Comparative genomics of Ascetosporea gives new insight into the evolutionary basis for animal parasitism in Rhizaria.

BACKGROUND: Ascetosporea (Endomyxa, Rhizaria) is a group of unicellular parasites infecting aquatic invertebrates. They are increasingly being recognized as widespread and important in marine environments, causing large annual losses in invertebrate aquaculture. Despite their importance, little molecular data of Ascetosporea exist, with only two genome assemblies published to date. Accordingly, the evolutionary origin of these parasites is unclear, including their phylogenetic position and the genomic adaptations that accompanied the transition from a free-living lifestyle to parasitism. Here, we sequenced and assembled three new ascetosporean genomes, as well as the genome of a closely related amphizoic species, to investigate the phylogeny, origin, and genomic adaptations to parasitism in Ascetosporea. RESULTS: Using a phylogenomic approach, we confirm the monophyly of Ascetosporea and show that Paramyxida group with Mikrocytida, with Haplosporida being sister to both groups. We report that the genomes of these parasites are relatively small (12-36 Mb) and gene-sparse (~ 2300-5200 genes), while containing surprisingly high amounts of non-coding sequence (~ 70-90% of the genomes). Performing gene-tree aware ancestral reconstruction of gene families, we demonstrate extensive gene losses at the origin of parasitism in Ascetosporea, primarily of metabolic functions, and little gene gain except on terminal branches. Finally, we highlight some functional gene classes that have undergone expansions during evolution of the group. CONCLUSIONS: We present important new genomic information from a lineage of enigmatic but important parasites of invertebrates and illuminate some of the genomic innovations accompanying the evolutionary transition to parasitism in this lineage. Our results and data provide a genetic basis for the development of control measures against these parasites.

Safety and Feasibility of Photodynamic Therapy for Percutaneous Image-guided Abdominopelvic Abscess Drainage: Phase 1 Trial.

Background Standard-of-care abscess management includes image-guided percutaneous drainage and antibiotics; however, cure rates vary, and concern for antibiotic-resistant bacteria is growing. Photodynamic therapy (PDT), which uses light-activated dyes to generate cytotoxic reactive oxygen species, could complement the standard of care by sterilizing the abscess at the time of drainage. Purpose To evaluate safety and feasibility of PDT with methylene blue (hereafter, MB-PDT) at the time of percutaneous abscess drainage. Materials and Methods This prospective, open-label, dose-escalation, first-in-humans, registered phase 1 clinical study of MB-PDT included participants who underwent percutaneous abdominal or pelvic abscess drainage with CT or US guidance from January 2015 to March 2020 and September 2022 to September 2023. Following drainage, MB-PDT was performed with laser illumination at a fluence rate of 20 mW/cm2, with fluence groups of 6, 12, 18, 24, 30, and 36 J/cm2 (n = 3 each). The primary outcome was safety, indicated by absence of fat embolism, MB escape, abscess wall damage, and need for surgery to remove optical fibers. Preliminary efficacy end points included the time to drainage catheter removal, drainage catheter output volume, and clinical symptom and fever duration. Relationships between fluence and outcomes were analyzed with Spearman correlation and linear regression analyses, and ordinary one-way analysis of variance was used for group comparisons. Results MB-PDT was safe and feasible in all 18 participants (mean age, 60.1 years ± 18.3 [SD]; 10 female), with no negative safety outcomes observed for any participant. No study-related adverse events were encountered, and the procedure did not increase reported pain (P = .1). Clinical symptom and fever duration was shorter in participants receiving higher fluences (30 and 36 J/cm2 vs 6 J/cm2) (P = .03). The presence of antibiotic-resistant bacteria was not predictive of clinical symptom and fever duration (ß = 0.13, P = .37). Conclusion MB-PDT was a safe and feasible adjunct to image-guided percutaneous abscess drainage. Clinical measures indicated a dose-dependent response to PDT. ClinicalTrials.gov registration no.: NCT02240498 © RSNA, 2024 Supplemental material is available for this article. See also the editorial by Johnston and Goldberg in this issue.

18S rDNA gene metabarcoding of microeukaryotes and epi-endophytes in the holobiome of seven species of large brown algae.

