SSU rRNA Gene Research Articles

Morpho-molecular description of a new HAB species, Pseudocochlodinium profundisulcus gen. et sp. nov., and its LSU rRNA gene based genetic diversity and geographical distribution

Harmful algal blooms (HABs) caused by an unknown dinoflagellate species have frequently occurred in the Pearl River Estuary, China Since 2006. These blooms were associated with severe water discoloration and economic losses, ranging from several km2 to 300 km2 with the maximum recorded cell density being 2.77 × 107 cells⋅L−1. This unknown dinoflagellate species was initially identified as Cochlodinium geminatum and subsequently reclassified as Polykrikos geminatus. However, after reviewing the original descriptions for Cochlodinium geminatum sensu Schütt (1895) and the genus Polykrikos, we considered this species is incongruent with their original descriptions. Further morphological examinations and particularly phylogenetic analyses based on the SSU and partial LSU rRNA genes of isolates and resting cysts from China and Japan prompted us to consider it a new species of a new genus. This new species was proposed to be Pseudocochlodinium profundisulcus gen. et sp. nov., based on its open comma-shaped apical structure complex (ASC), cingulum encircling the cell less than one and a half turns, a deep sulcus with a torsion of a half turn, either single cell or cell chain consisting of two cells with the same number of nuclei and zooids, the resting cyst bearing lobed ornaments, and the evolutionary distances from Polykrikos (and others) on the phylogenetic trees constructed using the concatenated SSU and partial LSU rRNA gene sequences. Metabarcoding investigation of surface sediment samples collected in China revealed that the species to be widely present along the entire Chinese coast with the highest abundance in the South China Sea. Further re-analysis of the Tara Oceans metabarcoding dataset targeting the SSU rRNA gene V9 domain suggested a global distribution of this new genus. Phylogenetic analyses on 46 OTUs (average length: ∼552 bases) of its LSU rRNA gene sequences (mainly D1-D2 domains) obtained from surface sediment samples revealed intraspecific genetic diversity of this species. Interestingly, based on the different distributions and the abundance of these OTUs along the coast of China, this species appeared to have expanded its distribution from the South China Sea to the northern Yellow Sea, or preferred a warm water habitat. We consider that the present work improves the taxonomy and provides important insights into the biogeography of Pseudocochlodinium profundisulcus.⋅

Prevalence of intestinal microsporidiosis by morphological and molecular methods in eastern Iran

Background and objectiveMicrosporidia are obligate intracellular pathogens. The parasite can infect a wide range of vertebrate and invertebrate hosts among all over the world. The current study was performed to evaluate the prevalence of intestinal microsporidiosis by staining and molecular methods in South Khorasan province of Iran. Materials and methodsFrom December 2019 to December 2020, 720 stool samples were stained by specific Weber Modified Trichrome (Chromotrope 2R) to detect Microsporidia spores. The DNA extraction was done by Kit and To amplify the SSU rRNA gene of Microsporidia Spp, the nested PCR was used with genus specific primers. Finally data analyzing were performed by chi square (X2) test, using SPSS software. ResultsFrom 720 stool samples, 25 (3.47%) samples were microscopically positive for intestinal Microsporidia infection. Out of 25 positive samples of Microsporidia in microscopic method, 22 samples were positive in PCR method. In this study, most of the molecularly approved patients were referred to the hospital (68.00%) and were in the age range of 30 to 50 years (40.80%). 92.00% of patients also had gastrointestinal symptoms and were infected in the summer (40.00%), (p < 0.05). ConclusionsThis study was the first evaluation of the prevalence of Microsporidia in South Khorasan province. Microsporidia infection is prevalent in the public population of eastern Iran. Statistical analysis of the results showed that factors such as education, age, gastrointestinal symptoms and season can be risk factors associated with parasitic infections.

Description of the marine predator Sericomyxa perlucida gen. et sp. nov., a cultivated representative of the deepest branching lineage of vampyrellid amoebae (Vampyrellida, Rhizaria).

