Capturing the population structure of microparasites: using ITS‐sequence data and a pooled DNA approach

The internal transcribed spacer (ITS) region of nuclear ribosomal DNA is a common marker not only for the molecular identification of different taxa and strains, but also for the analysis of population structure of wild microparasite communities. Importantly, the multicopy nature of this region allows the amplification of low‐quantity samples of the target DNA, a common problem in studies on unicellular, unculturable microparasites. We analysed ITS sequences from the protozoan parasite Caullerya mesnili (class Ichthyosporea) infecting waterflea (Daphnia) hosts, across several host population samples. We showed that analysing representative ITS‐types [as identified by statistical parsimony network analysis (SPN)] is a suitable method to address relevant polymorphism. The spatial patterns were consistent regardless of whether parasite DNA was extracted from individual hosts or pooled host samples. Remarkably, the efficiency in detecting different sequence types was even higher after sample pooling. As shown by simulations, an easily manageable number of sequences from pooled DNA samples are sufficient to resolve the spatial population structure in this system. In summary, the ITS region analysed from pooled DNA samples can provide valuable insights into the spatial and temporal dynamics of microparasites. Moreover, the application of SPN analysis is a good alternative to the well‐established neighbour‐joining method (NJ) for the identification of representative ITS‐types. SPN can even outperform NJ by joining most of the singleton sequences to representative sequence clusters.     

The Quantification of Representative Sequences pipeline for amplicon sequencing: case study on within‐population ITS1 sequence variation in a microparasite infecting Daphnia

Next generation sequencing (NGS) platforms are replacing traditional molecular biology protocols like cloning and Sanger sequencing. However, accuracy of NGS platforms has rarely been measured when quantifying relative frequencies of genotypes or taxa within populations. Here we developed a new bioinformatic pipeline (QRS) that pools similar sequence variants and estimates their frequencies in NGS data sets from populations or communities. We tested whether the estimated frequency of representative sequences, generated by 454 amplicon sequencing, differs significantly from that obtained by Sanger sequencing of cloned PCR products. This was performed by analysing sequence variation of the highly variable first internal transcribed spacer (ITS1) of the ichthyosporean Caullerya mesnili, a microparasite of cladocerans of the genus Daphnia. This analysis also serves as a case example of the usage of this pipeline to study within‐population variation. Additionally, a public Illumina data set was used to validate the pipeline on community‐level data. Overall, there was a good correspondence in absolute frequencies of C. mesnili ITS1 sequences obtained from Sanger and 454 platforms. Furthermore, analyses of molecular variance (amova ) revealed that population structure of C. mesnili differs across lakes and years independently of the sequencing platform. Our results support not only the usefulness of amplicon sequencing data for studies of within‐population structure but also the successful application of the QRS pipeline on Illumina‐generated data. The QRS pipeline is freely available together with its documentation under GNU Public Licence version 3 at http://code.google.com/p/quantification-representative-sequences .     

Temperature effects on parasite prevalence in a natural hybrid complex

Both host susceptibility and parasite infectivity commonly have a genetic basis, and can therefore be shaped by coevolution. However, these traits are often sensitive to environmental variation, resulting in genotype-by-environment interactions. We tested the influence of temperature on host–parasite genetic specificity in the Daphnia longispina hybrid complex, exposed to the protozoan parasite Caullerya mesnili. Infection rates were higher at low temperature. Furthermore, significant differences between host clones, but not between host taxa, and a host genotype-by-temperature interaction were observed.     

Transmission mode affects the population genetic structure of Daphnia parasites

Parasite life cycle variation can shape parasite evolution, by predisposing them towards different population genetic structures. We compared the population genetic structure of two co-occurring parasite species of Daphnia, to collect evidence for their expected transmission modes. The ichthyosporean Caullerya mesnili has a direct life cycle, whereas the microsporidian Berwaldia schaefernai is hypothesized to require passage through a secondary host. The parasites were collected from three geographically isolated Daphnia populations. The nucleotide variation in the internal transcribed spacer (ITS) region was assessed at the within-individual, within-population and among-population levels, using amova. We detected significant partitioning at all levels, except for a lack of among-population variation in Berwaldia. This was confirmed by neighbour-joining and principal component analyses; Caullerya populations were distinct from each other, while there was much overlap among parasite isolates representing different populations of Berwaldia. This all implies a higher amount of gene flow for Berwaldia, consistent with the hypothesized transmission mode.     

