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The West Kallaallit Nunaat (Greenland) coast, characterized by a variety of fjords, bays, and channels influenced by glacier melting and Atlantic and Arctic waters, is one of the most affected ecosystems by climate change. Here, we combine oceanography, optics, microscopy, high throughput sequencing, microalgal strain establishments, and state-of-the-art analytical methods to fully characterize the diversity, community composition, and toxin repertoire of the eukaryotic plankton members of the coast of the West Kalaallit Nunaat (Greenland). Results indicate that the West Kalaallit Nunaat (Greenland) coast is a complex and oceanographically challenging system, where the superimposition of water masses of different origins, the penetration of light and its repercussions, generate mainly vertical, rather than horizontal heterogeneity in nutrient concentration and plankton biomass. Nevertheless, our molecular data reveal a strong homogeneity and a high diversity in the plankton community along the Greenland coast. We confirmed the presence of five phycotoxin groups: domoic acid and paralytic shellfish toxins were most abundant along the transect from Qeqertarsuup Tunua (Disko Bay) to the northern Baffin Bay, while spirolides, yessotoxins and pectenotoxins were predominant in Nuup Kangerlua (GodthaabFjord) and Qeqertarsuup Tunua (Disko Bay). Concentrations of these phycotoxins correlate differently to temperature, salinity and nutrients, reflecting the ecological differences of their producers. Patterns of paralytic shellfish and spirolide toxins suggest the presence of genetically distinct populations of Alexandrium along the Western Kalaallit Nunaat (Greenland). Phytoplankton strains isolated during this oceanographic campaign resemble, in most cases, the toxin profiles of the respective field stations. Overall, this work shows the diversity and community composition of the plankton at the Western Kalaallit Nunaat coast and reveals a distinct spatial distribution of phycotoxins, with certain toxin groups restricted to specific areas. [ABSTRACT FROM AUTHOR]

Ticks are a common source for a variety of microorganisms especially those which are pathogenic or highly fatal. Hyalomma species are potential vectors for the CCHF virus. This study sought to identify Hyalomma spp. isolated form sheep and camel found in the Al- Samawah desert of Al-Muthanna Province. Sixty samples of ticks were used in this study, and each was subjected to PCR and partial gene sequencing using the 18S rRNA gene. The results from the analysis showed that 45 of 60 samples (75%) were identified as Hyalomma spp. While 17 PCR-purified products were chosen for sequencing, and the aligned data were used to build the phylogenetic tree. Uphill climbing the roof of phylogenetic analysis, the result reigned these isolates in this study as Hyalomma asiaticum related to several isolates in GenBank. This research has shown that these ticks of sheep and camel in the Al-Samawah desert of Al-Muthanna Province are commonly a marker of many viruses, including the CCHF virus. Further in-depth work ought to be completed as a priority for disease control purposes. [ABSTRACT FROM AUTHOR]

Harmful algal blooms (HABs) often occur in estuaries due to their unique environmental heterogeneity, posing significant environmental and human health risks. However, there is limited understanding of the community composition and community‐level change points (thresholds) of harmful microalgae in subtropical estuaries. This study explored harmful microalgae community structure and thresholds in the Dafengjiang River estuary using a metabarcoding approach. The results revealed 63 harmful microalgae species, and major species included Guinardia flaccida, Prorocentrum cordatum, Thalassiosira punctigera, Pseudo‐nitzschia galaxiae and T. gravida. Nonparametric change‐point analysis and threshold indicator taxa analysis (TITAN) showed threshold responses of harmful microalgae community structure to ammonium (57.5–60 μg·L−1), total phosphorus (27.8–28.5 μg·L−1) and dissolved inorganic phosphorus (14.5–28 μg·L−1) along the salinity gradient. Wider environmental thresholds were also found in hypersaline areas. Additionally, Pyrodinium bahamense, Pfiesteria piscicida, Skeletonema tropicum and T. punctigera were sensitive to environmental changes and thus could be used as bioindicators. Overall, our study unveiled diverse abrupt transitions of harmful microalgal communities, providing a risk assessment for human health and ecological safety in subtropical estuary ecosystems. [ABSTRACT FROM AUTHOR]

Ciliated microeukaryotes are insufficiently investigated despite their ubiquity and ecological significance. The morphology and morphogenesis of a new Stichotrichida species, Pseudosincirra binaria sp. nov., and the known Perisincirra paucicirrata Foissner et al., 2002, are here studied using live observations and protargol staining methods. The new species is characterized by having one buccal, one parabuccal and three frontal cirri, one frontoventral row extending to the posterior half of the cell, three left and two right marginal rows and three dorsal kineties with the left kinety conspicuously bipartite, along with one caudal cirrus at the rear end of each kinety. During morphogenesis, there exist five frontal-ventral cirral anlagen with anlagen IV and V forming a frontoventral row in the proter, and four cirral anlagen with only anlage IV generating a frontoventral row in the opisthe. The anlagen for marginal rows and dorsal kineties develop intrakinetally. The new population of Perisincirra paucicirrata corresponds well with other isolates regarding morphology and cell development. Phylogenetic analyses based on small subunit ribosomal gene sequence data revealed that both Perisincirra and Pseudosincirra are deeply clustered in the clade consisting of species from the genera Deviata and Heterodeviata, supporting the placement of both genera into the family Deviatidae Foissner, 2016. [ABSTRACT FROM AUTHOR]

The Yongle blue hole (YBH), situated in the South China Sea, represents a compelling subject of study in marine microbiology due to its unique redoxlayered microbial ecosystems. However, the diversity and ecology of microbial eukaryotes within the YBH remains underexplored. This study endeavors to bridge this gap through the application of the in situ microbial filtration and fixation (ISMIFF) device to collect 0.22-30 µm microbial samples from 21 water layers of YBH. Subsequent extraction of 18S rRNA metagenomic reads of 21 metagenomes and 10 metatranscriptomes facilitated a comprehensive analysis of community structures. Findings revealed a pronounced superiority in the diversity and richness of eukaryotic microorganisms in the oxic zone compared to its suboxic and anoxic counterparts. Notably, Dinophyceae and Maxillopoda emerged as the predominant taxa based on the analysis of the 18S rRNA reads for the V4 and V9 regions, which showed stratification In their relative abundance and suggested their potential role in the thermohalocline boundaries and oxic-anoxic interface. Specifically, In these eukaryotic microbial communities, Dinophyceae exhibited significant abundance at 20 m (20.01%) and 105 m (26.13%) water depths, while Maxillopoda was prevalent at 40 m (22.84%), 80 m (23.19%), and 100 m (15.42%) depths. A part of these organisms, identified as larvae and protists, were likely attracted by swarming chemosynthetic bacterial prey prevailing at the thermo-halocline boundaries and oxic-anoxic interface. Furthermore, the phylogenetic relationships of the major 18S operational taxonomic units (OTUs) showed a close adjacency to known species, except for three Dinophyceae OTUs. In conclusion, this study provides critical insights into the vertical distribution and transcriptional activity of <30-µm eukaryotic microbes, shedding light on the taxonomic novelty of eukaryotic microorganisms within the semi-enclosed blue holes. [ABSTRACT FROM AUTHOR]

The West Kallaallit Nunaat (Greenland) coast, characterized by a variety of fjords, bays, and channels influenced by glacier melting and Atlantic and Arctic waters, is one of the most affected ecosystems by climate change. Here, we combine oceanography, optics, microscopy, high throughput sequencing, microalgal strain establishments, and state-of-the-art analytical methods to fully characterize the diversity, community composition, and toxin repertoire of the eukaryotic plankton members of the coast of the West Kalaallit Nunaat (Greenland). Results indicate that the West Kalaallit Nunaat (Greenland) coast is a complex and oceanographically challenging system, where the superimposition of water masses of different origins, the penetration of light and its repercussions, generate mainly vertical, rather than horizontal heterogeneity in nutrient concentration and plankton biomass. Nevertheless, our molecular data reveal a strong homogeneity and a high diversity in the plankton community along the Greenland coast. We confirmed the presence of five phycotoxin groups: domoic acid and paralytic shellfish toxins were most abundant along the transect from Qeqertarsuup Tunua (Disko Bay) to the northern Baffin Bay, while spirolides, yessotoxins and pectenotoxins were predominant in Nuup Kangerlua (GodthaabFjord) and Qeqertarsuup Tunua (Disko Bay). Concentrations of these phycotoxins correlate differently to temperature, salinity and nutrients, reflecting the ecological differences of their producers. Patterns of paralytic shellfish and spirolide toxins suggest the presence of genetically distinct populations of Alexandrium along the Western Kalaallit Nunaat (Greenland). Phytoplankton strains isolated during this oceanographic campaign resemble, in most cases, the toxin profiles of the respective field stations. Overall, this work shows the diversity and community composition of the plankton at the Western Kalaallit Nunaat coast and reveals a distinct spatial distribution of phycotoxins, with certain toxin groups restricted to specific areas.