The vampyrellids (Vampyrellida, Rhizaria) are naked amoebae of considerable genetic diversity. Three families have been well-defined (Vampyrellidae, Leptophryidae, and Placopodidae), but most vampyrellid lineages detected by environmental sequencing are poorly known or completely uncharacterized. In the brackish sediment of Lake Bras D'Or, Nova Scotia, Canada, we discovered an amoeba with a vampyrellid-like life history that was morphologically dissimilar from previously known vampyrellid taxa. We established a culture of this amoeba, studied its feeding behavior and prey range specificity, and characterized it with molecular phylogenetic methods and light and electron microscopy. The amoeba was a generalist predator (i.e. eukaryotroph), devouring a range of marine microalgae, with a strong affinity for some benthic diatoms and Chroomonas. Interestingly, the amoeba varied its feeding strategy depending on the prey species. Small diatoms were engulfed whole, while larger species were fed on through extraction with an invading pseudopodium. The SSU rRNA gene phylogenies robustly placed the amoeba in the most basal, poorly described lineage ("clade C") of the Vampyrellida. Based on the phylogenetic position and the distinct morphology of the studied amoeba, we here describe it as Sericomyxa perlucida gen. et sp. nov., and establish the new vampyrellid family Sericomyxidae for "clade C."

Arbuscular mycorrhizal fungal communities colonising the roots of indigenous legumes of South Africa as revealed by high-throughput DNA metabarcoding

The important ecological role of arbuscular mycorrhizal (AM) fungi on plant communities necessitates an understanding of the diversity and the factors that shape the community composition of AM fungi associated with plants. Therefore, this study investigated the diversity and composition of arbuscular mycorrhizal fungal (AMF) communities colonising the roots of eleven indigenous legumes in two South African provinces using high-throughput sequencing of the partial SSU rRNA gene. The analysis revealed 172 operational taxonomic units (OTUs, at 97% sequence similarity) belonging to eight genera. Arbuscular mycorrhizal fungal community composition varied among legumes within and between provinces, with the genus Glomus dominant. The AMF OTU richness and Shannon-Wiener index were significantly correlated with potassium, copper, manganese, zinc, and silt content. Canonical correspondence analysis indicated that nitrates, pH, manganese, and organic C were the principal soil properties significantly influencing AMF community composition. Overall, findings from this study elucidate that soil factors are pertinent environmental cues that shape legume-AMF symbiosis in semi-arid ecosystems.

Occurrence of Giardia duodenalis in selected stations and tributary rivers of Laguna Lake, Philippines.

Fecal pollution is a major contributor to the progressive degradation of Laguna Lake, the Philippines' largest inland lake used for aquaculture, recreation, and as a source of irrigation and domestic water. Consequently, intestinal parasites may be present in this body of water, posing risks to water safety and human health. This study was able to detect the protozoan parasite, Giardia duodenalis, in three Laguna Lake stations and seven tributary rivers in a 13-month monitoring period by PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene of Giardia cysts concentrated from water samples. The pathogen was present in 37.7% of tributary samples (n = 69) and 16.7% of lake samples (n = 36). Notable frequencies of detection were observed in four tributary rivers -Bagumbayan, Taguig (66.7%); Santa Rosa, Laguna (55.6%); San Cristobal, Cabuyao, Laguna (44.4%); and Biñan, Laguna (42.9%). All test SSU rRNA gene sequences were identified as human-infective genotypes of G. duodenalis predominated by Assemblage A (94.1%). Furthermore, analysis of the glutamate dehydrogenase (gdh) gene revealed the possible presence of mixed genotypes in at least two samples. These results support the pressing need to include protozoan pathogen monitoring in Laguna Lake and its tributaries to prevent Giardia infection in humans and animals. This study also recommends microbial source tracking to identify fecal pollution sources and aid in regulation of waste discharges into the lake and its tributaries.

