New morphological and molecular data were generated for trematodes recovered from the intestines of the fish Pseudaspius hakonensis from two locations in the south of the Russian Far East. Morphologically, these trematodes are identical to Pseudozoogonoides ugui (Microphalloidea: Zoogonidae) from Japan. According to results of phylogenetic analysis based on 28S rDNA sequence data, P. ugui was closely related to Zoogonoides viviparus, and P. subaequiporus appears as a sister taxon to these two species. Genetic distance values, calculated based on both 28S rDNA and ITS2 rDNA, between P. ugui and Z. viviparus represents an interspecific differentiation level. Our results have an ambiguous explanation, indicating that the implication of the presence of one or two compact vitellarial aggregations for the differentiation of Zoogonoides and Pseudozoogonoides should be reconsidered or that our results open up the question of the taxonomical status of trematodes previously denoted as Z. viviparus and P. subaequiporus.

Dermoergasilus madagascarensis n. sp. is described from the gills of Paretroplus polyactis, an endemic cichlid fish in Madagascar, using a combined morphological (light microscopy and SEM) and molecular approach (partial 18S rDNA, 28S rDNA, and COI sequences). The new species is characterized mainly by possessing: (i) roughly pentagonal cephalosome; (ii) antennal endopodal segments covered with slightly inflated membrane; (iii) maxillule bearing 2 equally long outer setae and a minute inner seta; (iv) interpodal sternites of swimming legs ornamented with 3–4 rows of spinules; (v) genital segment and first abdominal somite both barrel-shaped; and (vi) a caudal ramus projecting into a digitiform process with inconspicuous terminal seta and bearing 3 terminal setae. The obtained DNA sequences of Malagasy species represent the first molecular data for species of Dermoergasilus. The 28S rDNA phylogeny showed the affiliation of D. madagascarensis n. sp. to Ergasilidae and its sister relationship with cosmopolitan Ergasilus sieboldi von Nordmann, 1832. The first checklist for all species of Dermoergasilus is provided.

During a survey of soil nematodes in Kirstenbosch National Botanical Garden in Cape Town, a population of plectid nematodes belonging to the genus Anaplectus was recovered and proved to be a species new to science. Anaplectus deconincki n. sp. is characterized by female body length (612–932 μm), b = 4.6–5.2, c = 12.8–18.0, c’ = 2.6–3.1, V = 51–54, and tail length (43–63 μm). Males are characterized by body length (779–956 μm), b = 4.8–5.6, c = 13.9–16.7, c’ = 2.2–2.5, spicule length 33–39 μm, gubernaculum length 10–12 μm, and tail length (56–65 μm). Discriminant analysis clearly separated A. deconincki n. sp. from the other related species of Aanaplectus. The phylogenetic analysis placed Anaplectus deconincki n. sp. in a clade with 1.00 posterior probability values with other Anaplectus. Partial sequences of the 18S and 28S regions of the ribosomal DNA gene were amplified for Anaplectus deconincki n. sp., and 18S rDNA showed 99% similarity with an unidentified Anaplectus (AJ966473) and A. porosus (MF622934) from Belgium. In addition, 28S rDNA showed a 93% similarity with A. porosus from Belgium (MF622938) and a 98% similarity with A. granulosus from Germany (MF325171). Measurements, illustrations, and light microscopy pictures for Anaplectus deconincki n. sp. are given.

A new species of dorylaimid nematode, Aporcelinus abeokutaensis sp. n., collected from a watermelon field in Nigeria, is described, illustrated and molecularly (D2–D3 28S ribosomal DNA) studied. It is characterized by its 1.18–1.52-mm-long body, lip region offset by weak constriction, 15.5–17.5 μm broad with perioral liplets, odontostyle 18–21.5 μm long at its dorsal side or 1.1–1.3 times the lip region diameter, neck 333–401 μm long, pharyngeal expansion occupying 45–51% of total neck length, uterus simple and 0.7–1.5 times the corresponding body diameter long, V = 48–53, tail conical with finely rounded tip (33–52 μm long, c = 26–41, c′ = 1.3–1.9) and a variably distinct dorsal concavity, and male unknown. Molecular analysis reveals that the new species grouped with other species of Aporcelinus in a highly supported clade, confirming the monophyly of the genus.