ABSTRACT Harmful algal blooms (HABs) often occur in estuaries due to their unique environmental heterogeneity, posing significant environmental and human health risks. However, there is limited understanding of the community composition and community‐level change points (thresholds) of harmful microalgae in subtropical estuaries. This study explored harmful microalgae community structure and thresholds in the Dafengjiang River estuary using a metabarcoding approach. The results revealed 63 harmful microalgae species, and major species included Guinardia flaccida, Prorocentrum cordatum, Thalassiosira punctigera, Pseudo‐nitzschia galaxiae and T. gravida. Nonparametric change‐point analysis and threshold indicator taxa analysis (TITAN) showed threshold responses of harmful microalgae community structure to ammonium (57.5–60 μg·L−1), total phosphorus (27.8–28.5 μg·L−1) and dissolved inorganic phosphorus (14.5–28 μg·L−1) along the salinity gradient. Wider environmental thresholds were also found in hypersaline areas. Additionally, Pyrodinium bahamense, Pfiesteria piscicida, Skeletonema tropicum and T. punctigera were sensitive to environmental changes and thus could be used as bioindicators. Overall, our study unveiled diverse abrupt transitions of harmful microalgal communities, providing a risk assessment for human health and ecological safety in subtropical estuary ecosystems.

Abstract Background Trichomonosis is a common infection in small animals, mostly manifesting in gastrointestinal symptoms such as diarrhea. Although oral trichomonads are also known, the species found colonizing the large intestine are more frequently detected protozoa. Methods In the present study, four wildcats, 94 domestic cats, and 25 dogs, originating from 18 different locations in Hungary, were investigated for the presence of oral and large intestinal trichomonads based on the 18S rRNA gene and ITS2. Results All oral swabs were negative by polymerase chain reaction (PCR). However, Tritrichomonas foetus was detected in a high proportion among tested domestic cats (13.8%) and dogs (16%), and Pentatrichomonas hominis only in two domestic cats. In addition, a novel Tritrichomonas genotype was identified in one cat, probably representing a new species that was shown to be phylogenetically most closely related to Tritrichomonas casperi described recently from mice. All positive dogs and half of the positive cats showed symptoms, and among cats, the most frequent breed was the Ragdoll. Conclusions With molecular methods, this study evaluated the prevalence of oral and intestinal trichomonads in clinical samples of dogs and cats from Hungary, providing the first evidence of T. foetus in dogs of this region. In contrast to literature data, P. hominis was more prevalent in cats than in dogs. Finally, a hitherto unknown large intestinal Tritrichomonas species (closely related to T. casperi) was shown to be present in a cat, raising two possibilities. First, this novel genotype might have been a rodent-associated pseudoparasite in the relevant cat. Otherwise, the cat was actually infected, thus suggesting the role of a predator–prey link in the evolution of this trichomonad. Graphical Abstract

Background: Trichomonosis is a common infection in small animals, mostly manifesting in gastrointestinal symptoms such as diarrhea. Although oral trichomonads are also known, the species found colonizing the large intestine are more frequently detected protozoa. Methods: In the present study, four wildcats, 94 domestic cats, and 25 dogs, originating from 18 different locations in Hungary, were investigated for the presence of oral and large intestinal trichomonads based on the 18S rRNA gene and ITS2. Results: All oral swabs were negative by polymerase chain reaction (PCR). However, Tritrichomonas foetus was detected in a high proportion among tested domestic cats (13.8%) and dogs (16%), and Pentatrichomonas hominis only in two domestic cats. In addition, a novel Tritrichomonas genotype was identified in one cat, probably representing a new species that was shown to be phylogenetically most closely related to Tritrichomonas casperi described recently from mice. All positive dogs and half of the positive cats showed symptoms, and among cats, the most frequent breed was the Ragdoll. Conclusions: With molecular methods, this study evaluated the prevalence of oral and intestinal trichomonads in clinical samples of dogs and cats from Hungary, providing the first evidence of T. foetus in dogs of this region. In contrast to literature data, P. hominis was more prevalent in cats than in dogs. Finally, a hitherto unknown large intestinal Tritrichomonas species (closely related to T. casperi) was shown to be present in a cat, raising two possibilities. First, this novel genotype might have been a rodent-associated pseudoparasite in the relevant cat. Otherwise, the cat was actually infected, thus suggesting the role of a predator–prey link in the evolution of this trichomonad. [ABSTRACT FROM AUTHOR]

DNA-based stable isotope probing (DNA-SIP) technology has been widely employed to trace microbes assimilating target substrates. However, the fractions with labelled universal genes are sometimes difficult to distinguish when detected by quantitative real-time PCR. In this experiment, three paddy soils (AQ, CZ, and NB) were amended with 0.1% glucose containing 13C at six levels, and DNA was then extracted after a 7-day incubation and subjected to isopycnic gradient centrifugation. The results showed that the amount of labelled DNA was notably related to the 13C-glucose percentage, while the separation spans of 18S rRNA and 16S rRNA genes between labelled and unlabelled treatments became notably clearer when the δ13C values of the total DNA were 90.9, 61.6, and 38.9‰ and 256.2, 104.5 and 126.1‰ in the AQ, CZ, and NB soils, respectively. Moreover, fractionated DNA was also labelled by determining the δ13C values while adding only 5 atom% 13C-glucose to the soil. The results suggest that the optimal labelling fractions were not always those fractions with the maximal gene abundance, and detecting the δ13C values of the total and fractionated DNA was beneficial in estimating the results of DNA-SIP. Key points: • Appropriate 13C-DNA amount was needed for DNA-SIP. • Detecting the 13C ratio of fractionated DNA directly was an assistant method for identifying the labelled fractions. • Fractions with the maximal 18S or 16S rRNA gene abundance always were not labelled. [ABSTRACT FROM AUTHOR]

Ciliates of the order Pleurostomatida play essential functions in microbial food webs from a variety of habitats and have been thought to possess a high level of diversity. Due to undersampling and often absent molecular data, the actual diversity and phylogenetic relationships within this group remain unclarified. To help address this deficiency, a survey of freshwater pleurostomatid ciliates was undertaken in Lake Weishan Wetland, northern China. Here, two new Amphileptus species, Amphileptus sinicus sp. nov. and Amphileptus piscinarius sp. nov., were investigated using modern morphological and molecular techniques. Amphileptus sinicus sp. nov. is characterized by possessing a comparatively large cell size of 330–490 μm, contractile vacuoles on both ventral and dorsal margins, and 8–10 left and 42–61 right kineties. Amphileptus piscinarius sp. nov. is characterized by possessing a cell size of 140–210 μm, a large distinctly developed apical extrusome group, 3–4 contractile vacuoles on the ventral margin, and 6–8 left and 24–28 right kineties. Phylogenetic results based on the 18S rRNA gene data of these two species group them with other congeners, with these data suggesting the genus Amphileptus is paraphyletic. [ABSTRACT FROM AUTHOR]

Unicellular eukaryotes that are capable of phago-mixotrophy in the ocean compete for inorganic nutrients and light with autotrophs, and for bacterial prey with heterotrophs. In this study, we ask what the overall prevalence of eukaryotic mixotrophs in the vast open ocean is, and how the availability of inorganic nutrients, light, and prey affects their relative success. We utilized the Tara Oceans eukaryotic 18S rRNA gene and environmental context variables dataset to conduct a large-scale field analysis. We also performed isolate-based culture experiments to verify growth and nutritional resource relationships for representative mixotrophic taxa. The field analysis suggested that the overall prevalence of mixotrophs were negatively correlated with nutrient concentrations and positively associated with light availability. Concentrations of heterotrophic bacteria as a single variable also presented a positive correlation with mixotrophic prevalence, but to a lesser extent. On the other hand, the culture experiments demonstrated a taxa-specific relationship between mixotrophic growth and nutrition resources, i.e., the growth of one group was significantly dependent on light availability, while the other group was less affected by light when they received sufficient prey. Both groups were capable of growing efficiently with low inorganic nutrients when receiving sufficient prey and light. Therefore, our field analysis and culture experiments both suggest that phago-mixotrophy for ocean eukaryotes is seemingly an efficient strategy to compensate for nutrient deficiency but unnecessary to compensate for light scarcity. This study collectively revealed a close relationship between abiotic and biotic nutritional resources and the prevalence of trophic strategies, shedding light on the importance of light and nutrients for determining the competitive success of mixotrophs versus autotrophic and heterotrophic eukaryotes in the ocean. [ABSTRACT FROM AUTHOR]