Brown algae (Phaeophyceae) are habitat-forming species in coastal ecosystems and include kelp forests and seaweed beds that support a wide diversity of marine life. Host-associated microbial communities are an integral part of phaeophyte biology, and whereas the bacterial microbial partners have received considerable attention, the microbial eukaryotes associated with brown algae have hardly been studied. Here, we used broadly targeted "pan-eukaryotic" primers (metabarcoding) to investigate brown algal-associated eukaryotes (the eukaryome). Using this approach, we aimed to investigate the eukaryome of seven large brown algae that are important and common species in coastal ecosystems. We also aimed to assess whether these macroalgae harbor novel eukaryotic diversity and to ascribe putative functional roles to the host-associated eukaryome based on taxonomic affiliation and phylogenetic placement. We detected a significant diversity of microeukaryotic and algal lineages associated with the brown algal species investigated. The operational taxonomic units (OTUs) were taxonomically assigned to 10 of the eukaryotic major supergroups, including taxonomic groups known to be associated with seaweeds as epibionts, endobionts, parasites, and commensals. Additionally, we revealed previously unrecorded sequence types, including novel phaeophyte OTUs, particularly in the Fucus spp. samples, that may represent fucoid genomic variants, sequencing artifacts, or undescribed epi-/endophytes. Our results provide baseline data and technical insights that will be useful for more comprehensive seaweed eukaryome studies investigating the evidently lineage-rich and functionally diverse symbionts of brown algae.

Modeling the Mechanical Microenvironment of Coiled Cerebral Aneurysms.

Successful occlusion of cerebral aneurysms using coil embolization is contingent upon stable thrombus formation, and the quality of the thrombus depends upon the biomechanical environment. The goal of this study was to investigate how coil embolization alters the mechanical micro-environment within the aneurysm dome. Inertialess particles were injected in three-dimensional, computational simulations of flow inside patient aneurysms using patient-specific boundary conditions. Coil embolization was simulated as a homogenous porous medium of known permeability and inertial constant. Lagrangian particle tracking was used to calculate the residence time and shear stress history for particles in the flow before and after treatment. The percentage of particles entering the aneurysm dome correlated with the neck surface area before and after treatment (pretreatment: R2 = 0.831, P < 0.001; post-treatment: R2 = 0.638, P < 0.001). There was an inverse relationship between the change in particles entering the dome and coil packing density (R2 = 0.600, P < 0.001). Following treatment, the particles with the longest residence times tended to remain within the dome even longer while accumulating lower shear stress. A significant correlation was observed between the treatment effect on residence time and the ratio of the neck surface area to porosity (R2 = 0.390, P = 0.007). The results of this study suggest that coil embolization triggers clot formation within the aneurysm dome via a low shear stress-mediated pathway. This hypothesis links independently observed findings from several benchtop and clinical studies, furthering our understanding of this treatment strategy.

A Novel Patient-Specific Computational Fluid Dynamics Study of the Activation of Primary Collateral Pathways in the Circle of Willis During Vasospasm.

The Circle of Willis (CoW) is a redundant network of blood vessels that perfuses the brain. The ringlike anatomy mitigates the negative effects of stroke by activating collateral pathways that help maintain physiological perfusion. Previous studies have investigated the activation of these pathways during embolic stroke and internal carotid artery occlusion. However, the role of collateral pathways during cerebral vasospasm-an involuntary constriction of blood vessels after subarachnoid hemorrhage-is not well-documented. This study presents a novel technique to create patient-specific computational fluid dynamics (CFD) simulations of the Circle of Willis before and during vasospasm. Computed tomographic angiography (CTA) scans are segmented to model the vasculature, and transcranial Doppler ultrasound (TCD) measurements of blood flow velocity are applied as boundary conditions. Bayesian analysis leverages information about the uncertainty in the measurements of vessel diameters and velocities to find an optimized parameter set that satisfies mass conservation and that is applied in the final simulation. With this optimized parameter set, the diameters, velocities, and flow rates fall within typical literature values. Virtual angiograms modeled using passive scalar transport agree closely with clinical angiography. A sensitivity analysis quantifies the changes in collateral flow rates with respect to changes in the inlet and outlet flow rates. This analysis can be applied in the future to a cohort of patients to investigate the relationship between the locations and severities of vasospasm, the patient-to-patient anatomical variability in the Circle of Willis, and the activation of collateral pathways.

Txikispora philomaios n. sp., n. g., a micro-eukaryotic pathogen of amphipods, reveals parasitism and hidden diversity in Class Filasterea.