Cyst-theca relationships of Spiniferites bentorii, S. hyperacanthus, S. ramosus, S. scabratus and molecular phylogenetics of Spiniferites and Tectatodinium (Gonyaulacales, Dinophyceae)

ABSTRACT It is well known that modern resting cysts with morphologies matching those of species of the fossil genus Spiniferites germinate into motile cells of the genus Gonyaulax. Different Spiniferites species have been connected to a single Gonyaulax species, raising the question of whether they are over-classified. Through germination experiments of cysts with the morphological features of four species of Spiniferites, viz. S. bentorii, S. hyperacanthus, S. ramosus and S. scabratus, we established cyst-theca relationships. Cysts with the morphology of S. bentorii gave rise to vegetative, motile cells of Gonyaulax nezaniae sp. nov., which is characterized by two stout antapical spines. Cysts with S. hyperacanthus and S. ramosus morphologies germinated into Gonyaulax whaseongensis and G. spinifera, respectively. Cysts with S. scabratus morphology lacked a ventral pore and were attributed to Gonyaulax cf. spinifera. Gene sequences for SSU, LSU and/or ITS-5.8S rRNA were obtained from these four species, and from cysts with the morphology of Spiniferites belerius, S. mirabilis, S. lazus, Spiniferites cf. bentorii and Tectatodinium pellitum. The maximum likelihood and Bayesian inference analyses based on LSU and SSU rRNA gene sequences revealed that cysts assignable to Spiniferites formed a polyphyletic group, intermingled with Tectatodinium, Bitectatodinium, Ataxiodinium and Impagidinium, whereas Gonyaulax species appeared as monophyletic. From our results we inferred the phylogenetic positions of S. bentorii, S. mirabilis, S. lazus, S. scabratus, Tectatodinium pellitum and Gonyaulax digitale for the first time, supporting the idea that Spiniferites species are not over-classified and each of them may correspond to different Gonyaulax species.

Clypifer cribrifer gen. nov., sp. nov. (Clypiferidae fam. nov., Pterocystida, Centroplasthelida), with notes on evolution of centrohelid siliceous coverings.

A new family, genus and species of centrohelid heliozoans, Clypifer cribrifer gen. nov., sp. nov. (Clypiferidae fam. nov.), from the Gulf of Aqaba (Israel) was studied with light and electron microscopy and SSU rRNA gene sequencing. Clypifer cribrifer has only one type of scales, partially running up the sides of the axopodia. Plate scales [0.8-2.3 (av. 1.5)×0.6-1.8 (av. 1.2) μm] are flat, elliptical or circular, fenestrated with holes of irregular shape and have a marginal rim and a very short axial rib. The cell diameter is 3.9-9.6 (av. 6.0) μm. Molecular phylogenetic analysis robustly places C. cribrifer in the C4 clade for which the new family Clypiferidae is proposed here. This position is confirmed with the short sequences in the panacanthocystid increased regions. The morphology of the new genus has similarities to the genus Raphidocystis. The probability that another Clypifer species was described under a different name in the centrohelid literature is discussed. Clypiferidae represent the second lineage of Pterocystida, which are characterized by the presence of only tangentially oriented plate scales of one type. Possible ways of evolution of the centrohelid siliceous coverings are also discussed.

Wide Genetic Diversity of Blastocystis in White-Tailed Deer (Odocoileus virginianus) from Maryland, USA.

Blastocystis is a gastrointestinal protist frequently reported in humans and animals worldwide. Wildlife populations, including deer, may serve as reservoirs of parasitic diseases for both humans and domestic animals, either through direct contact or through contamination of food or water resources. However, no studies of the occurrence and subtype distribution of Blastocystis in wildlife populations have been conducted in the United States. PCR and next generation amplicon sequencing were used to determine the occurrence and subtypes of Blastocystis in white-tailed deer (Odocoileus virginianus). Blastocystis was common, with 88.8% (71/80) of samples found to be positive. Twelve subtypes were identified, ten previously reported (ST1, ST3, ST4, ST10, ST14, ST21, and ST23–ST26) and two novel subtypes (ST30 and ST31). To confirm the validity of ST30 and ST31, MinION sequencing was used to obtain full-length SSU rRNA gene sequences, and phylogenetic and pairwise distance analyses were performed. ST10, ST14, and ST24 were the most commonly observed subtypes. Potentially zoonotic subtypes ST1, ST3, or ST4 were present in 8.5% of Blastocystis-positives. Mixed subtype infections were common (90.1% of Blastocystis-positives). This study is the first to subtype Blastocystis in white-tailed deer. White-tailed deer were found to be commonly infected/colonized with a wide diversity of subtypes, including two novel subtypes, zoonotic subtypes, and subtypes frequently reported in domestic animals. More studies in wildlife are needed to better understand their role in the transmission of Blastocystis.