Ciliated microeukaryotes are insufficiently investigated despite their ubiquity and ecological significance. The morphology and morphogenesis of a new Stichotrichida species, Pseudosincirra binaria sp. nov., and the known Perisincirra paucicirrata Foissner et al., 2002, are here studied using live observations and protargol staining methods. The new species is characterized by having one buccal, one parabuccal and three frontal cirri, one frontoventral row extending to the posterior half of the cell, three left and two right marginal rows and three dorsal kineties with the left kinety conspicuously bipartite, along with one caudal cirrus at the rear end of each kinety. During morphogenesis, there exist five frontal-ventral cirral anlagen with anlagen IV and V forming a frontoventral row in the proter, and four cirral anlagen with only anlage IV generating a frontoventral row in the opisthe. The anlagen for marginal rows and dorsal kineties develop intrakinetally. The new population of Perisincirra paucicirrata corresponds well with other isolates regarding morphology and cell development. Phylogenetic analyses based on small subunit ribosomal gene sequence data revealed that both Perisincirra and Pseudosincirra are deeply clustered in the clade consisting of species from the genera Deviata and Heterodeviata, supporting the placement of both genera into the family Deviatidae Foissner, 2016.

Introduction: High-throughput sequencing (HTS) provides an efficient and cost-effective way to generate large amounts of sequence data, providing a very powerful tool to analyze biodiversity of soil organisms. However, marker-based methods and the resulting datasets come with a range of challenges and disputes, including incomplete reference databases, controversial sequence similarity thresholds for delimitating taxa, and downstream compositional data analysis. Methods: Here, we use HTS data from a soil nematode biodiversity experiment to explore standardized HTS data processing procedures. We compared the taxonomic assignment performance of two main rDNA reference databases (SILVA and PR2). We tested whether the same ecological patterns are detected with Amplicon Sequence Variants (ASV; 100% similarity) versus classical Operational Taxonomic Units (OTU; 97% similarity). Further, we tested how different HTS data normalization methods affect the recovery of beta diversity patterns and the identification of differentially abundant taxa. Results: At this time, the SILVA 138 eukaryotic database performed better than the PR2 4.12 database, assigning more reads to family level and providing higher phylogenetic resolution. ASV- and OTU-based alpha and beta diversity of nematodes correlated closely, indicating that OTU-based studies represent useful reference points. For downstream data analyses, our results indicate that loss of data during subsampling under rarefaction-based methods might reduce the sensitivity of the method, e.g. underestimate the differences between nematode communities under different treatments, while the clr-transformation-based methods may overestimate effects. The Analysis of Compositions of Microbiome with Bias Correction approach (ANCOM-BC) retains all data and accounts for uneven sampling fractions for each sample, suggesting that this is currently the optimal method to analyze compositional data. Discussion: Overall, our study highlights the importance of comparing and selecting taxonomic reference databases before data analyses, and provides solid evidence for the similarity and comparability between OTU- and ASV-based nematode studies. Further, the results highlight the potential weakness of rarefaction-based and clr-transformation-based methods. We recommend future studies use ASV and that both the taxonomic reference databases and normalization strategies are carefully tested and selected before analyzing the data. [ABSTRACT FROM AUTHOR]

The genus Acanthamoeba comprises free-living amoebae distributed in a wide variety of environments. These amoebae are clinically significant, causing opportunistic infections in humans and other animals. Despite this, limited data on Acanthamoeba sequence types and alleles are available in Italy. In the present study, we analyzed all Acanthamoeba sequences deposited from Italy with new positive Acanthamoeba clinical samples from symptomatic AK cases, to provide an overview of the genetic variants' spatial patterns from different sources within the Italian context. A total of 137 Acanthamoeba sequences were obtained. Six sequence types were identified: T2/6, T3, T4, T11, T13, and T15. Only T4 and T15 were found in both sources. The Acanthamoeba T4 sequence type was found to be the most prevalent in all regions, accounting for 73% (100/137) of the Italian samples analyzed. The T4 sequence type demonstrated significant allelic diversity, with 30 distinct alleles from clinical and/or environmental samples. These outcomes enabled a better understanding of the distribution of Acanthamoeba isolates throughout Italy, reaffirming its well-recognized ubiquity. Acanthamoeba isolates analysis from keratitis, together with the environmental strains monitoring, might provide important information on different genotypes spreading. This might be useful to define the transmission pathways of human keratitis across different epidemiological scales. [ABSTRACT FROM AUTHOR]

Four species of dominant wild animals, namely, Prionailurus bengalensis euptilurus, Nyctereutes procyonoides koreensis, Hydropotes inermis argyropus, and Sus scrofa coreanus, are hosts of potential infectious agents, including helminths and protozoa. Therefore, it is necessary to analyze the infectious agents present in these wild animals to monitor and control the spread of pathogens. In the present study, fecal samples from 51 wild animals were collected from the mountains of Yangpyeong, Hoengseong, and Cheongyang in South Korea and metabarcoding of the V9 region of the 18S rRNA gene was performed to identify various parasite species that infect these wild animals. Genes from nematodes, such as Metastrongylus sp., Strongyloides spp., Ancylostoma sp., and Toxocara sp., were detected in the fecal samples from wild animals. In addition, platyhelminthes, including Spirometra sp., Echinostomatidae gen. sp., Alaria sp., Neodiplostomum sp., and Clonorchis sp., and protozoa, including Entamoeba sp., Blastocystis sp., Isospora sp., Tritrichomonas sp., Pentatrichomonas sp., and Cryptosporidium sp., were detected. In the present study, various parasites infecting wild animals were successfully identified using metabarcoding. Our technique may play a crucial role in monitoring parasites within wild animals, especially those causing zoonoses.

Objective:Acanthamoeba, one of the free-living amoeba, has been detected in many environmental samples, mainly in water, soil and air. Acanthamoeba keratitis and granulomatous amoebic encephalitis are among the most important clinical manifestations caused by Acanthamoeba. In this study, it was aimed to determine the sensitivity of the rapid loop mediated isothermal amplification (LAMP) test designed with primers specific to Acanthamoeba 18S rRNA gene to detect more rapidly the presence of Acanthamoeba in clinical and environmental samples.Methods:Acanthamoeba strain grown in culture was diluted in 200 μL as 1x106 trophozoites and DNA was isolated, and the amount of DNA was determined by Nano-Drop Spectrophotometer. The purified DNAs were diluted from 1000 pg to 0.001 pg and used in colorimetric and fluorescence-based LAMP reactions. The LAMP reaction mixture was incubated for 60 minutes at 63 °C in a total volume of 25 μL.Results:To determine the sensitivity of the test, positivity of Acanthamoeba genomic DNA was observed at 1, 10, 100 and 1000 pg/reaction in both colorimetric and fluorescence-based LAMP tests. The lowest analytical sensitivity of both calorimetric and fluorescent LAMP assay was determined as 1 pg/reaction. In addition, as a result of LAMP reaction applied with other parasite DNAs to evaluate the specificity of the test, no LAMP product was detected in calorimetric and 1% agarose gel electrophoresis, except for positive control, and the specificity of the test was determined as 100%.Conclusion:It has been demonstrated that the LAMP assay designed by targeting 18S rRNA gene of Acanthamoeba has a detection limit of 1 pg of genomic DNA. It is promising that LAMP test is more sensitive and faster than culture method, as well as simple, inexpensive and highly sensitive. For this reason, it is thought that developed test can be applied in the diagnosis of Acanthamoeba in environmental and clinical samples.

