Nuclear 18S Ribosomal RNA Gene Research Articles

Molecular characterisation of Amblyomma integrum circulating in southern India

Amblyomma integrum is a large gooseberry sized longirostrate tick (when fully repleted) found in India and Sri Lanka. In Kerala (India), this tick is commonly found in the forest and its fringe areas frequently infesting deer and hence it is locally known as “maan chellu / maanunny” (deer tick). In the present study, molecular characterisation and phylogenetic analysis of A. integrum collected from the area grazed by the sambar deer (Rusa unicolor) of Kerala, south India was performed using three molecular markers viz., the mitochondrial cytochrome c oxidase subunit 1 (COI), mitochondrial 16S ribosomal RNA, and nuclear 18S ribosomal RNA genes. Cytochrome c oxidase subunit 1 (COI) gene showed better resolving ability for elucidating the evolutionary relationship of A. integrum and identified two distinct clades, viz., A and B. The Tamil Nadu isolates of south India and Marayoor isolate 1 (from Idukki district of Kerala bordering with Tamil Nadu) belonged to clade A. Majority of Wayanad isolates from Kerala, occupied clade B. The intraspecific genetic distance among the A. integrum species ranged from 0.00 to 13.34%. Between clades A and B, the genetic distance observed was 11.49%. The clade B isolates were genetically close to A. geoemydae (GD: 1.22%). Morphological variations between the clades included darker exoskeletal coloration in clade A and distinct differences in the shape of basis capitulum. Further analysis using Assemble Species by Automatic Partitioning (ASAP) and Generalized Mixed Yule Coalescent (GMYC) provided additional insights. Assemble Species by Automatic Partitioning (ASAP) identified 26 Molecular Operational Taxonomic Units (MOTUs) at a threshold distance of 5.38%, supporting the species partition of A. integrum clade B. Generalized Mixed Yule Coalescent (GMYC) analysis retained the same species complex (A. integrum-geoemydae Complex) inferred from the ASAP analyses. It could be inferred from the present study that the A. integrum clades A and B could be two different putative pseudocryptic species.

Discovery of Encyclometra bungara (Digenea: Encyclometridae) in a new host (Enhydris enhydris) from Thailand and Cambodia through morphological and molecular identification.

The genus Encyclometra is one of the two genera in family Encyclometridae, known for parasitising the oesophagus, stomach and intestine of snakes. Among Encyclometra, the species present are: Encyclometra colubrimurorum, Encyclometra japonica, Encyclometra asymmetrica and Encyclometra bungara. Species discrimination within Encyclometra has predominantly relied on morphological differences, such as the length of the caeca and the position of the testes. Morphological overlaps exist among these species making species discrimination challenging. Additionally, the use of molecular information has been limited for Encyclometra. To determine the Encyclometra species infecting Enhydris enhydris from Thailand and Cambodia, morphological and molecular identification was conducted. Morphological characters and measurements were obtained from 30 Encyclometra adults, and they were compared with previous studies of other Encyclometra species. Novel sequences of E. bungara were generated using the nuclear 18S and 28S ribosomal RNA genes, and the mitochondrial cytochrome c oxidase subunit 1 gene. Our results revealed that the specimens could be morphologically identified as E. bungara, with support from molecular information obtained from the phylogenies of the 3 genetic markers employed. Molecular analysis showed that the Encyclometra specimens were distinct from E. colubrimurorum and E. japonica. Through morphological and molecular identification of the Encyclometra specimens found in E. enhydris from Thailand and Cambodia, we describe and provide a record of E. bungara in a new host and new locality. Additionally, novel molecular sequences were generated, revealing the phylogenetic position of E. bungara within the superfamily Gorgoderoidea.

Redescription and new record of Paracapillaria (Ophidiocapillaria) najae (Nematoda: Trichuroidea) in the monocled cobra Naja kaouthia from central Thailand: morphological and molecular insights.