Taxonomy and Molecular Phylogeny of Two New Species of Prostomatean Ciliates With Establishment of Foissnerophrys gen. n. (Alveolata, Ciliophora).

Prostomatean ciliates play important roles in the flow of material and energy in aquatic microbial food webs, and thus have attracted wide attention for over a century. Their taxonomy and systematics are, however, still poorly understood because of their relatively few taxonomically informative morphological characters. In this study, two new prostomateans, Lagynus binucleatus sp. n. and Foissnerophrys alveolata gen. n., sp. n., collected from a freshwater pool and the intertidal zone of a sandy beach, respectively, in Qingdao, China, are investigated using living observation, protargol staining, and SSU rRNA gene sequencing methods. The genus Lagynus is redefined, and the new species L. binucleatus sp. n. is established based on significant morphological differences with similar forms. Furthermore, a new genus, Foissnerophrys gen. n., is established based on a combination of morphological and molecular data with F. alveaolata sp. n. the type species by monotypy. The identities of intracellular prokaryotes of these two new species are discussed based on fluorescence in situ hybridization (FISH) data and newly obtained 16S rRNA gene sequences.

Consistency of Bacterial Communities in a Parasitic Worm: Variation Throughout the Life Cycle and Across Geographic Space.

Microbial communities within metazoans are increasingly linked with development, health and behaviour, possibly functioning as integrated evolutionary units with the animal in which they live. This would require microbial communities to show some consistency both ontogenetically (across life stages) and geographically (among populations). We characterise the bacteriome of the parasitic trematode Philophthalmus attenuatus, which undergoes major life cycle transitions, and test whether its bacteriome remains consistent on developmental and spatial scales. Based on sequencing the prokaryotic 16S SSU rRNA gene, we compared the parasite bacteriome (i) across three life stages (rediae in snails, cercariae exiting snails, adults in birds) in one locality and (ii) among three geographic localities for rediae only. We found that each life stage harbours a bacteriome different from that of its host (except the adult stage) and the external environment. Very few bacterial taxa were shared among life stages, suggesting substantial ontogenetic turnover in bacteriome composition. Rediae from the three different localities also had different bacteriomes, with dissimilarities increasing with geographical distance. However, rediae from different localities nevertheless shared more bacterial taxa than did different life stages from the same locality. Changes in the bacteriome along the parasite's developmental history but some degree of geographical stability within a given life stage point toward non-random, stage-specific acquisition, selection and/or propagation of bacteria.

Molecular prevalence and subtyping of Blastocystis sp. isolates in stray cats of İzmir, Turkey: First report of "ST4 allele 42" in cats.

Blastocystis sp. is one of the most frequently detected intestinal parasites in humans and can inhabit a wide range of animals. Close contact with animals is one of the transmission factors of Blastocystis sp. infection in humans. In this study, we aimed to investigate the molecular prevalence and subtypes of Blastocystis sp. in stray cats living in İzmir, Turkey. The PCR target- ing the barcode region in the SSU rRNA gene was performed with DNA samples isolated from feces (n:465) to investigate the presence of Blastocystis sp. PCR positive samples were sequen- ced for subtyping analysis. Among the samples analyzed, Blastocystis sp. DNA was detected in 17 (3.65%) of them and sequence data were obtained from only seven isolates. Phylogenetic analysis showed that seven Blastocystis sp. isolates clustered with the reference Blastocystis ST4 isolates. Similarity rates were between 83.22% and 99.25%. In addition, Blastocystis database results confirmed that all of these were "allele 42" corresponding to ST4. As a result, the present study shows for the first time the presence of "ST4 allele 42", the prevalent subtype in humans, in stray cats in İzmir, Turkey. This finding supports the notion that stray cats can be a source of Blastocystis sp. infection in humans.