IntroductionHigh-throughput sequencing (HTS) provides an efficient and cost-effective way to generate large amounts of sequence data, providing a very powerful tool to analyze biodiversity of soil organisms. However, marker-based methods and the resulting datasets come with a range of challenges and disputes, including incomplete reference databases, controversial sequence similarity thresholds for delimitating taxa, and downstream compositional data analysis. MethodsHere, we use HTS data from a soil nematode biodiversity experiment to explore standardized HTS data processing procedures. We compared the taxonomic assignment performance of two main rDNA reference databases (SILVA and PR2). We tested whether the same ecological patterns are detected with Amplicon Sequence Variants (ASV; 100% similarity) versus classical Operational Taxonomic Units (OTU; 97% similarity). Further, we tested how different HTS data normalization methods affect the recovery of beta diversity patterns and the identification of differentially abundant taxa.ResultsAt this time, the SILVA 138 eukaryotic database performed better than the PR2 4.12 database, assigning more reads to family level and providing higher phylogenetic resolution. ASV- and OTU-based alpha and beta diversity of nematodes correlated closely, indicating that OTU-based studies represent useful reference points. For downstream data analyses, our results indicate that loss of data during subsampling under rarefaction-based methods might reduce the sensitivity of the method, e.g. underestimate the differences between nematode communities under different treatments, while the clr-transformation-based methods may overestimate effects. The Analysis of Compositions of Microbiome with Bias Correction approach (ANCOM-BC) retains all data and accounts for uneven sampling fractions for each sample, suggesting that this is currently the optimal method to analyze compositional data.DiscussionOverall, our study highlights the importance of comparing and selecting taxonomic reference databases before data analyses, and provides solid evidence for the similarity and comparability between OTU- and ASV-based nematode studies. Further, the results highlight the potential weakness of rarefaction-based and clr-transformation-based methods. We recommend future studies use ASV and that both the taxonomic reference databases and normalization strategies are carefully tested and selected before analyzing the data.

Trypanosoma dionisii, for which only bat bugs (Cimicidae) had previously been demonstrated as vectors, was, for the first time, detected in the gamasine mite Steatonyssus periblepharus in Russia. The molecular phylogenetic analysis indicated that trypanosomes found in these mites belong to the "clade A" of T. dionisii, which, based on genetic distances, can be considered as a species separate from the sister clade B, and according to available data also has a distinct geographic distribution. The presence of developmental forms of T. dionisii resembling those previously described during the development of this trypanosome in cimicids suggests that S. periblepharus is a novel vector of the studied trypanosome. [ABSTRACT FROM AUTHOR]

The abyssal seafloor is a mosaic of highly diverse habitats that represent the least known marine ecosystems on Earth. Some regions enriched in natural resources, such as polymetallic nodules in the Clarion-Clipperton Zone (CCZ), attract much interest because of their huge commercial potential. Since nodule mining will be destructive, baseline data are necessary to measure its impact on benthic communities. Hence, we conducted an environmental DNA and RNA metabarcoding survey of CCZ biodiversity targeting microbial and meiofaunal eukaryotes that are the least known component of the deep-sea benthos. We analyzed two 18S rRNA gene regions targeting eukaryotes with a focus on Foraminifera (37F) and metazoans (V1V2), sequenced from 310 surface-sediment samples from the CCZ and other abyssal regions. Our results confirm huge unknown deep-sea biodiversity. Over 60% of benthic foraminiferal and almost a third of eukaryotic operational taxonomic units (OTUs) could not be assigned to a known taxon. Benthic Foraminifera are more common in CCZ samples than metazoans and dominated by clades that are only known from environmental surveys. The most striking results are the uniqueness of CCZ areas, both datasets being characterized by a high number of OTUs exclusive to the CCZ, as well as greater beta diversity compared to other abyssal regions. The alpha diversity in the CCZ is high and correlated with water depth and terrain complexity. Topography was important at a local scale, with communities at CCZ stations located in depressions more diverse and heterogeneous than those located on slopes. This could result from eDNA accumulation, justifying the interim use of eRNA for more accurate biomonitoring surveys. Our descriptions not only support previous findings and consolidate our general understanding of deep-sea ecosystems, but also provide a data resource inviting further taxon-specific and large-scale modeling studies. We foresee that metabarcoding will be useful for deep-sea biomonitoring efforts to consider the diversity of small taxa, but it must be validated based on ground truthing data or experimental studies.

Abstract Climatic changes are anticipated to have a detrimental effect on calcifying marine species. Calcareous red algae may be especially vulnerable to seasonal variations since they are common and essential biologically, but there is little research on the morpho-anatomical, and chemical characterization of such species. This study conducted the seasonal investigation of the three dominant Mediterranean calcified red algae. Morphological and 18S rRNA analysis confirmed the identification of collected species as Corallina officinalis, Jania rubens, and Amphiroa rigida. In general, C. officinalis was represented in the four seasons and flourishing maximum in autumn (70% of total species individuals). While J. rubens species was represented in winter, autumn, and spring and completely absent in summer. A. rigida was abundant only in the summer season by 40%. A full morphological and anatomical description of these species were examined, and their chemical compositions (carbohydrate, protein, lipid, pigments, and elements content) were assessed in different seasons, where carbohydrates were the dominant accumulates followed by proteins and lipids. Pearson correlation analysis confirmed a positive correlation between salinity level and nitrogenous nutrients of the seawater with the pigment contents (phycobiliproteins, carotenoids, and chlorophyll a) of the studied seaweeds. The results proved that calcified red algae were able to deposit a mixture of calcium carbonates such as calcite, vaterite, calcium oxalate, calcite-III I calcium carbonate, and aragonite in variable forms depending on the species.

Ciliates of the order Pleurostomatida play essential functions in microbial food webs from a variety of habitats and have been thought to possess a high level of diversity. Due to undersampling and often absent molecular data, the actual diversity and phylogenetic relationships within this group remain unclarified. To help address this deficiency, a survey of freshwater pleurostomatid ciliates was undertaken in Lake Weishan Wetland, northern China. Here, two new Amphileptus species, Amphileptus sinicus sp. nov. and Amphileptus piscinarius sp. nov., were investigated using modern morphological and molecular techniques. Amphileptus sinicus sp. nov. is characterized by possessing a comparatively large cell size of 330–490 μm, contractile vacuoles on both ventral and dorsal margins, and 8–10 left and 42–61 right kineties. Amphileptus piscinarius sp. nov. is characterized by possessing a cell size of 140–210 μm, a large distinctly developed apical extrusome group, 3–4 contractile vacuoles on the ventral margin, and 6–8 left and 24–28 right kineties. Phylogenetic results based on the 18S rRNA gene data of these two species group them with other congeners, with these data suggesting the genus Amphileptus is paraphyletic.

As a zoonotic parasite, Cryptosporidium spp. could cause severe diarrhea mainly in calves and children globally. Monitoring and prevention of Cryptosporidium spp.’s prevalence is of great significance in both economy and public health aspects. In this study, specific primers and probes were designed within the conserved region of 18S rRNA gene for Cryptosporidium spp. and recombinase polymerase amplification assays based on the fluorescence monitoring (real-time RPA) as well as combined with a lateral flow strip (LFS RPA) were developed. Both of the two RPA assays allowed the exponential amplification of the target fragment within 20 min. After incubation on a metal bath at 42 °C, the LFS RPA results were displayed on the lateral flow strip within 5 min while real-time RPA allowed the real-time observation of the results in Genie III at 39 °C. The RPA assays showed high specificity for Cryptosporidium spp. without any cross-reaction with other tested pathogens causing diarrhea in cattle. With the recombinant plasmid DNA containing the 18S rRNA gene of Cryptosporidium spp. serving as a template, the limit of detection for real-time RPA and LFS RPA assays were 14.6 and 12.7 copies/reaction, respectively. Moreover, the RPA assays were validated by testing diarrheic cattle fecal samples and compared with a real-time PCR. The positive ratio of Cryptosporidium spp. was 24.04 % (44/183) and 26.23 % (48/183) in both RPA assays and real-time PCR assay, respectively, and the kappa coefficient value was 0.942. The diagnostic specificity and diagnostic sensitivity of both RPA assays were 100 % and 91.67 %, respectively. Forty-one of 48 positive samples were successfully sequenced and four Cryptosporidium species were detected, including C. parvum (n = 20), C. andersoni (n = 17), C. bovis (n = 3) and C. ryanae (n = 1). The developed RPA assays are easy to operate and faster to obtain the detection results, and they are suiting for the point-of-care detection and facilitating the prevention and control of Cryptosporidium spp. infections.