Molecular and morphological characterization of a poorly known marine ciliate, Myoschiston duplicatum precht 1935: implications for phylogenetic relationships between three morphologically similar genera -- Zoothamnium, Myoschiston, and Zoothamnopsis (Ciliophora, Peritrichia, Zoothamniidae)

We studied the morphology and molecular phylogeny of Myoschiston duplicatum, a peritrich ciliate that has been recorded as an epibiont of crustaceans, but which we also identified on marine algae from Korea. The important morphological characteristics revealed by silver staining of Myoschiston species have not been described because they are rarely collected. Using morphological methods, we redescribed the type species of the genus, Myoschiston duplicatum, and provided an improved diagnosis of Myoschiston. In addition, the coding regions for nuclear small subunit (SSU) rRNA and internal transcribed spacer 1‐5.8S‐internal transcribed spacer 2 sequences were sequenced. Phylogenetic analyses that included available SSU rDNA sequences of peritrichs from GenBank strongly supported a position of M. duplicatum within the family Zoothamniidae. In addition, phylogenetic analyses were performed with single datasets (ITS1‐5.8S‐ITS2) and combined datasets (SSU rDNA + ITS1‐5.8S‐ITS2) to explore further the phylogenetic relationship in the family Zoothamniidae between the three morphologically similar genera—Zoothamnium, Myoschiston, and Zoothamnopsis.     

Persistence of host and parasite populations subject to experimental size-selective removal

Predators have the potential to limit the spread of pathogens not only by selecting infected prey but also by shaping prey demographics. We tested this idea with an epidemiological experiment in which we simulated variable levels of size-selective predation on zooplankton hosts and monitored the persistence of host and parasite populations. In the absence of simulated predation, the virulent protozoan Caullerya mesnili frequently drove its host Daphnia galeata to extinction. Uninfected control populations showed lower extinction rates and higher average densities than infected populations in the absence of simulated predation (all of the latter went extinct or remained infected). With a weak removal rate of the largest hosts, the proportion of populations in which the parasite drove the host to extinction decreased, while the number of populations in which the host persisted and the parasite went extinct increased. Host-parasite coexistence was also observed in some cases. With intermediate levels of removal, most of the parasite populations went extinct, while the host populations persisted. With an even higher removal rate, Daphnia were driven to extinction as well. Thus, variation in one factor, size-selective mortality, resulted in four different patterns of population dynamics. Our results highlight the potential role of predation in shaping the epidemiology and community structure of host-parasite systems.     

Molecular diversity of the syndinean genus Euduboscquella based on single-cell PCR analysis

The genus Euduboscquella is one of a few described genera within the syndinean dinoflagellates, an enigmatic lineage with abundant diversity in marine environmental clone libraries based on small subunit (SSU) rRNA. The region composed of the SSU through to the partial large subunit (LSU) rRNA was determined from 40 individual tintinnid ciliate loricae infected with Euduboscquella sampled from eight surface water sites in the Northern Hemisphere, producing seven distinct SSU sequences. The corresponding host SSU rRNA region was also amplified from eight host species. The SSU tree of Euduboscquella and syndinean group I sequences from environmental clones had seven well-supported clades and one poorly supported clade across data sets from 57 to 692 total sequences. The genus Euduboscquella consistently formed a supported monophyletic clade within a single subclade of group I sequences. For most parasites with identical SSU sequences, the more variable internal transcribed spacer (ITS) to LSU rRNA regions were polymorphic at 3 to 10 sites. However, in E. cachoni there was variation between ITS to LSU copies at up to 20 sites within an individual, while in a parasite of Tintinnopsis spp., variation between different individuals ranged up to 19 polymorphic sites. However, applying the compensatory base change model to the ITS2 sequences suggested no compensatory changes within or between individuals with the same SSU sequence, while one to four compensatory changes between individuals with similar but not identical SSU sequences were found. Comparisons between host and parasite phylogenies do not suggest a simple pattern of host or parasite specificity.     