The parasitic nematode Paracapillaria (Ophidiocapillaria) najae De, 1998, found in the Indian cobra Naja naja is redescribed and re-illustrated in the present study. The monocled cobra Naja kaouthia was discovered to be a new host for this parasite in central Thailand. A comprehensive description extending the morphological and molecular characteristics of the parasites is provided to aid species recognition in future studies. The morphometric characters of 41 parasites collected from 5 cobra specimens are compared with those described in the original studies. Phylogenetic analyses using mitochondrial cytochrome c oxidase subunit 1 and nuclear 18S ribosomal RNA genes were performed to provide novel information on the systematics of P. najae. Similar characteristics were observed in the examined nematode samples, despite being found in different hosts, confirming their identity as P. najae. The molecular genetic results support the species status of P. najae, indicating P. najae is well defined and separated from other related nematode species in the family Capillariidae. Morphological descriptions, genetic sequences, evolutionary relationships among capillariids and new host and distribution records of P. najae are discussed. Paracapillaria najae specimens found in the Thai cobra had some morphological variation, and sexual size dimorphism was also indicated. Paracapillaria najae was found to infect various cobra host species and appeared to be common throughout the Oriental regions, consistent with its hosts' distribution.

The Temnocephalidae (Platyhelminthes): molecular data illuminate the evolution of an ancient group of symbiotic flatworms

The Temnocephalidae sensu Van Steenkiste et al., 2021 is a diverse group of rhabdocoel flatworms that are ectosymbiotic mostly on freshwater crustaceans. The family is found on all inhabited continents except for N. America (north of Mexico) and Africa. Here, we explore the systematic implications of a molecular phylogeny inferred from partial sequences of the nuclear 18S and 28S ribosomal RNA genes. In agreement with previous views based on morphology, the earliest diverging group within the family was exemplified by the genus Scutariella (subfamily Scutariellinae) followed by the genus Didymorchis (Didymorchinae). The genus Dactylocephala from Madagascar, previously thought to be in the Temnocephalinae, then diverged as sister to the remaining temnocephalids. A new subfamily, Dactylocephalinae, is proposed to contain this genus. Remaining subfamilies were, in turn, Diceratocephalinae, Actinodactylellinae and Temnocephalinae. Further subdivision was apparent within the Temnocephalinae. One well-supported clade included the genera Temnohaswellia (eastern Australia and New Zealand), Temnomonticellia (Tasmania) and Temnocephala (South America). The widespread genus Temnosewellia (= Ts.) was not supported as monophyletic. One clade of Temnosewellia sensu lato included numerous species known primarily from crayfish of the genus Euastacus, found in eastern and south-eastern Australia. Two other clades circumscribed the taxa Ts. minor and Ts. dendyi. Identities and membership of these two clades are clouded by frequent misidentifications, the reasons for which are discussed here. A fourth clade of Temnosewellia s.l. included Ts. rouxi and other taxa from northern Australia and Asia (ranging at least from Japan to India). The diversity of temnocephalids in Australia, and especially in Asia, is greatly underappreciated. Some general comments are made about geographic distribution of temnocephalids, but detailed biogeographic analysis requires broader sampling. http://zoobank.org/urn:lsid:zoobank.org:pub:C8E6F83E-5A8E-4756-AAB9-1E65BBA7D9DE

Assigning Culicoides larvae to species using DNA barcoding of adult females and phylogenetic associations

BackgroundOrbivirus-induced hemorrhagic diseases cause high mortality in wild and captive white-tailed deer in North America. The role of different Culicoides species in Orbivirus transmission outside of areas of intensive animal production has not been established. At our study location, bluetongue virus (BTV) RNA-positive female Culicoides debilipalpis pools have been detected annually since 2012 when BTV transmission was noted in a captive deer herd. Identifying specific larval habitats of suspected vectors at active transmission sites is crucial both for identifying the source of the vectors and for subsequently planning intervention actions. Since C. debilipalpis larvae are known to develop in tree holes, this study was designed to use DNA barcoding to identify larvae collected from tree holes.MethodsAdult female Culicoides were collected using light or emergence traps and morphologically identified to 11 species. Culicoides sonorensis were also obtained from a laboratory colony. Substrate was collected from tree holes and flooded with water to harvest floating larvae. Total DNA from three to seven adult females per species and 19 larvae was extracted. Two loci of the nuclear 18S ribosomal RNA (rRNA) gene, one locus each of the mitochondrial cytochrome oxidase subunit I (COI) gene and the nuclear 28S rRNA gene were amplified using loci-specific primers.ResultsAll 61 adults were sequenced at each of the four loci under study. Since no single locus delineated all putative species and the COI locus yielded unreliable pseudogenes for two individuals of C. arboricola, sequences of all four loci were concatenated to maximize species separation and allow for larval association with identified adults. Sixteen larvae were clearly assigned to species based on DNA barcoding and phylogenetic results. Multiple larvae were assigned to each of the C. debilipalpis clade, the C. villosipennis clade, the C. arboricola clade and the C. nanus clade.ConclusionsOf the approximately 62 species described in the southeast USA, 21 have now been barcoded and sequences are publicly available. In this study, we constructed a database composed of species-specific sequences of adult Culicoides and then identified larvae to species by matching their corresponding sequences with adults. Since Culicoides larvae are difficult to identify, using DNA barcoding to facilitate larval habitat surveys can be a valuable tool.Graphical