The taxonomy of ruminant Trypanosoma theileri and its relatives (Kinetoplastida: Trypanosomatidae) is controversial, with recent phylogenetic studies segregating T. theileri in cattle and other ruminants worldwide into two major genetic lineages (the TthI and TthII clades) based on genetic markers. In the present study, T. theileri-like trypanosomes isolated from Honshu sika deer (Cervus nippon) in the western Japan (YMG isolate) were genetically characterized using a number of genetic markers. Sika deer trypanosomes of the YMG isolate were genetically different from the Trypanosoma sp. TSD1 isolate previously recorded from Hokkaido sika deer in northern Japan, with the former trypanosome isolate being genetically closer to European cervid trypanosomes and the bovine T. theileri TthII lineage. In contrast, the latter isolate exhibited greater relatedness to North American cervid trypanosomes and the bovine T. theileri TthI lineage, although a clear genetic distinction between these was apparent. Furthermore, trypanosomes in Honshu sika deer from the central part of Japan harboured additional genetic diversity and were closer to either TSD1 or YMG isolates, while distinct from known T. theileri-related genotypes. Importantly, cervids and wild ruminants worldwide might harbour divergent descendants of a T. theileri ancestor, which exhibit rigid host specificity to either bovines or cervid species.

The myxozoan genus Unicapsula Davis, 1924 (Myxosporea: Multivalvulida: Trilosporidae) is characterized as having one functional polar capsule (PC) and two rudimentary PCs in a three-valved myxospore. The plasmodia of Unicapsula spp. grow either in the myofibres or in the gills, oesophageal walls and urinary organs of marine fish. Few studies have investigated the taxonomy of Unicapsula spp. including the type species Unicapsula muscularis. Accordingly, the taxonomy of the genus was explored in the present study by using 15 new isolates of seven Unicapsula spp. (U. muscularis, U. galeata, U. andersenae, U. pyramidata, U. pflugfelderi, and two new species) that had formed pseudocysts in the trunk myofibres of commercial fish collected in southern China and Japan from November 2015 to January 2019. Two new species Unicapsula trigona n. sp., and Unicapsula motomurai n. sp. exhibited unique myxospore morphologies (semi-triangular and spherical myxospores, respectively) and 18S and 28S rDNA sequences that were distinct from those of the other Unicapsula spp. Phylogenetic analysis of the 18S and 28S rDNA sequences confirmed the monophyletic status of Unicapsula.

The naturalization of alien Reeves's muntjacs (Muntiacus reevesi) on Izu-Oshima Island, Tokyo, Japan, has proceeded intensively over the last five decades. To clarify whether the gastrointestinal helminths of these animals were brought from their original endemic area or were newly acquired in Japan, 32 Reeves's muntjacs trapped on the island were parasitologically examined. In addition to Gongylonema pulchrum in the oesophagus (34.4% prevalence), Chabaudstrongylus ninhae (Dróżdż, 1967) (Trichostrongylidae: Cooperiinae) and Oesophagostomum muntiacum Jian, 1989 (Chabertiidae: Oesophagostominae) were prevalent in the small (28.1%) and large (46.9%) intestines, respectively. For the first time, these trichostrongylid or chabertiid worms were genetically characterized based on partial nucleotide sequences of the nuclear ribosomal RNA gene (rDNA) and mitochondrial DNA cytochrome c oxidase subunit 1 gene (cox-1), and the phylogenetic relationships with other members of their family were explored. Since these two intestinal nematode species are inherent in muntjacs, this study demonstrates a new distribution of exotic helminth species in Japan in accordance with the naturalization of alien mammalian hosts. The molecular genetic data collected here could assist the taxonomic assessment of morphological variants in different Muntiacus spp. and/or of different geographical origins. Furthermore, our data may help to define the phylogenetic relationships among such isolates.