This study provides a morphological, ultrastructural, and phylogenetic characterization of a novel micro-eukaryotic parasite (2.3-2.6 µm) infecting amphipod genera Echinogammarus and Orchestia. Longitudinal studies across two years revealed that infection prevalence peaked in late April and May, reaching 64% in Echinogammarus sp. and 15% in Orchestia sp., but was seldom detected during the rest of the year. The parasite infected predominantly hemolymph, connective tissue, tegument, and gonad, although hepatopancreas and nervous tissue were affected in heavier infections, eliciting melanization and granuloma formation. Cell division occurred inside walled parasitic cysts, often within host hemocytes, resulting in hemolymph congestion. Small subunit (18S) rRNA gene phylogenies including related environmental sequences placed the novel parasite as a highly divergent lineage within Class Filasterea, which together with Choanoflagellatea represent the closest protistan relatives of Metazoa. We describe the new parasite as Txikispora philomaios n. sp. n. g., the first confirmed parasitic filasterean lineage, which otherwise comprises four free-living flagellates and a rarely observed endosymbiont of snails. Lineage-specific PCR probing of other hosts and surrounding environments only detected T. philomaios in the platyhelminth Procerodes sp. We expand the known diversity of Filasterea by targeted searches of metagenomic datasets, resulting in 13 previously unknown lineages from environmental samples.

Discovery of the parasite Marteilia cocosarum sp. nov. In common cockle (Cerastoderma edule) fisheries in Wales, UK and its comparison with Marteilia cochillia.

Diseases of bivalve molluscs caused by paramyxid parasites of the genus Marteilia have been linked to mass mortalities and the collapse of commercially important shellfish populations. Until recently, no Marteilia spp. have been detected in common cockle (Cerastoderma edule) populations in the British Isles. Molecular screening of cockles from ten sites on the Welsh coast indicates that a Marteilia parasite is widespread in Welsh C. edule populations, including major fisheries. Phylogenetic analysis of ribosomal DNA (rDNA) gene sequences from this parasite indicates that it is a closely related but different species to Marteilia cochillia, a parasite linked to mass mortality of C. edule fisheries in Spain, and that both are related to Marteilia octospora, for which we provide new rDNA sequence data. Preliminary light and transmission electron microscope (TEM) observations support this conclusion, indicating that the parasite from Wales is located primarily within areas of inflammation in the gills and the connective tissue of the digestive gland, whereas M. cochillia is found mainly within the epithelium of the digestive gland. The impact of infection by the new species, here described as Marteilia cocosarum n. sp., upon Welsh fisheries is currently unknown.

Infection cycle of Marteilia pararefringens in blue mussels Mytilus edulis in a heliothermic marine oyster lagoon in Norway.

Agapollen is a traditional heliothermic marine oyster lagoon in western Norway, representing the northernmost site of any Marteilia sp. protists detected in Europe. The semi-closed lagoon is a unique site to study the life cycle and development of M. pararefringens in naïve mussels. Two baskets with uninfected mussels were deployed in the lagoon outlet in May and October 2018, respectively, and sampled every 6 wk. The parasite was first detected in the mussels by PCR in early July and by histology in late August. By then, M. pararefringens had developed into mature stages, indicating a rapid development during mid-summer. Sporulation occurred during autumn. Mussels deployed in October never became infected, indicating that transmission was restricted to the warmest period of the year. Pronounced pathology was observed in infected mussels, including degenerated digestive tubules and infiltration of haemocytes. Mussel mortality was observed in the baskets, but whether this was due to infections of M. pararefringens or other environmental factors could not be determined. Plankton samples from the lagoon were also collected for PCR analysis. These samples, dominated by copepods, were positive for M. pararefringens in summer. In sorted samples, M. pararefringens was detected in the Acartia spp. and Paracartia grani fractions between July and October. These plankton copepods are therefore potentially involved in the life cycle of M. pararefringens.

High prevalence of Paramarteilia canceri infecting velvet swimming crabs Necora puber in Ireland.