High-throughput sequencing outperforms traditional morphological methods in Blue Catfish diet analysis and reveals novel insights into diet ecology.

Blue Catfish Ictalurus furcatus are an invasive, yet economically important species in the Chesapeake Bay. However, their impact on the trophic ecology of this system is not well understood. In order to provide in‐depth analysis of predation by Blue Catfish, we identified prey items using high‐throughput DNA sequencing (HTS) of entire gastrointestinal tracts from 134 samples using two genetic markers, mitochondrial cytochrome c oxidase I (COI) and the nuclear 18S ribosomal RNA gene. We compared our HTS results to a more traditional “hybrid” approach that coupled morphological identification with DNA barcoding. The hybrid study was conducted on additional Blue Catfish samples (n = 617 stomachs) collected from the same location and season in the previous year. Taxonomic representation with HTS vastly surpassed that achieved with the hybrid methodology in Blue Catfish. Significantly, our HTS study identified several instances of at‐risk and invasive species consumption not identified using the hybrid method, supporting the hypothesis that previous studies using morphological methods may greatly underestimate consumption of critical species. Finally, we report the novel finding that Blue Catfish diet diversity inversely correlates to daily flow rates, perhaps due to higher mobility and prey‐seeking behaviors exhibited during lower flow.

Metabarcoding of zooplankton diversity within the Chukchi Borderland, Arctic Ocean: improved resolution from multi-gene markers and region-specific DNA databases

DNA-based analyses have become powerful tools for characterizing the metazoan biodiversity of diverse marine ecosystems. Metabarcoding (i.e., large-scale taxonomic identification of complex samples via high-throughput sequencing of a DNA barcode region) frequently uses hypervariable regions of the nuclear eukaryotic 18S ribosomal RNA (rRNA) gene. However, species-level taxonomic identification is hampered by the conservative nature of the 18S gene in comparison to the mitochondrial cytochrome oxidase I (COI) barcode gene. Additionally, metabarcoding relies on reference DNA sequence databases for classification of millions of unknown sequence reads and molecular operational taxonomic units (OTUs); databases that are at present depauperate for marine zooplankton taxa. Here, we characterized the mesozooplankton community for the Chukchi Borderland (CBL) region, western Arctic Ocean, through metabarcoding analysis of the V4 and V9 hypervariable regions of 18S rRNA and a portion of COI. Characterization of zooplankton diversity for the epipelagic and upper mesopelagic layers (0–500 m) was based upon 17 metazoan taxonomic categories encompassing 24 orders in 14 classes. Taxonomic classification using the V4 and V9 markers was most reliable for orders, with copepods dominating OTU counts. To increase taxonomic resolution and allow detection of species, V4, V9, and COI OTUs were classified against DNA sequence databases for the Arctic Ocean for the subclass Copepoda. The geographic region-specific databases for 18S rRNA and COI resulted in the detection and identification of 6 genera and 49 species of copepods representing 23 families, a marked increase in the taxonomic classification of the 18S rRNA markers. The greatest copepod species diversity was captured with V4 (34 species) followed by COI (28 species) with the least copepod diversity detected by V9 (5 species). Our results demonstrate the power of using multiple gene markers, with DNA reference databases that are specific to the geographic region of interest, providing more accurate metabarcoding biodiversity measures for the copepod assemblages compared to universal metazoan sequence reference databases. Results from this study highlight the need for continued DNA barcode sequencing to increase species representation in the reference sequence databases that are crucial for accurate characterization of mesozooplankton communities.

