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The order Piroplasmida, including the genera Babesia, Cytauxzoon, and Theileria is often referred to as piroplasmids and comprises of dixenous hemoprotozoans transmitted by ticks to a mammalian or avian host. Although piroplasmid infections are usually asymptomatic in wild animals, in domestic animals, they cause serious or life-threatening consequences resulting in fatalities. Piroplasmids are particularly notorious for the enormous economic loss they cause worldwide in livestock production, the restrictions they pose on horse trade, and the negative health impact they have on dogs and cats. Furthermore, an increasing number of reported human babesiosis cases are of growing concern. Considerable international research and epidemiological studies are done to identify existing parasite species, reveal their phylogenetic relationships, and develop improved or new drugs and vaccines to mitigate their impact. In this review, we present a compilation of all piroplasmid species, isolates, and species complexes that infect domestic mammals and which have been well defined by molecular phylogenetic markers. Altogether, 57 taxonomic piroplasmid entities were compiled, comprising of 43 piroplasmid species, 12 well-defined isolates awaiting formal species description, and two species complexes that possibly mask additional species. The extrapolation of the finding of at least 57 piroplasmid species in only six domestic mammalian groups (cattle, sheep, goat, horse, dog, and cat) allows us to predict that a substantially higher number of piroplasmid parasites than vertebrate host species exist. Accordingly, the infection of a vertebrate host species by multiple piroplasmid species from the same and/or different phylogenetic lineages is commonly observed. Molecular phylogeny using 18S rRNA genes of piroplasmids infecting domestic mammals results in the formation of six clades, which emerge due to an anthropocentric research scope, but not due to a possibly assumed biological priority position. Scrutinizing the topology of inferred trees reveals stunning insights into some evolutionary patterns exhibited by this intriguing group of parasites. Contrary to expectations, diversification of parasite species appears to be dominated by host-parasite cospeciation (Fahrenholz’s rule), and, except for piroplasmids that segregate into Clade VI, host switching is rarely observed. When only domestic mammalian hosts are taken into account, Babesia sensu lato (s.l.) parasites of Clades I and II infect only dogs and cats, respectively, Cytauxzoon spp. placed into Clade III only infect cats, Theileria placed into Clade IV exclusively infect horses, wheras Theileria sensu stricto (s.s.) of Clade V infects only cattle and small ruminants. In contrast, Babesia s.s. parasites of Clade VI infect all farm and companion animal species. We outline how the unique ability of transovarial transmission of Babesia s.s. piroplasmids of Clade VI facilitates species diversification by host switching to other host vertebrate species. Finally, a deterioration of sequence fidelity in databases is observed which will likely lead to an increased risk of artifactual research in this area. Possible measures to reverse and/or avoid this threat are discussed.

Background Several fungi-specific primers target the 18S rRNA gene sequence, one of the prominent markers for fungal classification. The design of most primers goes back to the last decades. Since then, the number of sequences in public databases increased leading to the discovery of new fungal groups and changes in fungal taxonomy. However, no reevaluation of primers was carried out and relevant information on most primers is missing. With this study, we aimed to develop an 18S rRNA gene sequence primer toolkit allowing an easy selection of the best primer pair appropriate for different sequencing platforms, research aims (biodiversity assessment versus isolate classification) and target groups. Results We performed an intensive literature research, reshuffled existing primers into new pairs, designed new Illumina-primers, and annealing blocking oligonucleotides. A final number of 439 primer pairs were subjected to in silico PCRs. Best primer pairs were selected and experimentally tested. The most promising primer pair with a small amplicon size, nu-SSU-1333-5′/nu-SSU-1647-3′ (FF390/FR-1), was successful in describing fungal communities by Illumina sequencing. Results were confirmed by a simultaneous metagenomics and eukaryote-specific primer approach. Co-amplification occurred in all sample types but was effectively reduced by blocking oligonucleotides. Conclusions The compiled data revealed the presence of an enormous diversity of fungal 18S rRNA gene primer pairs in terms of fungal coverage, phylum spectrum and co-amplification. Therefore, the primer pair has to be carefully selected to fulfill the requirements of the individual research projects. The presented primer toolkit offers comprehensive lists of 164 primers, 439 primer combinations, 4 blocking oligonucleotides, and top primer pairs holding all relevant information including primer’s characteristics and performance to facilitate primer pair selection.