A new coccidian species, Isospora elliotae n. sp., from the Australian magpie Gymnorhina tibicen (Latham, 1801) in Western Australia, is described and characterized morphologically and molecularly. Microscopic analysis of a faecal sample identified subspheroidal oocysts (n = 20), 20–22 × 18–20 (20.7 × 18.7); length/width (L/W) ratio 1.05–1.14 (1.10). Wall bi‐layered, 1.0–1.3 (1.2) thick, outer layer smooth, c. 2/3 of total thickness. Micropyle and oocyst residuum absent, but usually two polar granules are present. Sporocysts (n = 28) ovoidal, 12–13 × 9–11 (12.6 × 9.7); L/W ratio 1.22–1.35 (1.30). Stieda body present, flattened to half‐moon‐shaped, c. 0.5 deep × 2.0 wide; sub‐Stieda indistinct or barely discernible, c. 1.0 deep × 2.5 wide; para‐Stieda body absent; sporocyst residuum present, composed of granules dispersed among the sporozoites. Sporozoites vermiform, with anterior and posterior refractile bodies and nucleus. Segments of three gene loci (18S rRNA, 28S rRNA and COI) were sequenced and I. elliotae n. sp. exhibited 99.8% genetic similarity to Isospora sp. MAH‐2013a (KF648870) followed by 99.7% genetic similarity to Isospora neochmiae (Yang, Brice & Ryan, 2016) (KT224380) at the 18S rRNA gene locus. It shared 97.0% genetic similarity with an unnamed Isospora sp. (AY283852) at the 28S rRNA gene locus and it also shared the highest genetic similarity of 99.8% with the unnamed Isospora sp. from an American crow (OL999120) at the COI gene locus. Based on morphological and molecular data, this isolate is a new species named as I. elliotae n. sp. [ABSTRACT FROM AUTHOR]

We present here the first detailed description of the seasonal patterns in bacterial community composition (BCC) in shelf waters off the Ría de Vigo (Spain), based on monthly samplings during 2 years. Moreover, we studied the relationship between bacterial and small-sized eukaryotic community composition to identify potential biotic interactions among components of these two communities. Bacterial operational taxonomic unit (OTU) richness and diversity systematically peaked in autumn–winter, likely related to low resource availability during this period. BCC showed seasonal and vertical patterns, with Rhodobacteraceae and Flavobacteriaceae families dominating in surface waters, and SAR11 clade dominating at the base of the photic zone (30 m depth). BCC variability was significantly explained by environmental variables (e.g., temperature of water, solar radiation, or dissolved organic matter). Interestingly, a strong and significant correlation was found between BCC and small-sized eukaryotic community composition (ECC), which suggests that biotic interactions may play a major role as structuring factors of the microbial plankton in this productive area. In addition, co-occurrence network analyses revealed strong and significant, mostly positive, associations between bacteria and small-sized phytoplankton. Positive associations likely result from mutualistic relationships (e.g., between Dinophyceae and Rhodobacteraceae), while some negative correlations suggest antagonistic interactions (e.g., between Pseudo-nitzchia sp. and SAR11). These results support the key role of biotic interactions as structuring factors of the small-sized eukaryotic community, mostly driven by positive associations between small-sized phytoplankton and bacteria. [ABSTRACT FROM AUTHOR]

Abstract Background The subgenus Pholeoixodes contains Ixodes species typically associated with birds that nest in cavities or with carnivorous mammals that are burrow-dwelling. Among ticks infesting the latter, Ixodes rugicollis is regarded as the rarest species in the western Palearctic. Despite the unique morphology of this species, its identification (especially of subadult stages) is difficult, and molecular-phylogenetic data to offer other diagnostic methods and a better understanding of its taxonomy are not available. Methods In this study, a female and a male of I. rugicollis were collected in Romania. The female was compared morphologically to another female of this species collected in France and to the lectotype of Ixodes cornutus (from Tajikistan), which has similar morphology and host association. Following DNA extraction, two mitochondrial (cytochrome c oxidase subunit I: cox1 and the 16S rRNA gene) and two nuclear genetic markers (18S and 28S rRNA genes) of I. rugicollis were amplified and analyzed in a phylogenetic context. Results Females of I. rugicollis and I. cornutus differed in the shape of their palps, scutum and areae porosae and the size of peritremes, but they were similar in palpal setal length, dental formula and arrangement of anal setae. Measurements of two I. rugicollis females examined were not less different from each other than from I. cornutus. Phylogenetically, I. rugicollis clustered with other members of its subgenus. The topology of all trees showed the position of bat-associated tick species of the subgenus Eschatocephalus among Pholeoixodes species. Conclusions For the first time to our knowledge, this study provides high-resolution digital pictures of male and female I. rugicollis as well as corresponding molecular data. Morphological comparison of this species with I. cornutus could not resolve uncertainties in the validity of the latter species, which can only be accomplished after collecting new specimens of I. cornutus and consequent molecular comparisons. This study includes the first comprehensive molecular-phylogenetic analysis of western Palearctic Pholeoixodes species based on both nuclear and mitochondrial genetic markers and including I. rugicollis. The results of these confirm the phylogenetic position of subgenus Eschatocephalus within Pholeoixodes, justifying the need to merge them to comply with the taxonomic criterion of monophyly. Graphical Abstract

The microbial community plays an important role in the biogeochemical cycles in water aquatic ecosystems, and it is regulated by environmental variables. However, the relationships between microbial keystone taxa and water variables, which play a pivotal role in aquatic ecosystems, has not been clarified in detail. We analyzed the seasonal variation in microbial communities and co-occurrence network in the representative areas taking Lake Dongqian as an example. Both pro- and eukaryotic community compositions were more affected by seasons than by sites, and the prokaryotes were more strongly impacted by seasons than the eukaryotes. Total nitrogen, pH, temperature, chemical oxygen demand, dissolved oxygen and chlorophyll a significantly affected the prokaryotic community, while the eukaryotic community was significantly influenced by total nitrogen, ammonia, pH, temperature and dissolved oxygen. The eukaryotic network was more complex than that of prokaryotes, whereas the number of eukaryotic keystone taxa was less than that of prokaryotes. The prokaryotic keystone taxa belonged mainly to Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Bacteroidetes. It is noteworthy that some of the keystone taxa involved in nitrogen cycling are significantly related to total nitrogen, ammonia, temperature and chlorophyll a, including Polaromonas, Albidiferax, SM1A02 and Leptolyngbya so on. And the eukaryotic keystone taxa were found in Ascomycota, Choanoflagellida and Heterophryidae. The mutualistic pattern between pro- and eukaryotes was more evident than the competitive pattern. Therefore, it suggests that keystone taxa could be as bio-indicators of aquatic ecosystems. [ABSTRACT FROM AUTHOR]

Cockatiels (Nymphicus hollandicus) are among the most commonly sold psittacines pets. The aim of this study was to evaluate the occurrence of Cryptosporidium spp. in domestic N. hollandicus and identify risk factors for this infection. We collected fecal samples from 100 domestic cockatiels in the city of Araçatuba, São Paulo, Brazil. Feces from birds of both genders and older than two months were collected. Owners were asked to complete a questionnaire to identify how they handle and care for their birds. Based on nested PCR targeting the 18S rRNA gene, the prevalence of Cryptosporidium spp. in the cockatiels sampled was 9.00%, 6.00% based on Malachite green staining, 5.00% based on modified Kinyoun straining, and 7.00% when the Malachite green was combined with Kinyoun. Applying multivariate logistic regression to test the association between Cryptosporidium proventriculi positivity and potential predictors showed that gastrointestinal alterations was a significant predictor (p < 0.01). Amplicons from five samples were sequenced successfully and showed 100% similarity with C. proventriculi. In summary, this study demonstrates the occurrence of C. proventriculi in captive cockatiels. [ABSTRACT FROM AUTHOR]

