. Two operational taxonomic units (OTUs) were determined based on the mt genome sequences, OTU 2 (Csm 1 and Csm 2) and OTU 3 (Csm 3 and Csm 4). Sequence analysis showed the divergence between OTU 2 and OTU 3 was 8.9–10.7%. Pairwise comparisons of 12 protein coding genes between OTU 2 and OTU 3 showed a difference of 3.0–13.3% at the nucleotide level and 0–6.7% at the amino acid level. Phylogenetic analysis showed the separation of Cs. minutus isolates from the same host into different distinct clades based on mt genomes. Comparisons of partial mt cox1, nad5, and cytb and ITS2 sequences from 20 Cs. minutus isolates from the same host and the same geographical location with other Cs. minutus sequences available in GenBank revealed significant nucleotide differences. Phylogenetic analysis showed a separation of Cs. minutus into three distinct clades. Thus, the comparative and phylogenetic analyses of mtDNA datasets indicated that Cs. minutus represents a complex of at least three species. Our results have further confirmed the existence of a cryptic Cs. minutus species, and provides a reference for the taxonomical, population genetics, and systematics studies of other cyathostomin species.