Abstract We investigated if activity of the pre-infective juveniles (J2s) of root-knot nematodes is linked to the recruitment of a specific microbiome on the nematode surface and/or to the composition of the surrounding microbiota. For this, we determined the J2 activity (active vs. non-motile, which referred to dead and immobile J2s) upon a 3-day incubation in soil suspensions and studied the composition of bacteria, protists, and fungi present on the nematode surface and in the suspensions using amplicon sequencing of the 16S/18S rRNA genes, and ITS region. We also amended suspensions with Pseudomonas protegens strain CHA0 to study its effects on J2 activity and microbial composition. The J2 activity was suppressed in soil suspensions, but increased when suspensions were amended with P. protegens CHA0. The active and non-motile J2s differed in the composition of surface-attached bacteria, which was altered by the presence of P. protegens CHA0 in the soil suspensions. The bacterial genera Algoriphagus, Pedobacter, and Bdellovibrio were enriched on active J2s and may have protected the J2s against antagonists. The incubation time appeared short for attachment of fungi and protists. Altogether, our study is a step forward in disentangling the complex nematode-microbe interactions in soil for more successful nematode control. Active and non-motile root-knot nematode juveniles carry differently composed microbiomes on their surfaces.

Although many primers have been proposed for arbuscular mycorrhizal fungi (AMF)–specific sequencing, only a few studies have evaluated suitable primer pairs for MiSeq analysis. Here, we analyzed AMF community compositions using several primer pairs and evaluated which pair can detect a more diverse AMF community. Three AMF-specific primer pairs targeting the 18S and 28S rRNA genes and a fungal universal primer pair targeting the ITS region were examined using soil DNA as the template. The effect of nested PCR was also evaluated. Only a few AMF sequences were obtained from a fungal universal primer targeting the ITS region. On the other hand, use of AMF-specific primers improved AMF detection rates (35–98%). Particularly, almost all of the sequences obtained with the nested PCR were AMF-related sequences. However, the AMF community compositions were differentiated among the primer pairs. In addition, the number of AMF taxa and diversity decreased with the nested PCR, suggesting that it affected AMF community analysis.

Brownfields are unused sites that contain hazardous substances due to previous commercial or industrial use. The sites are inhospitable for many organisms, but some fungi and microbes can tolerate and thrive in the nutrient-depleted and contaminated soils. However, few studies have characterized the impacts of long-term contamination on soil microbiome composition and diversity at brownfields. This study focuses on an urban brownfield—a former rail yard in Los Angeles that is contaminated with heavy metals, volatile organic compounds, and petroleum-derived pollutants. We anticipate that heavy metals and organic pollutants will shape soil microbiome diversity and that several candidate fungi and bacteria will be tolerant to the contaminants. We sequence three gene markers (16S ribosomal RNA, 18S ribosomal RNA, and the fungal internal transcribed spacer (FITS)) in 55 soil samples collected at five depths to (1) profile the composition of the soil microbiome across depths; (2) determine the extent to which hazardous chemicals predict microbiome variation; and (3) identify microbial taxonomic groups that may metabolize these contaminants. Detected contaminants in the samples included heavy metals, petroleum hydrocarbons, polycyclic aromatic hydrocarbons, and volatile organic compounds. Bacterial, eukaryotic, and fungal communities all varied with depth and with concentrations of arsenic, chromium, cobalt, and lead. 18S rRNA microbiome richness and fungal richness were positively correlated with lead and cobalt levels, respectively. Furthermore, bacterial Paenibacillus and Iamia , eukaryotic Actinochloris , and fungal Alternaria were enriched in contaminated soils compared to uncontaminated soils and represent taxa of interest for future bioremediation research. Based on our results, we recommend incorporating DNA-based multi-marker microbial community profiling at multiple sites and depths in brownfield site assessment standard methods and restoration.

The intestinal parasite communities of vertebrates are shaped by complex interactions among biological, environmental, and anthropogenic factors. However, such dynamics remain poorly characterized in endangered the Chinese Alligator (Alligator sinensis). Here, we conducted a high‐throughput 18S rRNA amplicon survey of intestinal parasites in 149 fecal samples from the critically endangered Chinese alligators, spanning two geographic localities, four feeding regimens, three developmental stages, and 3 months. We identified 1524 eukaryotic parasite OTUs and 6606 nematode OTUs, revealing significant geographic variation in parasite composition, with higher richness in Wuhu than in Xuancheng populations. PERMANOVA further confirmed significant locality‐level differences in parasite community structure for both eukaryotic and nematode assemblages. Feeding regimen strongly influenced parasite diversity and community composition, with nematode communities exhibiting particularly pronounced responses, showing the lowest abundance and diversity in alligators maintained on a standardized catfish‐carp diet and highest in those fed diverse prey. Eukaryotic parasite richness declined markedly with host age, while nematode diversity showed pronounced depression in August. At finer taxonomic resolution, multiple parasite phyla and genera exhibited dynamic, factor‐specific responses to locality, diet, age, and sampling month, highlighting strong ecological filtering effects. Linear Discriminant Analysis Effect Size analysis uncovered specific parasite taxa associated with distinct factors. Our results firstly demonstrate the structure of parasite communities in A. sinensis, and clarified how they are shaped by interacting ecological and management factors, providing critical baseline data for future research. Our results firstly demonstrate the structure of parasite communities in A. sinensis and clarify how they are shaped by interacting ecological and management factors, providing critical baseline data for future research.