The velvet swimming crab Necora puber has been fished in Ireland since the early 1980s and contributes significant income to smaller fishing vessels. From 2016 onwards, reduced landings have been reported. We undertook a full pathological investigation of crabs from fishing grounds at 3 sites on the west (Galway), southwest (Castletownbere) and east (Howth) coasts of Ireland. Histopathology, transmission electron microscopy and molecular taxonomic and phylogenetic analyses showed high prevalence and infection level of Paramarteilia canceri, previously only reported from the edible crab Cancer pagurus. This study provides the first molecular data for P. canceri, and shows its phylogenetic position in the order Paramyxida (Rhizaria). Other parasites and symbionts detected in the crabs were also noted, including widespread but low co-infection with Hematodinium sp. and a microsporidian consistent with the Ameson and Nadelspora genera. This is the first histological record of Hematodinium sp. in velvet crabs from Ireland. Four N. puber individuals across 2 sites were co-infected by P. canceri and Hematodinium sp. At one site, 3 velvet crabs infected with P. canceri were co-infected with the first microsporidian recorded from this host; the microsporidian 18S sequence was almost identical to Ameson pulvis, known to infect European shore crabs Carcinus maenas. The study provides a comprehensive phylogenetic analysis of this and all other available Ameson and Nadelspora 18S sequences. Together, these findings provide a baseline for further investigations of N. puber populations along the coast of Ireland.

Identifying Potential Hosts of Short-Branch Microsporidia.

Microsporidia are obligate parasites that are closely related to Fungi. While the widely known "long-branch" Microsporidia infect mostly metazoans, the hosts of "short-branch" Microsporidia are only partially characterized or not known at all. Here, we used network analyses from Neotropical rainforest soil metabarcoding data, to infer co-occurrences between environmental lineages of short-branch microsporidians and their potential hosts. We found significant co-occurrences with several taxa, especially with Apicomplexa, Cercozoa, and Fungi, as well as some Metazoa. Our results are the first step to identify potential hosts of the environmental lineages of short-branch microsporidians, which can be targeted in future molecular and microscopic studies.

Understanding the role of the shrimp gut microbiome in health and disease.

With rapid increases in the global shrimp aquaculture sector, a focus on animal health during production becomes ever more important. Animal productivity is intimately linked to health, and the gut microbiome is becoming increasingly recognised as an important driver of cultivation success. The microbes that colonise the gut, commonly referred to as the gut microbiota or the gut microbiome, interact with their host and contribute to a number of key host processes, including digestion and immunity. Gut microbiome manipulation therefore represents an attractive proposition for aquaculture and has been suggested as a possible alternative to the use of broad-spectrum antibiotics in the management of disease, which is a major limitation of growth in this sector. Microbiota supplementation has also demonstrated positive effects on growth and survival of several different commercial species, including shrimp. Development of appropriate gut supplements, however, requires prior knowledge of the host microbiome. Little is known about the gut microbiota of the aquatic invertebrates, but penaeid shrimp are perhaps more studied than most. Here, we review current knowledge of information reported on the shrimp gut microbiota, highlighting the most frequently observed taxa and emphasizing the dominance of Proteobacteria within this community. We discuss involvement of the microbiome in the regulation of shrimp health and disease and describe how the gut microbiota changes with the introduction of several economically important shrimp pathogens. Finally, we explore evidence of microbiome supplementation and consider its role in the future of penaeid shrimp production.

Direct electrical stimulation of the amygdala enhances declarative memory in humans.

Emotional events are often remembered better than neutral events, a benefit that many studies have hypothesized to depend on the amygdala's interactions with memory systems. These studies have indicated that the amygdala can modulate memory-consolidation processes in other brain regions such as the hippocampus and perirhinal cortex. Indeed, rodent studies have demonstrated that direct activation of the amygdala can enhance memory consolidation even during nonemotional events. However, the premise that the amygdala causally enhances declarative memory has not been directly tested in humans. Here we tested whether brief electrical stimulation to the amygdala could enhance declarative memory for specific images of neutral objects without eliciting a subjective emotional response. Fourteen epilepsy patients undergoing monitoring of seizures via intracranial depth electrodes viewed a series of neutral object images, half of which were immediately followed by brief, low-amplitude electrical stimulation to the amygdala. Amygdala stimulation elicited no subjective emotional response but led to reliably improved memory compared with control images when patients were given a recognition-memory test the next day. Neuronal oscillations in the amygdala, hippocampus, and perirhinal cortex during this next-day memory test indicated that a neural correlate of the memory enhancement was increased theta and gamma oscillatory interactions between these regions, consistent with the idea that the amygdala prioritizes consolidation by engaging other memory regions. These results show that the amygdala can initiate endogenous memory prioritization processes in the absence of emotional input, addressing a fundamental question and opening a path to future therapies.

Revised Taxonomy and Expanded Biodiversity of the Phytomyxea (Rhizaria, Endomyxa).