Life on an island: the phylogenetic placement of Loveridgeana and Afrotropical Sphaerophoria (Diptera: Syrphidae) inferred from molecular characters

Phylogenetic relationships of the Sphaerophoria lineage (Sphaerophoria Le Peletier & Audinet-Serville and related genera) were inferred based on molecular characters, with the specific aim to infer the phylogenetic placement of the Afrotropical Sphaerophoria species and Loveridgeana beattiei van Doesburg & van Doesburg. Three molecular markers were used, i.e., the mitochondrial protein-coding gene cytochrome c oxidase subunit I (COI) and the nuclear 28S and 18S ribosomal RNA genes. The Sphaerophoria lineage genera Exallandra Vockeroth and Loveridgeana were resolved within the genus Sphaerophoria, and the Indomalayan Eosphaerophoria Frey was placed sister to Citrogramma Vockeroth, both related to a large species radiation from the New World. Fazia Shannon and Allograpta Osten Sacken were recovered as non-monophyletic. Our results recovered two different Fazia clades with dissimilar natural history resulted from our analyses, and Allograpta species were resolved into two clades, one with Nearctic and Neotropical species and a second clade with species from Oceanian, Indomalayan and Afrotropical Regions. Exallandra is considered a subgenus of Sphaerophoria, S. (Exallandra) stat. rev., and Sphaerophoria cinctifacies (Speiser) n. comb. a member of this subgenus together with S. loewii Zetterstedt. A new Sphaerophoria subgenus is designated S. (Loveridgeana) stat. rev. to include S. beattiei n. comb. and the South African species, i.e., S. quadrituberculata Bezzi, S. retrocurva Hull, and S. aff. retrocurva. Based on their phylogenetic distinctiveness, functional traits, and ecological relevance we do recommend further ecological study and protection efforts for this Afrotropical group of pollinators.

Evaluation and utility of mitochondrial ribosomal genes for molecular systematics of parasitic nematodes

BackgroundMolecular advances have accelerated our understanding of nematode systematics and taxonomy. However, comparative analyzes between various genetic markers have led to discrepancies in nematode phylogenies. This study aimed to evaluate the suitability of using mitochondrial 12S and 16S ribosomal RNA genes for nematode molecular systematics.MethodsTo study the suitability of mitochondrial 12S and 16S ribosomal RNA genes as genetic markers for nematode molecular systematics, we compared them with the other commonly used genetic markers, nuclear internal transcribed spacer 1 and 2 regions, nuclear 18S and 28S ribosomal RNA genes, and mitochondrial cytochrome c oxidase subunit 1 gene. After that, phylum-wide primers for mitochondrial 12S and 16S ribosomal RNA genes were designed, and parasitic nematodes of humans and animals from 75 taxa with 21 representative species were inferred through phylogenetic analyzes. Phylogenetic analyzes were carried out using maximum likelihood and Bayesian inference algorithms.ResultsThe phylogenetic relationships of nematodes based on the mitochondrial 12S rRNA gene supported the monophyly of nematodes in clades I, IV, and V, reinforcing the potential of this gene as a genetic marker for nematode systematics. In contrast, the mitochondrial 16S rRNA gene only supported the monophyly of clades I and V, providing evidence that the 12S rRNA gene is more suitable for nematode molecular systematics. In this study, subclades of clade III containing various nematode families were not monophyletic when the 16S or 12S rRNA gene was used as the genetic marker. This is similar to the phylogenetic relationship revealed by previous studies using whole mitochondrial genomes as genetic markers.ConclusionsThis study supports the use of the 12S rRNA gene as a genetic marker for studying the molecular systematics of nematodes to understand intra-phyla relationships. Phylum-wide primers for nematodes using mitochondrial ribosomal genes were prepared, which may enhance future studies. Furthermore, sufficient genetic variation in the mitochondrial 12S and 16S rRNA genes between species also allowed for accurate taxonomy to species level, revealing the potential of these two genes as genetic markers for DNA barcoding.

Molecular data provide new insights into the phylogeny of Cladonotinae (Orthoptera: Tetrigoidea) from China with the description of a new genus and species.