A phylogenetic framework to investigate the microsporidian communities through metabarcoding and its application to lake ecosystems.

Microsporidia are obligate intracellular eukaryotic parasites known to parasitize many species of the animal kingdom as well as some protists. However, their diversity is underestimated, in part as a consequence of the failure of 'universal' primers to detect them in metabarcoding studies. Besides, due to the inconsistency between taxonomy and phylogenetic data, available databases may assign incorrectly sequences obtained with high-throughput sequencing. In this work, we developed a comprehensive reference database which positions microsporidian SSU rRNA gene sequences within a coherent ranked phylogenetic framework. We used this phylogenetic framework to study the microsporidian diversity in lacustrine ecosystems, focusing on < 150 μm planktonic size fractions. Our analysis shows a high diversity of Microsporidia, with the identification of 1531 OTUs distributed within seven clades, of which 76% were affiliated to clade IV2 and 20% to clade I (nomenclature presented hereby). About a quarter of the obtained sequences shared less than 85% identity to the closest known species, which might represent undescribed genera or families infecting small hosts. Variations in the abundance of Microsporidia were recorded between the two lakes sampled and across the sampling period, which might be explained by spatio-temporal variations of their potential hosts such as microeukaryotes and metazooplankton.

Full-Length SSU rRNA Gene Sequencing Allows Species-Level Detection of Bacteria, Archaea, and Yeasts Present in Milk.

Full-length SSU rRNA gene sequencing allows species-level identification of the microorganisms present in milk samples. Here, we used bulk-tank raw milk samples of two German dairies and detected, using this method, a great diversity of bacteria, archaea, and yeasts within the samples. Moreover, the species-level classification was improved in comparison to short amplicon sequencing. Therefore, we anticipate that this approach might be useful for the detection of possible mastitis-causing species, as well as for the control of spoilage-associated microorganisms. In a proof of concept, we showed that we were able to identify several putative mastitis-causing or mastitis-associated species such as Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli and Staphylococcus aureus, as well as several Candida species. Overall, the presented full-length approach for the sequencing of SSU rRNA is easy to conduct, able to be standardized, and allows the screening of microorganisms in labs with Illumina sequencing machines.

Ubiquity of Euglena mutabilis Population in Three Ecologically Distinct Acidic Habitats in Southwestern Japan

Three strains of Euglena mutabilis were isolated from sediments in acidic inland water systems (pH = 3.4–4.7), in Southwestern Japan—acid mine drainage in Sensui (Fukuoka), cold sulfidic spring in Bougatsuru (Oita), and a temporal pool in the Ebinokogen volcanic area (Miyazaki). All strains grew well in acidic media at pH 3.07. Phylogenetic analysis among these three strains showed high similarities to plastid SSU and nuclear SSU rRNA gene sequences (99.86% and 99.76%, respectively). They were closely related to the cultured isolates from other highly acidic habitats (pH = 2.0–5.9). Concentration of sulfate, aluminum, calcium, and iron had 7–70 fold of differences among the three studied habitats. Our results imply that the rRNA genes of E. mutabilis have compensated for their low genetic diversity by adapting to a wide pH range, as well as various water chemistry of habitats.