Phytomyxea (phytomyxids) is a group of obligate biotrophic pathogens belonging to the Rhizaria. Some phytomyxids are well studied and include known plant pathogens such as Plasmodiophora brassicae, the causal agent of clubroot disease. Despite this economic importance, the taxonomy and biodiversity of this group are largely cryptic, with many species described in the premolecular area. Some of these species were key for establishing the morphotaxonomic concepts that define most genera to this day, but systematic efforts to include and integrate those species into molecular studies are still lacking. The aim of this study was to expand our understanding of phytomyxid biodiversity in terrestrial environments. Thirty-eight environmental samples from habitats in which novel and known diversity of Phytomyxea was expected were analysed. We were able to generate 18S rRNA sequences from Ligniera verrucosa, a species which is well defined based on ultrastructure. Phylogenetic analyses of the collected sequences rendered the genera Lignera, Plasmodiophora and Spongospora polyphyletic, and identified two novel and apparently diverse lineages (clade 17, clade 18). Based on these findings and on data from previous studies, we formally establish the new genera Pseudoligniera n. gen. for L. verrucosa,Hillenburgia n. gen. for Spongospora nasturtii and revert Plasmodiophora diplantherae to its original name Ostenfeldiella diplantherae.

Phylogenetic Estimation of Community Composition and Novel Eukaryotic Lineages in Base Mine Lake: An Oil Sands Tailings Reclamation Site in Northern Alberta.

Reclamation of anthropogenically impacted environments is a critical issue worldwide. In the oil sands extraction industry of Alberta, reclamation of mining-impacted areas, especially areas affected by tailings waste, is an important aspect of the mining life cycle. A reclamation technique currently under study is water-capping, where tailings are capped by water to create an end-pit lake (EPL). Base Mine Lake (BML) is the first full-scale end-pit lake in the Alberta oil sands region. In this study, we sequenced eukaryotic 18S rRNA genes recovered from 92 samples of Base Mine Lake water in a comprehensive sampling programme covering the ice-free period of 2015. The 565 operational taxonomic units (OTUs) generated revealed a dynamic and diverse community including abundant Microsporidia, Ciliata and Cercozoa, though 41% of OTUs were not classifiable below the phylum level by comparison to 18S rRNA databases. Phylogenetic analysis of five heterotrophic phyla (Cercozoa, Fungi, Ciliata, Amoebozoa and Excavata) revealed substantial novel diversity, with many clusters of OTUs that were more similar to each other than to any reference sequence. All of these groups are entirely or mostly heterotrophic, as a relatively small number of definitively photosynthetic clades were amplified from the BML samples.

Diagnosis and prevalence of two new species of haplosporidians infecting shore crabs Carcinus maenas: Haplosporidium carcini n. sp., and H. cranc n. sp.

This study provides a morphological and phylogenetic characterization of two novel species of the order Haplosporida (Haplosporidium carcini n. sp., and H. cranc n. sp.) infecting the common shore crab Carcinus maenas collected at one location in Swansea Bay, South Wales, UK. Both parasites were observed in the haemolymph, gills and hepatopancreas. The prevalence of clinical infections (i.e. parasites seen directly in fresh haemolymph preparations) was low, at ~1%, whereas subclinical levels, detected by polymerase chain reaction, were slightly higher at ~2%. Although no spores were found in any of the infected crabs examined histologically (n = 334), the morphology of monokaryotic and dikaryotic unicellular stages of the parasites enabled differentiation between the two new species. Phylogenetic analyses of the new species based on the small subunit (SSU) rDNA gene placed H. cranc in a clade of otherwise uncharacterized environmental sequences from marine samples, and H. carcini in a clade with other crustacean-associated lineages.

Ephemeral detection of Bonamia exitiosa (Haplosporida) in adult and larval European flat oysters Ostrea edulis in the Solent, United Kingdom.