Digenea Carus, 1863 represent a highly diverse group of parasitic platyhelminths that infect all major vertebrate groups as definitive hosts. Morphology is the cornerstone of digenean systematics, but molecular markers have been instrumental in searching for a stable classification system of the subclass and in establishing more accurate species limits. The first comprehensive molecular phylogenetic tree of Digenea published in 2003 used two nuclear rRNA genes (ssrDNA = 18S rDNA and lsrDNA = 28S rDNA) and was based on 163 taxa representing 77 nominal families, resulting in a widely accepted phylogenetic classification. The genetic library for the 28S rRNA gene has increased steadily over the last 15 years because this marker possesses a strong phylogenetic signal to resolve sister-group relationships among species and to infer phylogenetic relationships at higher levels of the taxonomic hierarchy. Here, we have updated the database of 18S and 28S rRNA genes until December 2017, we have added newly generated 28S rDNA sequences and we have reassessed phylogenetic relationships to test the current higher-level classification of digeneans (at the subordinal and subfamilial levels). The new dataset consisted of 1077 digenean taxa allocated to 106 nominal families for 28S and 419 taxa in 98 families for 18S. Overall, the results were consistent with previous higher-level classification schemes, and most superfamilies and suborders were recovered as monophyletic assemblages. With the advancement of next-generation sequencing (NGS) technologies, new phylogenetic hypotheses from complete mitochondrial genomes have been proposed, although their power to resolve deep levels of trees remains controversial. Since data from NGS methods are replacing other widely used markers for phylogenetic analyses, it is timely to reassess the phylogenetic relationships of digeneans with conventional nuclear rRNA genes, and to use the new analysis to test the performance of genomic information gathered from NGS, e.g. mitogenomes, to infer higher-level relationships of this group of parasitic platyhelminths.

The gullet worms, classical Gongylonema pulchrum and newly differentiated Gongylonema nepalensis, are prevalent in various mammals in Japan and Sardinia, Italy, respectively. The former species is cosmopolitan in distribution, dwelling in the mucosa of the upper digestive tract of a variety of domestic and wild mammals, and also humans. At present, the geographical distribution of G. nepalensis is known in Nepal and Sardinia, with the nematode having been recorded from the oesophagus of water buffaloes (Nepal), cattle, sheep, goats and wild mouflon (Sardinia). To clarify their natural transmission cycles among domestic and wild mammals, the present study analysed the ribosomal RNA gene (rDNA) and mitochondrial cytochrome c oxidase subunit 1 gene (cox1) of worms of various origins: G. pulchrum worms from sika deer, wild boars, Japanese macaques, and feral alien Reeves's muntjacs in Japan, and G. nepalensis worms from a red fox and a wild boar in Sardinia. Although the internal transcribed spacer (ITS) regions of rDNA and partial cox1 nucleotide sequences of G. pulchrum from native wild mammals in Japan were distinct from those of the worms in cattle, the worms from feral alien Reeves's muntjacs showed the cattle-type ITS genotype and cox1 cattle-I and II haplotypes. The rDNA and cox1 nucleotide sequences of G. nepalensis from a red fox in Sardinia were almost identical to those of the worms from domestic and wild ruminants on the island. The ecological interaction between domestic and wild mammals and their susceptibility to different Gongylonema spp. must be considered when trying to elucidate this spirurid's transmission dynamics in nature.

Mosquitoes’ importance as vectors of pathogens that drive disease underscores the importance of precise and comparable methods of taxa identification among their species. While several molecular targets have been used to study mosquitoes since the initiation of PCR in the 1980s, its application to mosquito identification took off in the early 1990s. This review follows the research's recent journey into the use of mitochondrial DNA (mtDNA) cytochrome oxidase 1 (COI or COX1) as a DNA barcode target for mosquito species identification – a target whose utility for discriminating mosquitoes is now escalating. The pros and cons of using a mitochondrial genome target are discussed with a broad sweep of the mosquito literature suggesting that nuclear introgressions of mtDNA sequences appear to be uncommon and that the COI works well for distantly related taxa and shows encouraging utility in discriminating more closely related species such as cryptic/sibling species groups. However, the utility of COI in discriminating some closely related groups can be problematic and investigators are advised to proceed with caution as problems with incomplete lineage sorting and introgression events can result in indistinguishable COI sequences appearing in reproductively independent populations. In these – if not all – cases, it is advisable to run a nuclear marker alongside the mtDNA and thus the utility of the ribosomal DNA – and in particular the internal transcribed spacer 2 – is also briefly discussed as a useful counterpoint to the COI.