Considerable effort has been devoted to elucidating the phylogenetic relationships of tetrigides. However, there is still no commonly accepted phylogenetic hypothesis. Therefore, the phylogenetic relationships among some subfamilies remain unclear; e.g., Cladonotinae is a controversial group, in which the phylogenetic relationships between genera and the boundaries of some of the included genera are unclear, causing some of the taxa to be difficult to identify. Therefore, an in-depth phylogenetic analysis of Cladonotinae is urgently needed. In this study, a robust phylogenetic framework for the tetrigides was reconstructed based on the combined mitochondrial cytochrome oxidase subunit I (COI), 16S ribosomal RNA (16S rRNA), and nuclear 18S ribosomal RNA (18S rRNA) gene sequences of 25 species belonging to 16 genera of Tetrigoidea from China, which included 13 species from 8 genera of Cladonotinae. Phylogenetic inferences were performed using the combined dataset and Bayesian inference (BI) and Maximum Parsimony (MP) methods, and the phylogenetic tree of Cladonotinae was reconstructed. All inferences based on the results of the present study supported the Cladonotinae subfamily as a polyphyletic group; within the Cladonotinae subfamily, Tetradinodula, and Tuberfemurus were closely related to Tetriginae, while Austrohancockia and Gibbotettix showed a close relationship to the Scelimenidae subfamily. Additionally, a new genus and new species of the Cladonotinae subfamily are described and illustrated: Hainantettix Deng, gen. nov. and Hainantettix strictivertex Deng, sp. nov.

A new species of the genus Sarsamphiascus Huys, 2009 (Copepoda: Harpacticoida: Miraciidae) from a sublittoral zone of Hawaii.

A new species of Sarsamphiascus Huys, 2009 was collected from sandy sediments of Hawaii at 12 –18 m depth. While the new species, Sarsamphiascus hawaiiensis sp. nov., is morphologically most closely related to S. kawamurai (Ueda & Nagai, 2005), the two species can be distinguished by the combination of the following morphological characteristics: elongated segments of the antennule in the new species, type of outer setae of the P5 exopod (bare in S. kawamurai), position of the inner seta of the P5 exopod in both sexes (more proximal in S. kawamurai), length and type of the setae of female P6 (shorter and bare in S. kawamurai). This is the first species of Sarsamphiascus from Hawaii to be discovered. Molecular analyses of mitochondrial cytochrome c oxidase subunit I (mtCOI) and nuclear 18S ribosomal RNA (18S rRNA) genes confirmed that S. hawaiiensis and S. kawamurai are distinct species.

Exploring Environmental DNA (eDNA) to Assess Biodiversity of Hard Substratum Faunal Communities on the Lucky Strike Vent Field (Mid-Atlantic Ridge) and Investigate Recolonization Dynamics After an Induced Disturbance

Deep hydrothermal vent ecosystems face physical disturbances from naturally occurring volcanic and tectonic activities and are at increasing risk of mineral resource exploitation, raising concerns about the resilience of biological communities. Following destructive events, efficient and rapidly applicable surveys of organisms are required to monitor the state, evolution and recovery of these ecosystems to their baseline status. In this study, we explored the eDNA approach as a tool to assess biodiversity of communities associated with deep-sea hard substrata and tracked the recolonization dynamics of benthic communities living on the Montsegur edifice within the Lucky Strike vent field (Mid-Atlantic Ridge), after an induced disturbance. Hard substratum samples were collected prior to and one year after the disturbance, for environmental DNA (eDNA) metabarcoding using one marker of the mitochondrial Cytochrome Oxidase I (COI) gene and three markers of the nuclear 18S ribosomal RNA (18S) gene. We also generated a DNA barcoding inventory that consisted of taxa physically collected and morphologically identified. This inventory contained amplified barcodes from COI, 18S and the nuclear ribosomal RNA 28S gene. The resulting sequence information from the COI and 18S were used for eDNA taxonomic assignment. The eDNA datasets uncovered a high diversity of metazoan OTUs. Baseline data collected at the start of the experiment identified higher OTU richness at sites peripheral to the active edifice, as well as inactive sites. One year following the initial disturbance, analysis of recolonization data found no statistical difference in presence/absence from baseline communities. The eDNA protocols provide a reproducible strategy to quickly assess biodiversity associated with deep sea hard substratum, enabling comparisons across various habitats. To follow recolonization dynamics at small spatial scales however, we recommend an approach that uses both molecular and morphological-based traditional methods. Finally, we present original data on the “unseen” diversity of the fauna inhabiting the poorly studied inactive sites, locations that are targeted by commercial mining. Continued monitoring of these sites is currently ongoing and will bring new insight on recovery potential over time, with the ultimate goal of informing conservation and management decisions in relation to the protection of hydrothermal vent ecosystems.