Climatic changes are anticipated to have a detrimental effect on calcifying marine species. Calcareous red algae may be especially vulnerable to seasonal variations since they are common and essential biologically, but there is little research on the morpho-anatomical, and chemical characterization of such species. This study conducted the seasonal investigation of the three dominant Mediterranean calcified red algae. Morphological and 18S rRNA analysis confirmed the identification of collected species as Corallina officinalis, Jania rubens, and Amphiroa rigida. In general, C. officinalis was represented in the four seasons and flourishing maximum in autumn (70% of total species individuals). While J. rubens species was represented in winter, autumn, and spring and completely absent in summer. A. rigida was abundant only in the summer season by 40%. A full morphological and anatomical description of these species were examined, and their chemical compositions (carbohydrate, protein, lipid, pigments, and elements content) were assessed in different seasons, where carbohydrates were the dominant accumulates followed by proteins and lipids. Pearson correlation analysis confirmed a positive correlation between salinity level and nitrogenous nutrients of the seawater with the pigment contents (phycobiliproteins, carotenoids, and chlorophyll a) of the studied seaweeds. The results proved that calcified red algae were able to deposit a mixture of calcium carbonates such as calcite, vaterite, calcium oxalate, calcite-III I calcium carbonate, and aragonite in variable forms depending on the species. Highlights: The abundance and diversity of the Rhodophyta species are threatened by global climate changes, rising sea levels, and stronger tidal waves. Shoreline hardening had direct impacts on the abundance and distribution of native species. Calcified species can be used as biomarker species for assessing climate changes. The morphological and 18S rRNA analysis confirmed the identified species. Only three species of red calcified algae viz. Corallina officinalis, Jania rubens, and Amphiroa rigida were recorded during seasons of autumn 2019−summer 2020. [ABSTRACT FROM AUTHOR]

Background: Cryptosporidium spp., Cystoisospora belli and Cyclospora cayetanensis are common intestinal coccidian parasites causing gastroenteritis. The clinical presentation caused by each parasite is indistinguishable from each other. Uniplex polymerase chain reaction (PCR) for these three groups of intestinal coccidian parasites was developed by us in our laboratory. Thereafter, we planned to develop a single-run multiplex polymerase chain reaction (mPCR) assay to detect Cryptosporidium spp., C. belli and C. cayetanensis simultaneously from a stool sample and described it here as coccidian mPCR. Methods: New primers for C. belli and C. cayetanensis were designed and uniplex PCRs were standardized. The coccidian mPCR was standardized with known positive DNA control isolates. It was validated with 58 known positive and 58 known negative stool samples, which were previously identified by uniplex PCR. Results: The coccidian mPCR was standardized with earlier primers designed by us for Cryptosporidium spp. and C. cayetanensis, and a newly designed primer for the internal transcribed spacer-1 (ITS-1) gene for C. belli. The coccidian mPCR was 92.1% sensitive for Cryptosporidium spp., and 100% sensitive for C. belli and C. cayetanensis each, when tested on 116 known samples. It was 100% specific for all intestinal coccidian parasites. Two representative PCR products of the newly designed ITS-1 primer for C. belli were sequenced and submitted to the GenBank, which best match with the sequences of C. belli. Conclusion: A highly sensitive, specific, cost-effective, indigenous, single-run coccidian mPCR has been developed, which can simultaneously detect Cryptosporidium spp., C. belli and C. cayetanensis. [ABSTRACT FROM AUTHOR]

Abstract A new coccidian species, Isospora elliotae n. sp., from the Australian magpie Gymnorhina tibicen (Latham, 1801) in Western Australia, is described and characterized morphologically and molecularly. Microscopic analysis of a faecal sample identified subspheroidal oocysts (n = 20), 20–22 × 18–20 (20.7 × 18.7); length/width (L/W) ratio 1.05–1.14 (1.10). Wall bi‐layered, 1.0–1.3 (1.2) thick, outer layer smooth, c. 2/3 of total thickness. Micropyle and oocyst residuum absent, but usually two polar granules are present. Sporocysts (n = 28) ovoidal, 12–13 × 9–11 (12.6 × 9.7); L/W ratio 1.22–1.35 (1.30). Stieda body present, flattened to half‐moon‐shaped, c. 0.5 deep × 2.0 wide; sub‐Stieda indistinct or barely discernible, c. 1.0 deep × 2.5 wide; para‐Stieda body absent; sporocyst residuum present, composed of granules dispersed among the sporozoites. Sporozoites vermiform, with anterior and posterior refractile bodies and nucleus. Segments of three gene loci (18S rRNA, 28S rRNA and COI) were sequenced and I. elliotae n. sp. exhibited 99.8% genetic similarity to Isospora sp. MAH‐2013a (KF648870) followed by 99.7% genetic similarity to Isospora neochmiae (Yang, Brice & Ryan, 2016) (KT224380) at the 18S rRNA gene locus. It shared 97.0% genetic similarity with an unnamed Isospora sp. (AY283852) at the 28S rRNA gene locus and it also shared the highest genetic similarity of 99.8% with the unnamed Isospora sp. from an American crow (OL999120) at the COI gene locus. Based on morphological and molecular data, this isolate is a new species named as I. elliotae n. sp.

The genus Acanthamoeba comprises free-living amoebae distributed in a wide variety of environments. These amoebae are clinically significant, causing opportunistic infections in humans and other animals. Despite this, limited data on Acanthamoeba sequence types and alleles are available in Italy. In the present study, we analyzed all Acanthamoeba sequences deposited from Italy with new positive Acanthamoeba clinical samples from symptomatic AK cases, to provide an overview of the genetic variants’ spatial patterns from different sources within the Italian context. A total of 137 Acanthamoeba sequences were obtained. Six sequence types were identified: T2/6, T3, T4, T11, T13, and T15. Only T4 and T15 were found in both sources. The Acanthamoeba T4 sequence type was found to be the most prevalent in all regions, accounting for 73% (100/137) of the Italian samples analyzed. The T4 sequence type demonstrated significant allelic diversity, with 30 distinct alleles from clinical and/or environmental samples. These outcomes enabled a better understanding of the distribution of Acanthamoeba isolates throughout Italy, reaffirming its well-recognized ubiquity. Acanthamoeba isolates analysis from keratitis, together with the environmental strains monitoring, might provide important information on different genotypes spreading. This might be useful to define the transmission pathways of human keratitis across different epidemiological scales.

During a nematological survey in Iraq, in the Bashika area, Ninevah province, an anguinid nematode population was isolated from galls of infected barley plants. The morphological characteristics indicated that the recovered species is identical to Anguina tritici. The barley population of A. tritici was molecularly characterized by sequencing two ribosomal regions (ITS and 18S rRNA genes), and their phylogenetic analyses revealed the newly generated sequences are in sister relation to corresponding sequences of A. tritici from wheat in the Bayesian tree, providing further evidence that the host plant can contribute to the separation of new isolates of plant parasitic nematodes.

Background: The subgenus Pholeoixodes contains Ixodes species typically associated with birds that nest in cavities or with carnivorous mammals that are burrow-dwelling. Among ticks infesting the latter, Ixodes rugicollis is regarded as the rarest species in the western Palearctic. Despite the unique morphology of this species, its identification (especially of subadult stages) is difficult, and molecular-phylogenetic data to offer other diagnostic methods and a better understanding of its taxonomy are not available. Methods: In this study, a female and a male of I. rugicollis were collected in Romania. The female was compared morphologically to another female of this species collected in France and to the lectotype of Ixodes cornutus (from Tajikistan), which has similar morphology and host association. Following DNA extraction, two mitochondrial (cytochrome c oxidase subunit I: cox1 and the 16S rRNA gene) and two nuclear genetic markers (18S and 28S rRNA genes) of I. rugicollis were amplified and analyzed in a phylogenetic context. Results: Females of I. rugicollis and I. cornutus differed in the shape of their palps, scutum and areae porosae and the size of peritremes, but they were similar in palpal setal length, dental formula and arrangement of anal setae. Measurements of two I. rugicollis females examined were not less different from each other than from I. cornutus. Phylogenetically, I. rugicollis clustered with other members of its subgenus. The topology of all trees showed the position of bat-associated tick species of the subgenus Eschatocephalus among Pholeoixodes species. Conclusions: For the first time to our knowledge, this study provides high-resolution digital pictures of male and female I. rugicollis as well as corresponding molecular data. Morphological comparison of this species with I. cornutus could not resolve uncertainties in the validity of the latter species, which can only be accomplished after collecting new specimens of I. cornutus and consequent molecular comparisons. This study includes the first comprehensive molecular-phylogenetic analysis of western Palearctic Pholeoixodes species based on both nuclear and mitochondrial genetic markers and including I. rugicollis. The results of these confirm the phylogenetic position of subgenus Eschatocephalus within Pholeoixodes, justifying the need to merge them to comply with the taxonomic criterion of monophyly. [ABSTRACT FROM AUTHOR]