Abstract Cyanobacterial mats are commonly reported as hotspots of microbial diversity across polar environments. These thick, multilayered microbial communities provide a refuge from extreme environmental conditions, with many species able to grow and coexist despite the low allochthonous nutrient inputs. The visibly dominant phototrophic biomass is dependent on internal nutrient recycling by heterotrophic organisms within the mats; however, the specific contribution of heterotrophic protists remains little explored. In this study, mat community diversity was examined along a latitudinal gradient (55–83°N), spanning subarctic taiga, tundra, polar desert, and the High Arctic ice shelves. The prokaryotic and eukaryotic communities were targeted, respectively, by V4 16S ribosomal RNA (rRNA) and V9 18S rRNA gene amplicon high-throughput sequencing. Prokaryotic and eukaryotic richness decreased, in tandem with decreasing temperatures and shorter seasons of light availability, from the subarctic to the High Arctic. Taxonomy-based annotation of the protist community revealed diverse phototrophic, mixotrophic, and heterotrophic genera in all mat communities, with fewer parasitic taxa in High Arctic communities. Co-occurrence network analysis identified greater heterogeneity in eukaryotic than prokaryotic community structure among cyanobacterial mats across the Canadian Arctic. Our findings highlight the sensitivity of microbial eukaryotes to environmental gradients across northern high latitudes. Microbial diversity in freshwater microbial mats declines with decreasing temperature and light availability, from the subarctic to High Arctic, highlighting a sensitivity to environmental gradients and climate change.

The small subunit 18S rRNA (18S) gene is the most commonly used marker for taxonomic identification in eukaryotes. However, protists may harbor substantial variation in their 18S gene copy number, which can lead to a rapid decline in concordance between 18S gene sequences and actual organismal abundances. Here we used a computational method to estimate 18S gene copy number in seven species of marine eukaryotic phytoplankton and found strong interspecies and strain-level differences across and within the examined species. Our results emphasize that variations in 18S gene copy number need to be taken into consideration and that corrections can improve the accuracy of quantitative eukaryotic microbial community profiles.

Abstract Snow algae blooms often form green or red coloured patches in melting alpine and polar snowfields worldwide, yet little is known about their biology, biogeography, and species diversity. We investigated eight isolates collected from red snow in northern Norway, using a combination of morphology, 18S rRNA gene and internal transcribed spacer 2 (ITS2) genetic markers. Phylogenetic and ITS2 rRNA secondary structure analyses assigned six isolates to the species Raphidonema nivale, Deuterostichococcus epilithicus, Chloromonas reticulata, and Xanthonema bristolianum. Two novel isolates belonging to the family Stichococcaceae (ARK-S05-19) and the genus Chloromonas (ARK-S08-19) were identified as potentially new species. In laboratory cultivation, differences in the growth rate and fatty acid profiles were observed between the strains. Chlorophyta were characterized by abundant C18:3n-3 fatty-acids with increases in C18:1n-9 in the stationary phase, whilst Xanthonema (Ochrophyta) was characterized by a large proportion of C20:5n-3, with increases in C16:1n-7 in the stationary phase. In a further experiment, lipid droplet formation was studied in C. reticulata at the single-cell level using imaging flow cytometry. Our study establishes new cultures of snow algae, reveals novel data on their biodiversity and biogeography, and provides an initial characterization of physiological traits that shape natural communities and their ecophysiological properties. Norwegian red snow microbiomes host phylogenetically diverse culturable microalgae that exhibit different growth strategies and accumulate lipids under physiological stress.

We studied the 16S and 18S rRNA genes of the bacterial, protist, and fungal microbiomes of 131 samples collected in 14 ephemeral small inland lakes located in the endorheic area of the Monegros Desert (NE Spain). The sampling covered different temporal flooding/desiccation cycles that created natural salinity gradients between 0.1% (w/v) and salt saturation. We aimed to test the hypothesis of a lack of competitive advantage for microorganisms using the “salt-in” strategy in highly fluctuating hypersaline environments where temperature and salinity transitions widely vary within short time periods, as in ephemeral inland lakes. Overall, 5653 bacterial zOTUs and 2658 eukaryal zOTUs were detected heterogeneously distributed with significant variations on taxonomy and general energy-yielding metabolisms and trophic strategies along the gradient. We observed a more diverse bacterial assembly than initially expected at extreme salinities and a lack of dominance of a few “salt-in” organisms. Microbial thresholds were unveiled for these highly fluctuating hypersaline environments with high selective pressures. We conclude that the extremely high dynamism observed in the ephemeral lakes of Monegros may have given a competitive advantage for more versatile (\"salt-out\") organisms compared to those better adapted to stable high salinities usually more common in solar salterns. Ephemeral inland saline lakes offered a well-suited natural framework for highly detailed evolutionary and ecological studies.