The haplosporidian parasite Bonamia exitiosa was detected using PCR in four adult and six larval brood samples of the European flat oyster Ostrea edulis from the Solent, UK. This represents the second reported detection of this parasite along the south coast of England. Adult oysters were collected and preserved from seabed populations or restoration broodstock cages between 2015 and 2018. The larvae within brooding adults sampled during 2017 and 2018 were also preserved. Molecular analysis of all samples was performed in 2019. The DNA of B. exitiosa was confirmed to be present within the gill tissue of one oyster within the Portsmouth wild fishery seabed population (n = 48), sampled in November 2015; the congeneric parasite Bonamia ostreae was not detected in this individual. This is the earliest record of B. exitiosa in the Solent. Concurrent presence of both B. ostreae and B. exitiosa, determined by DNA presence, was confirmed in the gill and heart tissue of three mature individuals from broodstock cages sampled in October 2017 (n = 99), two from a location on the River Hamble and one from the Camber Dock in Portsmouth Harbour. B. exitiosa was not detected in the November 2018 broodstock populations. A total of six larval broods were positive for B. exitiosa, with five also positive for B. ostreae. None of the brooding adults were positive for B. exitiosa suggesting that horizontal transmission from the surrounding environment to the brooding larvae is occurring. Further sampling of broodstock populations conducted by the Fish Health Inspectorate at the Centre for Environment, Fisheries and Aquaculture Science in June 2019 did not detect infection of O. edulis by B. exitiosa. These findings together suggest that the pathogen has not currently established in the area.

Medicolegal issues in abusive head trauma for the pediatric neurosurgeon.

The purpose of this article is to serve as a rational guide for the pediatric neurosurgeon in navigating common medicolegal issues that arise in the management of abusive head trauma (AHT). Many of these issues may be unfamiliar or unpleasant to surgeons focused on addressing disease. The authors begin with a brief history on the origins of the diagnosis of AHT and the controversy surrounding it, highlighting some of the facets of the diagnosis that make it particularly unique in pediatric neurosurgery. They then review some special medical considerations in these patients through the perspective of the neurosurgeon and provide several examples as illustration. The authors discuss how to appropriately document these cases in the medical record for expected legal review, and last, they provide an overview of the legal process through which the neurosurgeon may be called to provide testimony.

Awake Mapping of the Auditory Cortex during Tumor Resection in an Aspiring Musical Performer: A Case Report.

INTRODUCTION: Preoperative functional MRI (fMRI) and intraoperative awake cortical mapping are established strategies to identify and preserve critical language structures during neurosurgery. There is growing appreciation for the need to similarly identify and preserve eloquent tissue critical for music production. CASE REPORT: A 19-year-old female musician, with a 3- to 4-year history of events concerning for musicogenic seizures, was found to have a right posterior temporal tumor, concerning for a low-grade glial neoplasm. Preoperative fMRI assessing passive and active musical tasks localized areas of activation directly adjacent to the tumor margin. Cortical stimulation during various musical tasks did not identify eloquent tissue near the surgical site. A gross total tumor resection was achieved without disruption of singing ability. At 9-month follow-up, the patient continued to have preserved musical ability with full resolution of seizures and without evidence of residual lesion or recurrence. CONCLUSION: A novel strategy for performing an awake craniotomy, incorporating preoperative fMRI data for music processing with intraoperative cortical stimulation, interpreted with the assistance of a musician expert and facilitated gross total resection of the patient's tumor without comprising her musical abilities.

Trauma on the high seas: an overview of recreational water use injuries.

Recreational water use (RWU) injuries span from superficial lacerations to even death. Given the global popularity of RWU, radiologists should be aware of the common mechanisms and key imaging findings related to injuries in this setting. The goal of this article is to depict common RWU injuries and their emergent radiographic findings, which may have both important surgical and management implications. We present a broad review with case illustrations of these injuries seen at our level 1 trauma center showing the breadth of injury that can occur, general mechanisms and sample imaging findings.

Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes.

This revision of the classification of eukaryotes follows that of Adl et al., 2012 [J. Euk. Microbiol. 59(5)] and retains an emphasis on protists. Changes since have improved the resolution of many nodes in phylogenetic analyses. For some clades even families are being clearly resolved. As we had predicted, environmental sampling in the intervening years has massively increased the genetic information at hand. Consequently, we have discovered novel clades, exciting new genera and uncovered a massive species level diversity beyond the morphological species descriptions. Several clades known from environmental samples only have now found their home. Sampling soils, deeper marine waters and the deep sea will continue to fill us with surprises. The main changes in this revision are the confirmation that eukaryotes form at least two domains, the loss of monophyly in the Excavata, robust support for the Haptista and Cryptista. We provide suggested primer sets for DNA sequences from environmental samples that are effective for each clade. We have provided a guide to trophic functional guilds in an appendix, to facilitate the interpretation of environmental samples, and a standardized taxonomic guide for East Asian users.