Prevalence and molecular subtyping of Blastocystis in patients with Clostridium difficile infection, Singapore

BackgroundBlastocystis is a common anaerobic colonic protist in humans with controversial pathogenicity. Clostridium difficile (C. difficile) is the commonest cause of infectious diarrhea in healthcare settings. The prevalence and subtype (ST) characteristics of Blastocystis in patients with C. difficile infection (CDI) are rarely documented. Therefore, the present study was conducted to investigate the prevalence and subtype characteristics of Blastocystis in patients with suspicion of CDI in Singapore.MethodsFecal samples were collected from 248 patients presenting with suspected CDI from a single tertiary hospital in Singapore. C. difficile was diagnosed through positive glutamate dehydrogenase (GDH) with or without toxin A/B using enzyme immunoassay methods. The prevalence and subtype genetic characteristics of Blastocystis were determined by polymerase chain reaction (PCR) amplification and analysis of the barcode region of the SSU rRNA gene.ResultsThe proportion of C. difficile in patients with healthcare-associated diarrhea in this study was 44% (109/248). Among the 109 C. difficile-positive patients, 59 (54.1%, 59/109) tested positive for toxigenic C. difficile, which was considered CDI. Based on the sequence analyses of the barcode region of the SSU rRNA gene, 10.1% (25/248) of the patients were found to be Blastocystis-positive, and three subtypes were identified: ST7 (64%, 16/25), ST1 (20%, 5/25), and ST3 (16%, 4/25). Remarkably, we found five patients with Blastocystis and C. difficile coinfection, and further subtype analysis showed two with ST7, two with ST1, and one with ST3.ConclusionsTo the best of our knowledge, this is the first study to investigate the subtype distributions of Blastocystis in patients with CDI in Singapore. We found ST7 to be the predominant subtype in diarrheal patients. The pathogenicity of ST7 has been strongly suggested in previous in vitro and mouse model experiments, further confirming its potential pathogenicity to humans.Graphical

First report of Blastocystis infection in Pallas's squirrels (Callosciurus erythraeus) in China.

Blastocystis, an intestinal anaerobic protist with high genetic diversity, inhabits a variety of hosts worldwide, including rodents. However, there have been few studies on squirrel Blastocystis infections in China to date. Herein, 171 fecal samples from Pallas's squirrels (Callosciurus erythraeus) sold as pets were collected to investigate the prevalence and genetic characteristics of Blastocystis. A total of 10 Blastocystis-positive samples (10/171, 5.9%) were obtained by PCR amplification and DNA sequencing of the barcode region of the SSU rRNA gene. Blastocystis subtype analysis revealed four known subtypes, namely, ST1, ST3, ST5 and ST6, with ST5 and ST6 being predominant. Phylogenetic analysis was performed to identify each subtype. To our knowledge, this study is the first to explore Blastocystis infection in Pallas's squirrels, expanding the host range of this parasite. Moreover, multiple zoonotic subtypes were found in Pallas's squirrels, suggesting that these animals may serve as reservoirs for pathogens of human Blastocystis infections.

Comprehensive single-PCR 16S and 18S rRNA community analysis validated with mock communities, and estimation of sequencing bias against 18S.

Universal primers for SSU rRNA genes allow profiling of natural communities by simultaneously amplifying templates from Bacteria, Archaea, and Eukaryota in a single PCR reaction. Despite the potential to show relative abundance for all rRNA genes, universal primers are rarely used, due to various concerns including amplicon length variation and its effect on bioinformatic pipelines. We thus developed 16S and 18S rRNA mock communities and a bioinformatic pipeline to validate this approach. Using these mocks, we show that universal primers (515Y/926R) outperformed eukaryote-specific V4 primers in observed versus expected abundance correlations (slope=0.88 vs. 0.67-0.79), and mock community members with single mismatches to the primer were strongly underestimated (threefold to eightfold). Using field samples, both primers yielded similar 18S beta-diversity patterns (Mantel test, p< 0.001) but differences in relative proportions of many rarer taxa. To test for length biases, we mixed mock communities (16S+18S) before PCR and found a twofold underestimation of 18S sequences due to sequencing bias. Correcting for the twofold underestimation, we estimate that, in Southern California field samples (1.2-80 μm), there were averages of 35% 18S, 28% chloroplast 16S, and 37% prokaryote 16S rRNA genes. These data demonstrate the potential for universal primers to generate comprehensive microbiome profiles.