CULTIVATION‐INDEPENDENT SPECIES IDENTIFICATION OF DINOBRYON SPECIES (CHRYSOPHYCEAE) BY MEANS OF MULTIPLEX SINGLE‐CELL PCR1

With the discovery of a high molecular diversity of protists, a discrepancy between morphological and molecular species richness estimates became apparent. Solving the current concerns requires a comparative analysis of different sequences combined with morphological analyses of single cells originating from preserved field samples. We refined a single‐cell PCR (SC‐PCR) protocol for analyzing cells from field samples preserved with Lugol’s iodine solution. We linked microscopic screening with multiplex PCR targeting the SSU rDNA, internal transcribed spacer 1 (ITS1), 5.8S rDNA, internal transcribed spacer 2 (ITS2), and the mitochondrial cytochrome oxidase 1 (CO1) in a single PCR reaction. Using this method, we investigated the intraspecific molecular variation in Dinobryon populations originating from two lakes in the Salzkammergut area of Austria. All investigated genetic markers showed two separated clusters within the investigated populations of Dinobryon divergens O. E. Imhof, indicating a reproductive isolation of the two coexisting populations. Based on these findings, we describe a lineage, which is morphologically similar to D. divergens but, based on the molecular data, is reproductively isolated.     

Phylogeography of the freshwater raphidophyte Gonyostomum semen confirms a recent expansion in northern Europe by a single haplotype

Gonyostmum semen is a freshwater raphidophyte that has increased in occurrence and abundance in several countries in northern Europe since the 1980s. More recently, the species has expanded rapidly also in north‐eastern Europe, and it is frequently referred to as invasive. To better understand the species history, we have explored the phylogeography of G. semen using strains from northern Europe, United States, and Japan. Three regions of the ribosomal RNA gene (small subunit [SSU], internal transcribed spacer [ITS] and large subunit [LSU]) and one mitochondrial DNA marker (cox1) were analyzed. The SSU and partial LSU sequences were identical in all strains, confirming that they belong to the same species. The ITS region differentiated the American from the other strains, but showed high intra‐strain variability. In contrast, the mitochondrial marker cox1 showed distinct differences between the European, American, and Japanese strains. Interestingly, only one cox1 haplotype was detected in European strains. The overall low diversity and weak geographic structure within northern European strains supported the hypothesis of a recent invasion of new lakes by G. semen. Our data also show that the invasive northern European lineage is genetically distinct from the lineages from the other continents. Finally, we concluded that the mitochondrial cox1 was the most useful marker in determining large‐scale biogeographic patterns in this species.     

Revision of the kelp family Alariaceae and the taxonomic affinities of Lessoniopsis Reinke (Laminariales,Phaeophyta)

The morphologically diverse members of the Laminariales are separated into three families, the Alariaceae, Laminariaceae and Lessoniaceae, on the basis of developmental features exhibited at the stipeblade transition zone. We have investigated the relationships of lessoniacean taxa with those of alariacean and laminariacean affinity. This was done by completing phylogenetic analyses of 3 small-subunit (SSU) rRNA gene (113 bp), first internal transcribed spacer (ITS 1), 5.8S rRNA gene and second internal transcribed spacer (ITS2)(12 bp) sequence data from a variety of taxa. In summary, the Alariaceae and Lessoniaceae are polyphyletic. We present a restricted view of the Alariaceae, including only Alaria and Pterygophora of the genera usually placed in this family, and transfer the monotypic genus Lessoniopsis to this group. Current theories of kelp evolution and biogeography must be reconsidered in view of our data.     

Brandtia ciliaticola gen. et sp. nov. (Chlorellaceae,Trebouxiophyceae) a common symbiotic green coccoid of various ciliate species

Many freshwater protists harbor unicellular green algae within their cells and these host‐symbiont relationships slowly are becoming better understood. Recently, we reported that several ciliate species shared a single species of symbiotic algae. Nonetheless, the algae from different host ciliates were each distinguishable by their different genotypes, and these host‐algal genotype combinations remained unchanged throughout a 15‐month period of sampling from natural populations. The same algal species had been reported as the shared symbiont of several ciliates from a remote lake. Consequently, this alga appears to play a key role in ciliate‐algae symbioses. In the present study, we successfully isolated the algae from ciliate cells and established unialgal cultures. This species is herein named Brandtia ciliaticola gen. et sp. nov. and has typical ‘Chlorella‐like’ morphology, being a spherical autosporic coccoid with a single chloroplast containing a pyrenoid. The alga belongs to the Chlorella‐clade in Chlorellaceae (Trebouxiophyceae), but it is not strongly connected to any of the other genera in this group. In addition to this phylogenetic distinctiveness, a unique compensatory base change in the SSU rRNA gene is decisive in distinguishing this genus. Sequences of SSU‐ITS (internal transcribed spacer) rDNA for each isolate were compared to those obtained previously from the same host ciliate. Consistent algal genotypes were recovered from each host, which strongly suggests that B. ciliaticola has established a persistent symbiosis in each ciliate species.     