Somatic cell nuclear transfer (SCNT) is an important technique for life science research. However, most SCNT embryos fail to develop to term due to undefined reprogramming defects. Here, we show that abnormal Xi occurs in somatic cell NT blastocysts, whereas in female blastocysts derived from cumulus cell nuclear transfer, both X chromosomes were inactive. H3K27me3 removal by Kdm6a mRNA overexpression could significantly improve preimplantation development of NT embryos, and even reached a 70.2% blastocyst rate of cleaved embryos compared with the 38.5% rate of the control. H3K27me3 levels were significantly reduced in blastomeres from cloned blastocysts after overexpression of Kdm6a. qPCR indicated that rDNA transcription increased in both NT embryos and 293T cells after overexpression of Kdm6a. Our findings demonstrate that overexpression of Kdm6a improved the development of cloned mouse embryos by reducing H3K27me3 and increasing rDNA transcription.

Neck samples from 54 badgers and 32 tongue samples of the same badgers (Meles meles), collected in the Lothians and Borders regions of Scotland, were tested using polymerase chain reactions (PCRs) directed against the 18S ribosomal DNA and the internal transcribed spacer (ITS1) region of protozoan parasites of the family Sarcocystidae. Positive results were obtained from 36/54 (67%) neck and 24/32 (75%) tongue samples using an 18S rDNA PCR. A 468 base pair consensus sequence that was generated from the 18S rDNA PCR amplicons (KX229728) showed 100% identity to Sarcocystis lutrae. The ITS1 PCR results revealed that 12/20 (60%) neck and 10/20 (50%) tongue samples were positive for Sarcocystidae DNA. A 1074 bp consensus sequence was generated from the ITS1 PCR amplicons (KX431307) and showed 100% identity to S. lutrae. Multiple sequence alignments and phylogenetic analysis support the finding that the rDNA found in badgers is identical to that of S. lutrae. This parasite has not been previously reported in badgers or in the UK. Sarcocystis lutrae has previously only been detected in tongue, skeletal muscle and diaphragm samples of the Eurasian otter (Lutra lutra) in Norway and potentially in the Arctic fox (Vulpes lagopus).

Thrips palmi and Frankliniella occidentalis (order Thysanoptera) are thrips species that represent major plant pests. They are polyphagous insects capable of adversely affecting crop production. As such, in the European Union, these thrips species should be regulated as quarantine organisms. T. palmi and F. occidentalis can cause considerable damage to susceptible plants by feeding on them and transmitting several viruses responsible for serious plant diseases. Successful pest control strategies are based on an early, fast, and reliable diagnosis, which precedes the selection of appropriate steps to limit the effects of harmful organisms. We herein describe a novel diagnostic approach that enables the sensitive and species-specific detection (and differentiation) of these pests in a duplex polymerase chain reaction assay, which was adapted for both standard and real-time quantitative assays. Our method is based on the amplification of a 5.8S-internal transcribed spacer 2 ribosomal DNA fragment that is conserved between T. palmi and F. occidentalis.

The oyster Crassostrea gasar is a species widely used as food and a source of income for the local population of the estuaries of Northeast Brazil. Perkinsus marinus and Perkinsus olseni are deleterious parasites for oyster farming and were recently detected in Brazil. In this study, a histopathologic survey of the oyster C. gasar cultured in the estuary of the River Mamanguape (Paraíba State) was performed. Adult oysters were collected in December 2011 and March, May, August and October 2012 and processed for histology and Perkinsus sp. identification by molecular analyses. Histopathological analysis revealed the presence of parasitic organisms including viral gametocytic hypertrophy, prokaryote-like colonies, protozoans (Perkinsus sp. and Nematopsis sp.) and metazoans (Tylocephalum sp. and cestodes). Other commensal organisms were also detected (the protozoan Ancistrocoma sp. and the turbellarian Urastoma sp.). The protozoan parasite Perkinsus sp. had the highest overall prevalence among the symbiotic organisms studied (48·9%), followed by Nematopsis sp. (36·3%). The other organisms were only sporadically observed. Only the protozoan Perkinsus sp. caused alterations in the oysters’ infected organs. Molecular analyses confirmed the presence of P. marinus, P. olseni and Perkinsus beihaiensis infecting the oyster C. gasar. This is the first report of P. beihaiensis in this oyster species.