Morphology, morphogenesis and phylogeny of Anteglaucoma orientalis n. sp. (Ciliophora, Oligohymenophorea), a freshwater hymenostomatid from northeastern China

A new hymenostomatid ciliate, Anteglaucoma orientalis n. sp., isolated from a freshwater pond in Harbin, northeastern China, was investigated using live observation and silver staining methods. Anteglaucoma orientalis is characterized as follows: size in vivo about 50–60 × 30–35 μm; oval body shape; buccal area occupies about 25% of body length; 28–36 somatic kineties; membranelle 1 having six or seven basal body rows, membranelle 2 five to seven rows, and membranelle 3 three rows; single macronucleus with one micronucleus attached. Morphogenesis of the genus Anteglaucoma is revealed for the first time. The main events during binary fission are as follows: morphogenesis begins with proliferation of kinetosomes in the middle part of postoral kinety 1, and kinetosomes of this primordial field multiply and organize to finally form the paroral membrane and membranelles 1–3 of the opisthe; the parental apparatus in the proter does not take part in the stomatogenetic process. Phylogenetic analyses based on SSU rRNA gene sequences show that Anteglaucoma orientalis n. sp. clusters with the type species, A. harbinensis Pan et al., 2017, with full support.

Mind the Gap: New Full-Length Sequences of Blastocystis Subtypes Generated via Oxford Nanopore Minion Sequencing Allow for Comparisons between Full-Length and Partial Sequences of the Small Subunit of the Ribosomal RNA Gene.

Blastocystis is a common food- and water-borne intestinal protist parasite of humans and many other animals. Blastocystis comprises multiple subtypes (STs) based on variability within the small subunit ribosomal (SSU rRNA) RNA gene. Though full-length reference sequences of the SSU rRNA gene are a current requirement to name a novel Blastocystis subtype, full-length reference sequences are not currently available for all subtypes. In the present study, Oxford Nanopore MinION long-read sequencing was employed to generate full-length SSU rRNA sequences for seven new Blastocystis subtypes for which no full-length references currently exist: ST21, ST23, ST24, ST25, ST26, ST27, and ST28. Phylogenetic analyses and pairwise distance matrixes were used to compare full-length and partial sequences of the two regions that are most commonly used for subtyping. Analyses included Blastocystis nucleotide sequences obtained in this study (ST21 and ST23–ST28) and existing subtypes for which full-length reference sequences were available (ST1–ST17 and ST29). The relationships and sequence variance between new and existing subtypes observed in analyses of different portions of the SSU rRNA gene are discussed. The full-length SSU rRNA reference sequences generated in this study provide essential new data to study and understand the relationships between the genetic complexity of Blastocystis and its host specificity, pathogenicity, and epidemiology.

Global Trends of Benthic Bacterial Diversity and Community Composition Along Organic Enrichment Gradients of Salmon Farms.

The analysis of benthic bacterial community structure has emerged as a powerful alternative to traditional microscopy-based taxonomic approaches to monitor aquaculture disturbance in coastal environments. However, local bacterial diversity and community composition vary with season, biogeographic region, hydrology, sediment texture, and aquafarm-specific parameters. Therefore, without an understanding of the inherent variation contained within community complexes, bacterial diversity surveys conducted at individual farms, countries, or specific seasons may not be able to infer global universal pictures of bacterial community diversity and composition at different degrees of aquaculture disturbance. We have analyzed environmental DNA (eDNA) metabarcodes (V3–V4 region of the hypervariable SSU rRNA gene) of 138 samples of different farms located in different major salmon-producing countries. For these samples, we identified universal bacterial core taxa that indicate high, moderate, and low aquaculture impact, regardless of sampling season, sampled country, seafloor substrate type, or local farming and environmental conditions. We also discuss bacterial taxon groups that are specific for individual local conditions. We then link the metabolic properties of the identified bacterial taxon groups to benthic processes, which provides a better understanding of universal benthic ecosystem function(ing) of coastal aquaculture sites. Our results may further guide the continuing development of a practical and generic bacterial eDNA-based environmental monitoring approach.