A new <Emphasis Type="Italic">Coniosporium</Emphasis> species from historical marble monuments

The aim of this investigation was to use morphological and molecular genetic techniques to characterize strains of newly discovered biodeteriorative fungal species originating from antique marble monuments in the Mediterranean basin (Side, Antalya, Turkey). The strains studied were isolated from the lid of a sarcophagus in the Side Museum. Black colonies were picked up and purified by one to several transfers onto culture media. Their morphology was characterized, and DNA was extracted, and amplified. Novelty of the selected species is supported by sequencing of the small ribosomal subunit (18 S) and internal transcribed spacer (ITS) regions. The black meristematic species is placed in the genus Coniosporium. Judging from SSU phylogeny data, this species is supposed to belong to the Chaetothyriales. Analysis of nucleotide sequences did not show a degree of similarity to any sequence in Genbank that would allow to assign it to one of the already existing species. The new species was denominated as C. sümbülii.     

Multiple Independent Losses of Photosynthesis and Differing EvolutionaryRates in the Genus Cryptomonas (Cryptophyceae): Combined Phylogenetic Analyses of DNA Sequences of the Nuclear and the Nucleomorph Ribosomal Operons

In this study, evidence for at least three independent losses of photosynthesis in the freshwater cryptophyte genus Cryptomonas is presented. The phylogeny of the genus was inferred by molecular phylogenetic analyses of the nuclear internal transcribed spacer 2 (nuclear ITS2), partial nuclear large subunit ribosomal DNA (LSU rDNA), and nucleomorph small subunit ribosomal DNA (SSU rDNA, NM). Both concatenated and single data sets were used. In all data sets, the colorless Cryptomonas strains formed three different lineages, always supported by high bootstrap values (maximum parsimony, neighbor joining and maximum likelihood) and posterior probabilities (Bayesian analyses). The three leukoplast-bearing lineages displayed differing degrees of accelerated evolutionary rates in nuclear and nucleomorph rDNA. Also an increase in A+T-content in highly variable regions of the nucleomorph SSU rDNA was observed in one of the leukoplast-bearing lineages.This article contains three online-only supplementary tables.Reviewing Editor: Dr. Yves Van de Peer     

Symbiodinium diversity in the soft coral Heteroxenia sp. and its nudibranch predator Phyllodesmium lizardensis

We examined the diversity of the photosynthetic dinoflagellate, Symbiodinium, over a 2-year period in two invertebrates from Australia’s Northern Great Barrier Reef: the nudibranch Phyllodesmium lizardensis and an octocoral of the genus Heteroxenia. In years one and two, we used denaturing gradient gel electrophoresis with internal transcribed spacer 2 (ITS2) region amplicons and identified two nearly identical genotypes of clade C (C64 and a variant) in all samples of each species. We examined the secondary structure of both sequences and found that each had predicted ∆G values within the range of stable free energy values for Symbiodinium ITS2 sequences. In year two, we also used real-time quantitative polymerase chain reaction assays (qPCR) with clade-specific internal transcribed spacer 1 primers to determine whether there were cryptic clades (A, B, and/or D) associated with either host in addition to clade C. qPCR revealed that clades B, C, and D were present in all animals of both species and that all but two nudibranch samples also harbored clade A. These findings suggest that there may be more flexibility in this host/symbiont interaction than has previously been assumed.

     

First Characterization in China of Encephalitozoon cuniculi in the Blue Fox (Alopex lagopus)

Encephalitozoon cuniculi is a microsporidian parasite that infects a wide range of vertebrates, including primates. It has recently emerged as an opportunistic parasite of patients infected with the human immunodeficiency virus. The blue fox (Alopex lagopus; also known as the arctic fox) is one of the most susceptible species for encephalitozoonosis. Here, we report an outbreak of encephalitozoonosis at a fox farm in China. The isolated parasites displayed the typical morphology of E. cuniculi as assessed by Masson's trichrome staining. Analysis of the internal transcribed spacer sequence indicated that the isolated parasite is a genotype III strain of E. cuniculi. Furthermore, phylogenetic analysis of the PTP1 gene verifies classification of this new strain (termed LN‐1) with other genotype III E. cuniculi strains, though the PTP3 and SWP1 sequences diverge from the reference strain. This is the first report of encephalitozoonosis in farmed blue foxes in China.     