Many lichens have extensive distributional ranges covering several climatic zones and are able to colonize extreme habitats, including high alpine and polar regions. Cladonia borealis, one of the dominant lichen species on King George Island, is a cosmopolitan species inhabiting polar, subpolar, and alpine areas. It is usually found on soil, humus, and mosses, and is morphologically highly diverse. To understand the phylogeographic history of C. borealis on King George Island, we compared specimens from there with specimens from Norway and Chile. We conducted phylogenetic and haplotype network analyses of the partial SSU, ITS1-5.8S-ITS2, and partial LSU rDNA sequences including intron sequences in LSU rRNA genes. Nuclear rDNA locus of C. borealis from King George Island was separated into two monophyletic lineages. It is suggested that they originated in multiple independent introduction events after long-distance dispersal from other continents.

Four distinctive morphological types can be found among living Globocassidulina in surface sediments of Admiralty Bay (King George Island, South Shetland Islands). The molecular analysis of the SSU and ITS rDNA indicates that they are monospecific and belong to Globocassidulina biora, except for minute forms from deeper than 200 m water depth which probably represent G. subglobosa. The morphological types of G. biora that show doubled or branched apertures, varied test size and shape as well as colour of cytoplasm, represent populations at different stages of ontogenetic development. However, the variability among large G. biora from the same locations is difficult to comprehend. It seems probable that G. biora is the only recent, large, shallow water Globocassidulina represented throughout the Antarctica, while G. crassa is typical for the Magellan region.

We applied a combined molecular and morphological approach to resolve the taxonomic status of Saccocoelium spp. parasitizing sympatric mullets (Mugilidae) in the Mediterranean. Eight morphotypes of Saccocoelium were distinguished by means of multivariate statistical analyses: 2 of Saccocoelium obesum ex Liza spp.; 4 of S. tensum ex Liza spp.; and 2 (S. cephali and Saccocoelium sp.) ex Mugil cephalus. Sequences of the 28S and ITS2 rRNA gene regions were obtained for a total of 21 isolates of these morphotypes. Combining sequence data analysis with a detailed morphological and multivariate morphometric study of the specimens allowed the demonstration of cryptic diversity thus rejecting the hypothesis of a single species of Saccocoelium infecting sympatric mullets in the Mediterranean. Comparative sequence analysis revealed 4 unique genotypes, thus corroborating the distinct species status of Saccocoelium obesum, S. tensum and S. cephali and a new cryptic species ex Liza aurata and L. saliens recognized by its consistent morphological differentiation and genetic divergence. However, in spite of their sharp morphological difference the 2 morphotypes from M. cephalus showed no molecular differentiation and 4 morphotypes of S. tensum were genetically identical. This wide intraspecific morphological variation within S. tensum and S. cephali suggests that delimiting species of Saccocoelium using solely morphological criteria will be misleading.

The identity of Anisakis type II larvae with adult A. physeteris was confirmed by comparison of restriction fragment length polymorphisms (RFLPs) of 25S ribosomal DNA (rDNA). Patterns of RFLPs in larvae were almost identical with those in adult worms. Directly labelled 25S rDNA might serve as an appropriate probe with highly specific activity for examining RFLPs of larvae and adult worms.

Restriction fragment length polymorphisms (RFLPs) of ribosomal DNA (rDNA) were compared between Diphyllobothrium latum and D. nihonkaiense using seven kinds of restriction endonucleases. No intra-specific variation in restriction fragment profiles was shown within both species of Diphyllobothrium. Digestion of the genomic DNA with three endonucleases, Smal, Hinfl and Hhal, provided one or two different bands between two species, although the hybridization patterns generated with the others, Hindlll, Xbal, Styl and Haelll, were the same in both. RFLPs in the digested profiles with Smal, Hinfl and Hhal could be used as species-specific markers even if only fragments of strobilae with morphological similarity were available. Other cestodes, Spirometra erinacei and Taenia saginata, used as controls showed quite different restriction fragment patterns with all the enzymes used.

rRNA and a heterologous cloned rDNA probe have been used to detect the rRNA genes of Eimeria species which infe the chicken, and has allowed the isolation and preliminary characterization of cloned rDNA sequences from a genomic DNA library of Eimeria tenella. It is demonstrated that rRNA and rDNA probes can be used to identify individual Eimeria species by the restriction fragment patterns detected after Southern hybridization. In addition, studies have shown that the large and small subunit rRNAs are expressed throughout sporulation.