Abstract Background Among live wild mammals adapted to urban and peri-urban habitats in Europe, members of the families Felidae, Mustelidae and Sciuridae deserve special attention as pathogen reservoirs because all of these families include members that are kept as pets. We report here the results of our study on two important groups of tick-borne protozoan parasites in ticks and tissues of wild cats, mustelids and red squirrels. Methods DNA was extracted from the tissues of carnivores (wild cats, mustelids; n = 16) and red squirrels (n = 4), as well as from ixodid ticks (n = 89) collected from these hosts. These DNA extracts were screened for piroplasms and Hepatozoon spp. using conventional PCR analysis and sequencing. In addition, 53 pooled samples of 259 questing Haemaphysalis concinna ticks were evaluated for the presence of Hepatozoon DNA, followed by phylogenetic analyses. Results One wild cat was found to be coinfected with Cytauxzoon europaeus and a new genotype of Hepatozoon felis, and two additional wild cats were infected with H. felis from a different phylogenetic group. In mustelids, Hepatozoon martis and two further Hepatozoon genotypes were detected. The latter clustered separately, close to others reported from eastern Asia. In addition, Hepatozoon sciuri was detected in red squirrels. Morphologic and molecular analyses verified eight tick species. One wild cat was infected with a H. felis genotype that was significantly different from that in Ixodes ricinus females infesting this cat. Only three pools of questing H. concinna nymphs tested positive for Hepatozoon, one of which contained H. martis. Conclusions This study provides the first evidence of the occurrence of any Cytauxzoon species and of three Hepatozoon species in Hungary. In addition to H. martis, two further mustelid-associated Hepatozoon genotypes were detected, one of which was new in terms of phylogenetic and broader geographical contexts. This may be the first indication that H. felis genotypes from both of its phylogenetic groups occur in Europe. This also appears to be the first evidence of H. felis and C. europaeus coinfection in felids in Europe, and of autochthonous H. felis infection in wild cats north of the Mediterranean Basin. New tick–host associations were also observed in this study. Based on the results, H. felis and H. martis might survive transstadially in I. ricinus and H. concinna, respectively. Graphical Abstract

Trypanosoma dionisii, for which only bat bugs (Cimicidae) had previously been demonstrated as vectors, was, for the first time, detected in the gamasine mite Steatonyssus periblepharus in Russia. The molecular phylogenetic analysis indicated that trypanosomes found in these mites belong to the “clade A” of T. dionisii, which, based on genetic distances, can be considered as a species separate from the sister clade B, and according to available data also has a distinct geographic distribution. The presence of developmental forms of T. dionisii resembling those previously described during the development of this trypanosome in cimicids suggests that S. periblepharus is a novel vector of the studied trypanosome.

Abstract How does the evolution of bioinformatics tools impact the biological interpretation of high‐throughput sequencing datasets? For eukaryotic metabarcoding studies, in particular, researchers often rely on tools originally developed for the analysis of 16S ribosomal RNA (rRNA) datasets. Such tools do not adequately account for the complexity of eukaryotic genomes, the ubiquity of intragenomic variation in eukaryotic metabarcoding loci, or the differential evolutionary rates observed across eukaryotic genes and taxa. Recently, metabarcoding workflows have shifted away from the use of operational taxonomic units (OTUs) toward delimitation of amplicon sequence variants (ASVs). We assessed how the choice of bioinformatics algorithm impacts the downstream biological conclusions that are drawn from eukaryotic 18S rRNA metabarcoding studies. We focused on four workflows including UCLUST and VSearch algorithms for OTU clustering, and DADA2 and Deblur algorithms for ASV delimitation. We used two 18S rRNA datasets to further evaluate whether dataset complexity had a major impact on the statistical trends and ecological metrics: a “high complexity” (HC) environmental dataset generated from community DNA in Arctic marine sediments, and a “low complexity” (LC) dataset representing individually barcoded nematodes. Our results indicate that ASV algorithms produce more biologically realistic metabarcoding outputs, with DADA2 being the most consistent and accurate pipeline regardless of dataset complexity. In contrast, OTU clustering algorithms inflate the metabarcoding‐derived estimates of biodiversity, consistently returning a high proportion of “rare” molecular operational taxonomic units (MOTUs) that appear to represent computational artifacts and sequencing errors. However, species‐specific MOTUs with high relative abundance are often recovered regardless of the bioinformatics approach. We also found high concordance across pipelines for downstream ecological analysis based on beta‐diversity and alpha‐diversity comparisons that utilize taxonomic assignment information. Analyses of LC datasets and rare MOTUs are especially sensitive to the choice of algorithms and better software tools may be needed to address these scenarios.

Babesia gibsoni is a tick-borne apicomplexan protozoan causing canine babesiosis. This parasite has diploid sexual reproduction in ticks, during which genetic exchanges can occur leading to increased genetic diversity, which is an important factor in adapting to environmental changes. Exploring the genetic variation of B. gibsoni population can provide a foundation for understanding the patterns of disease transmission and developing babesiosis control strategies. Partial 18S rRNA fragment sequences were obtained from 11 B. gibsoni isolates collected from different regions in China and 117 publicly available sequences were from 12 geographical areas including China. The genetic variation, demographic expansion and population structure were examined. A total of 34 haplotypes were identified among B. gibsoni populations. Analysis of molecular variance, pairwise Fst and structure analysis showed that high genetic variation within populations, low genetic differentiation and obvious mixture haplotype were apparent in a single continent, but higher genetic differentiation was detected across different continents. Neutrality tests implied that B. gibsoni populations had experienced population extension. These findings will contribute to understand the genetics and evolution of B. gibsoni and will be useful for formulating effective management strategies to prevent and control this parasite.

The Southwest Atlantic Ocean (SWAO) is a spatially dynamic region with a remarkably high primary productivity. An exhaustive identification of the members of its phytoplankton community is a key step to understand the processes that sustain this ecosystem. Here, we provide a community composition analysis of eukaryotic phytoplankton in four SWAO sectors. We gathered 18S rRNA gene metabarcoding data and complemented it with confocal microscopy images, both from the Tara Oceans expedition in late spring 2010. Our work showed local and regional variation across three different size fractions that reflect the complexity of this region. Diversity decreased along temperature and latitudinal gradients, but also showed intricate patterns of occurrences across samples, suggesting that multiple factors shape the community structure. Samples resembled communities from other temperate regions and showed an increasing influence by cold waters from the Southern Ocean towards higher latitudes. These results complement previous regional studies that used other methods such as microscopy or pigment analysis. Our study contributes to the beginning of genomic-based surveys of plankton communities in the SW Atlantic and calls for further work in the region to enhance ecosystem monitoring and projections in the context of global change. [ABSTRACT FROM AUTHOR]

Mycoplankton are an important component of marine ecosystems and play a key role in material cycling and energy flow in marine ecosystems. In this study, high-throughput sequencing of the 18S rDNA gene was employed to investigate the community structure of mycoplankton during summer and winter and their response to environmental changes in the Dafengjiang River estuary in the Beibu Gulf, Guangxi. The mycoplanktonic community was generally dominated by Ascomycota, Basidiomycota, and Cryptomycota. However, there were significant seasonal differences in the α-diversity of the mycoplanktonic community (p < 0.05). Random forest modeling also revealed that Paramicrosporidium, Parengyodontium, Arthrinium, Paramycosphaerella, Pestalotia, and Talaromyces were the most effective bioindicators of environmental changes. Spearman's correlation analysis and distance-based redundancy analysis suggested that the trophic status, chemical oxygen demand, dissolved oxygen, and salinity were the key environmental factors regulating the mycoplanktonic community structure. Variation partitioning analysis also found that nutrient levels were the main driver of the β-diversity of the mycoplanktonic community, showing a greater effect than the water quality parameters. In conclusion, this study revealed the mycoplanktonic community structure and its key drivers in the Dafengjiang River estuary, thus providing a theoretical reference for ecological environmental monitoring and resource management in the Beibu Gulf. [ABSTRACT FROM AUTHOR]