Gymnophallid digenean Parvatrema duboisi uses Manila clam as the first and second intermediate host

To identify the metacercariae of a gymnophallid trematode in the Manila clam Ruditapes philippinarum from the Ariake Sea, experimental infection and molecular analysis were conducted. Based on the morphology of adult worms obtained from experimentally infected mice, the parasite was identified as Parvatrema duboisi. Comparison of internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) sequences for metacercariae and sporocysts from Manila clams and adult worms collected from wild wigeon Anas penelope showed minor differences ranging from 0 to 0.8%. These data strongly suggest that in the Ariake Sea, the parasite has a lifecycle using the Manila clam as the first and second intermediate hosts and wigeon as the definitive host.     

A new meristematic fungus,Pseudotaeniolina globosa

A new species of Pseudotaeniolina, a genus of anamorphic, melanized fungi with meristematic development, is described. The species is compared to morphologically similar taxa among which are Trimmatostroma and Coniosporium. Its novelty is supported by SSU (small subunit) and ITS (internal transcribed spacer) rDNA sequence data. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molecular,biochemical and morphological data suggest an affiliation of Spongiochrysis hawaiiensis with the Trentepohliales (Ulvophyceae,Chlorophyta)

The aeroterrestrial, unicellular green alga Spongiochrysis hawaiiensis had been included in the ulvophycean order Cladophorales based on small subunit (SSU) rDNA sequence data, and represents so far the only fully terrestrial member of this order. Other characteristics of S. hawaiiensis that are atypical for Cladophorales include the presence of large amounts of carotenoids and a budding‐like mode of cell division. As the position of this terrestrial, unicellular alga in an order of aquatic, multicellular green algae is unusual, we re‐evaluated the phylogenetic relationships of this enigmatic organism based on supplementary SSU rDNA sequences as well as novel large ribosomal subunit (LSU) rDNA and internal transcribed spacer (ITS rDNA) sequences. Additionally, we examined several morphological characters of S. hawaiiensis, as well as low molecular weight carbohydrate (LMWC) patterns of S. hawaiiensis and members of the Cladophorales and Trentepohliales as potential chemotaxonomic markers. We found S. hawaiiensis to be uninucleate. The analysis of the LMWC content detected the presence of the polyol erythritol in S. hawaiiensis and in the Trentepohliales, while this compound was missing in the Cladophorales. The phylogenetic analyses of the novel sequences placed S. hawaiiensis in the terrestrial Trentepohliales. This placement is supported by the aeroterrestrial habitat, the presence of large amounts of carotenoids, the uninucleate cells, and the presence of the polyol erythritol as a protective compound against water loss.     

Characterisation of anisakid nematodes with zoonotic potential by nuclear ribosomal dna sequences

Larvae of three species of anisakid nematode from fish, Anisakis simplex, Hysterothylacium aduncum and Contracaecum osculatum, were characterised genetically using a molecular approach. The nuclear ribosomal DNA region spanning the first internal transcribed spacer, the 5.8S gene and the second internal transcribed spacer was amplified and sequenced. The lengths of the first and second internal transcribed spacer sequences of the three species ranged from 392 to 449 bp and 262 to 347 bp, respectively, whereas the 5.8S sequence was 157 bp. For the three species, the G+C contents for the three regions of ribosomal DNA ranged from 42.4 to 52.2%. While no intraspecific variation was detected in the second internal transcribed spacer or 5.8S sequence of any species examined, one polymorphic nucleotide position was detected in the first internal transcribed spacer sequence for A. simplex and H. aduncum. The extent of sequence differences in the first (34–45%) and second (50–53%) internal transcribed spacers among the species was greater than in the 5.8S gene (3–5%). Based on the sequence differences, PCR-based restriction fragment length polymorphism and single-strand conformation polymorphism methods were established for the unequivocal delineation of the three species. These methods should provide valuable tools for studying the life-cycle, transmission pattern (s) and population structure of each of the three anisakid nematodes examined herein, and for the diagnosis of anisakiasis in humans and animals.