An effective method for identification of three mussel species and their hybrids based on SNPs

We described here the successful identification of three economic aquaculture mussel species in China and their hybrids by high resolution melting curve analysis (HRM) of single nucleotide polymorphisms (SNPs). The nuclear 18S ribosomal RNA gene sequences from Mytilus edulis, Mytilus coruscus, and Perna viridis were aligned and amplified. In total, 8 SNP sites were detected from the 79-bp amplicon. A total of 90 samples from the three mussel species were clearly separated, hybrids from M. edulis and M. coruscus could also be clearly identified. The melting profiles from HRM analysis were found to be distinct across the species tested. This new identification method will be a useful tool for discrimination of the three commercially important mussel species and their hybrids.

Morphological and molecular genetic analysis of Synhimantus (Synhimantus) laticeps (Rudolphi, 1819) (Nematoda, Acuariidae) from the barn owl (Tyto alba) and the common kestrel (Falco tinnunculus) in Austria

Summary In the framework of the biodiversity initiative and barcoding project “Austrian Barcode of Life” (ABOL) post mortem examinations of the gastro-intestinal tracts of different species of wild birds were carried out and several adult helminths were retrieved. In the gizzard of two barn owls (Tyto alba) and one common kestrel (Falco tinnuculus) acuariid nematodes belonging to the species Synhimantus (Synhimantus) laticeps (Rudolphi, 1819) were discovered. This report illustrates the identification of this parasitic nematode by morphometric comparison and scanning electron microscopic photographs. Furthermore, genetic identification of individual parasites based on a fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene and the nuclear 18S ribosomal RNA gene was carried out. This report constitutes the first COI-based DNA barcoding of S. (S.) laticeps and its first record in the barn owl (Tyto alba) in Austria.

High-Resolution Melting of 18S rDNA sequences (18S-HRM) for discrimination of bivalve’s species at early juvenile stage: application to a spat survey

It is particularly difficult to identify bivalve species at early development stages (larvae and spat) by morphological examination. Targeting genetic marker commonly used for species identification, we developed a cheap and user friendly tool for bivalve identification through High-Resolution Melting (HRM) analysis. Based on the polymorphism of the nuclear 18S ribosomal RNA gene among 11 orders of bivalve, the 18S-HRM method allowed discriminating species of the Ostreida, Mytilida and Pectinida orders. In this study, this method proved to be a very sensitive tool able to authenticate one species that differed by only one mutation from another one. We applied the method by identifying bivalve species in spat collections from different benthic substrates moored at subtidal and intertidal areas in euhaline conditions. This field survey showed that the settlement of the invasive species Crassostrea gigas was limited vertically to intertidal substrates. In conclusion, the 18S-HRM analysis is a fast, closed-tube method with high resolution and cost effectiveness, and is thus a good alternative method to identify bivalve species. This HRM assay can by applied for species identification of bivalves in the context of ecological management as well as food traceability for bivalves with economical interests.

Molecular phylogenetic relationships of halacarid mites suggest the reevaluation of traditional subfamily classification

Molecular phylogenetic relationships were examined for marine mites in the subfamilies Halacarinae, Copidognathinae, Rhombognathinae and Halixodinae from the aquatic mite family Halacaridae by using nuclear 18S and 28S ribosomal RNA gene sequences. The analysis revealed incongruences between the clustering obtained in the phylogenetic trees and the current morphological classification in the subfamilies Halacarinae and Rhombognathinae (based on 18S sequences) and in the Rhombognathinae (based on both 28S and combined 18S and 28S sequences). By contrast, the tree clustering was consistent with the morphological taxonomic positions in the Copidognathinae and Halixodinae subfamilies. It can be concluded that molecular phylogenetic analysis challenges the current classifications of Halacarinae and Rhombognathinae subfamilies. Accordingly diagnostic characters for their classification should be reevaluated.

Update on Acanthamoeba jacobsi genotype T15, including full-length 18S rDNA molecular phylogeny.