Abstract Background The microbiome affects the health of plants and animals, including humans, and has many biological, ecological, and evolutionary consequences. Microbiome studies typically rely on sequencing ribosomal 16S RNA gene fragments, which serve as taxonomic markers for prokaryotic communities; however, for eukaryotic microbes this approach is compromised, because 18S rRNA gene sequences from microbial eukaryotes are swamped by contaminating host rRNA gene sequences. Results To overcome this problem, we developed CRISPR-Cas Selective Amplicon Sequencing (CCSAS), a high-resolution and efficient approach for characterizing eukaryotic microbiomes. CCSAS uses taxon-specific single-guide RNA (sgRNA) to direct Cas9 to cut 18S rRNA gene sequences of the host, while leaving protistan and fungal sequences intact. We validated the specificity of the sgRNA on ten model organisms and an artificially constructed (mock) community of nine protistan and fungal pathogens. The results showed that > 96.5% of host rRNA gene amplicons were cleaved, while 18S rRNA gene sequences from protists and fungi were unaffected. When used to assess the eukaryotic microbiome of oyster spat from a hatchery, CCSAS revealed a diverse community of eukaryotic microbes, typically with much less contamination from oyster 18S rRNA gene sequences than other methods using non-metazoan or blocking primers. However, each method revealed taxonomic groups that were not detected using the other methods, showing that a single approach is unlikely to uncover the entire eukaryotic microbiome in complex communities. To facilitate the application of CCSAS, we designed taxon-specific sgRNA for ~16,000 metazoan and plant taxa, making CCSAS widely available for characterizing eukaryotic microbiomes that have largely been neglected. Conclusion CCSAS provides a high-through-put and cost-effective approach for resolving the eukaryotic microbiome of metazoa and plants with minimal contamination from host 18S rRNA gene sequences. Video Abstract

Trichomonas gallinae is a geographically widespread protozoan parasite of birds. In this study, oropharyngeal swab samples were collected in Hungary and Romania from 99 columbiform birds, including 76 feral pigeons (Columba livia domestica: 42 kept for racing, 32 with urban and two with rural habitat), four common wood pigeons (C. palumbus), 16 ring doves (Streptopelia risoria) and three Eurasian collared doves (S. decaocto). These samples were analyzed for the presence of T. gallinae using molecular methods. Racing feral pigeons had significantly higher prevalence of T. gallinae infection than urban feral pigeons. The rate of PCR-positivity was the highest among wood pigeons and ring doves. Based on 18S rRNA gene, T. gallinae was the most heterogenous among racing feral pigeons sampled in a trading-breeding place. Clinical signs were associated with only one 18S rRNA gene subtype. The most divergent 18S rRNA gene subtype, Trichomonas sp. Hu-TG37 clustered with T. canistomae and T. tenax and represents probably a new species. To our knowledge, this is the first report on the genetic diversity of T. gallinae in the southeastern European region. The results suggest that most detected T. gallinae 18S rRNA gene subtypes are not host-specific and do not cause clinical signs. The highest number of 18S rRNA gene subtypes was demonstrated among racing feral pigeons. Significantly more captive than free-living columbiform birds had T. gallinae infection. These data highlight the importance of epizootic monitoring of the genetic diversity and presence of T. gallinae in trading-breeding places of pigeons and doves.

Body size is an important ecological trait, but it has been poorly explored in microbial communities. Here, we examined the effect of cell size on coastal eukaryotic communities across a size continuum of 0.2–3 (pico-), 3–20 (nano-), and 20–200 μm (micro-sized), which were characterized via high-throughput sequencing based on the V4 region of the 18S rRNA gene. We found that, at the alpha diversity level, there was a decreasing trend across the pico-, nano-, and micro-sized eukaryotic communities regarding both amplicon sequence variant (ASV) richness and Shannon index. At the beta diversity level, the three categories were significantly different, and these were accompanied by a relatively high local contribution to beta diversity in contrasting freshwater and seawater locations. The community variations observed for the microbial eukaryotes could largely be explained by the environmental effects which decreased between the pico- (40.5%), nano- (37.3%), and micro-sized (27.3%) fractions. These environmental effects were mainly contributed by several ASV modules showing opposing responses to environmental conditions. This might partly indicate the coalescence of the freshwater and seawater groups of microbial eukaryotes. In summary, our findings suggest that the cell size of microbial eukaryotes is a phylogenetically conserved trait, which is tightly associated with biogeographic patterns.

In this survey, six species of Brachionus genus are indexed as new record in the south Iraqi water resource at the first time in Iraq depending on the sequence of 18S rRNA gene, these species were registered as a new record in Iraq and the other two species which are HH1 and HH2 were registered in the National Center for Biotechnology Information (NCBI), The European Nucleotide Archive (ENA) and DNA Data Bank of Japan (DDBJ) sequentially. [ABSTRACT FROM AUTHOR]

Abstract Background Hepatozoon canis is a protozoan transmitted to dogs and other wild carnivores by the ingestion of ticks containing mature oocysts and is considered the principal cause of canine hepatozoonosis in the world. Here, we examined ribosomal RNA 18S gene sequence variation to determine the genetic differences and phylogeographic diversity of H. canis from various geographical areas around the world. Methods We used 550 publicly available sequences of H. canis from 46 countries to assess haplotype relationships, geographical structure, genetic diversity indices, and relationships among populations. We performed neutrality tests and pairwise comparisons of fixation index (F ST) values between groups and pairwise comparisons of F ST values between populations. To determine whether populations are structured, analyses of molecular variance (AMOVAs) and spatial analysis of molecular variance (SAMOVA) were performed. Results The dataset of H. canis yielded 76 haplotypes. Differentiation among populations indicated that there is no phylogeographical structure (G ST = 0.302 ± 0.0475). Moreover, when samples were grouped by continents a significant F ST was obtained, meaning that populations were genetically differentiated. The AMOVA showed that 57.4% of the genetic variation was explained by differences within populations when all locations were treated as a single group and revealed that there is no population structure when populations are grouped into two, three, and four groups (F CT, p > 0.05), suggesting that dispersal between populations is high. SAMOVA revealed significant F CT values for groups K = 5. The Tajima’s D and Fu’s Fs show that populations have undergone recent expansion, and the mismatch distribution analysis showed population expansion (multimodal distribution). Conclusions The current molecular data confirmed that H. canis does not show phylogeographic or population structure. The haplotypes exhibit low genetic differentiation, suggesting a recent expansion due to gene flow among populations. These results provide pivotal information required for future detailed population genetic analysis or to establish control strategies of this parasite. Graphical abstract

The Southwest Atlantic Ocean (SWAO) is a spatially dynamic region with a remarkably high primary productivity. An exhaustive identification of the members of its phytoplankton community is a key step to understand the processes that sustain this ecosystem. Here, we provide a community composition analysis of eukaryotic phytoplankton in four SWAO sectors. We gathered 18S rRNA gene metabarcoding data and complemented it with confocal microscopy images, both from the Tara Oceans expedition in late spring 2010. Our work showed local and regional variation across three different size fractions that reflect the complexity of this region. Diversity decreased along temperature and latitudinal gradients, but also showed intricate patterns of occurrences across samples, suggesting that multiple factors shape the community structure. Samples resembled communities from other temperate regions and showed an increasing influence by cold waters from the Southern Ocean towards higher latitudes. These results complement previous regional studies that used other methods such as microscopy or pigment analysis. Our study contributes to the beginning of genomic-based surveys of plankton communities in the SW Atlantic and calls for further work in the region to enhance ecosystem monitoring and projections in the context of global change. Ph: Marin LE ROUX (polaRYSE - Fondation Tara Ocean)

Amoeboid protists, an assemblage of organisms belonging to different phylogenetic lineages, have drawn increasing attention due to their crucial ecological roles in various environments and their potential health risks. Currently, 18S rRNA gene sequencing is widely applied for the detection of amoebae. However, it is not clear which is the best primer pair for 18S rRNA gene amplification in amoebae. This study compared the four most commonly used primer pairs for revealing the diversity, composition, core species, and community assembly processes of amoebae in water and sediments. We found that the choice of primers artificially influences the detection of community composition of amoebae. We also found that short-read fragments may lead to mismatches in taxonomy and were not suitable for phylogenetic analyses. In contrast, full-length primers could detect the highest number of amoeba lineages and annotate 80% of reads belonging to amoebae to known species. However, full-length primers did not detect as many amoeba species as V4 primers. Moreover, we showed that beta diversity and community assembly determination were largely unaffected by primer choice, but different primers could influence our interpretations of the ecological process underlying stochasticity and determinism. This study indicates that full-length read sequencing and V4 region Illumina sequencing are suitable for profiling amoeba diversity in the environment.