Free-living amoebae of the genus Acanthamoeba are worldwide present in natural and artificial environments, and are also clinically important, as causative agents of diseases in humans and other animals. Acanthamoeba comprises several species, historically assigned to one of the three groups based on their cyst morphology, but presently recognized as at least 20 genotypes (T1-T20) on the basis of their nuclear 18S ribosomal RNA (rRNA) gene (18S rDNA) sequences. While strain identification may usually be achieved targeting short (<500bp) 18S ribosomal DNA (rDNA) fragments, the use of full-length gene sequences (>2200bp) is necessary for correct genotype description and reliable molecular phylogenetic inference. The genotype T15, corresponding to Acanthamoeba jacobsi, is the only genotype described on the basis of partial sequences (~1500bp). While this feature does not prevent the correct identification of the strains, having only partial sequences renders the genotype T15 not completely defined and may furthermore affect its position in the Acanthamoeba molecular tree. Here, we complete this gap, by obtaining full-length 18S rDNA sequences from eight A. jacobsi strains, genotype T15. Morphologies and physiological features of isolated strains are reported. Molecular phylogeny based on full 18S rDNA confirms some previous suggestions for a genetic link between T15 and T13, T16, and T19, with T19 as sister-group to T15.

Euduboscquella costata n. sp. (Dinoflagellata, Syndinea), an Intracellular Parasite of the Ciliate Schmidingerella arcuata: Morphology, Molecular Phylogeny, Life Cycle, Prevalence, and Infection Intensity.

The syndinean dinoflagellate Euduboscquella costata n. sp., an intracellular parasite of the tintinnid ciliate Schmidingerella arcuata, was discovered from Korean coastal water in November of 2013. Euduboscquella costata parasitized in about 62% of the host population, with infection intensity (= number of trophonts in a single host cell) ranging from 1 to 8. Based on morphology and nuclear 18S ribosomal RNA gene sequences, the parasite is new to science. Euduboscquella costata n. sp. had an infection cycle typical of the genus, but had morphological and developmental features that distinguished it from congeneric species. These features include: (1) episome of the trophont with 25-40 grooves converging toward the center of the shield; (2) a narrow, funnel-shaped lamina pharyngea extending from the margin of the episomal shield to the nucleus; (3) persistence of grooves during extracellular development (sporogenesis); (4) a single food vacuole during sporogenesis; (5) separation of sporocytes early in sporogenesis, regardless of type of spore formed; and (6) dinospore size (ca. 14μm in length) and shape (bulbous episome with narrower, tapering hyposome). After sporogenesis, E. costata produced four different types of spore that showed completely identical 18S rRNA gene sequences. The gene sequence was completely identical with a previously reported population, Euduboscquella sp. ex S. arcuata, from Assawoman Bay, USA, indicating that the two populations are likely conspecific. Favella ehrenbergii, a widely recorded tintinnid known to host Euduboscquella spp., co-occurred with S. arcuata, but was not infected by E. costata in field samples or during short-term, cross-infection experiments.

Molecular Identification of Ptychodera flava (Hemichordata: Enteropneusta): Reconsideration in Light of Nucleotide Polymorphism in the 18S Ribosomal RNA Gene.

Seven nuclear and mitochondrial DNA markers were examined in 12 specimens of Ptychodera flava, a model acorn worm used in molecular biology, collected in Japan from three local populations with different modes of living. A comparison of intraspecific results did not show genetically isolated populations despite the species' enclave habitats and asexual reproduction. Moreover, both the nuclear 18S ribosomal RNA gene and mitochondrial 16S ribosomal RNA gene sequences were identical to those from Moorea in French Polynesia, nearly 10,000 kilometers away from Japan. I also provide the first definitive information regarding polymorphisms in 18S ribosomal RNA gene, the external transcribed spacer (ETS), internal transcribed spacers (ITS), and mitochondrial cytochrome c oxidase subunit 1 (mtCO1) sequence in hemichordates using newly designed primer sets, and I show both high larval vagility and certain criteria for the molecular identification of this species.

The systematic position and phylogenetic relationships of Asiobaccha Violovitsh (Diptera, Syrphidae)

The placement and phylogenetic relationships of Asiobaccha were explored using molecular evidence. The mitochondrial protein-coding gene cytochrome c oxidase subunit I (COI) and the nuclear 28S and 18S ribosomal RNA genes were analysed using parsimony, Bayesian inference and Maximum Likelihood. Two alignments approaches were used for rRNA genes: a multiple sequence alignment software, MAFFT, and their secondary structure. The present results do not place Asiobaccha close to Baccha or Allobaccha, which are placed in different evolutionary lineages, but close to Episyrphus and Meliscaeva. The relationship among these three genera is not fully resolved. Morphological characters are discussed and Asiobaccha stat. rev. is proposed as a valid genus.