Amyloodinium ocellatum is a protozoan parasite that causes amyloodiniosis in marine and brackish water fish, threatening global aquaculture. The present study investigates the morphology and ultrastructure of the free-living stages of A. ocellatum (tomont and dinospore) using light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Dinospores measured 13.03-19.66 μm in length, 12.32-18.71 μm in width, and were laterally flattened. Dinospores had a transverse flagellum for propulsion and a longitudinal flagellum for direction control. The cyst wall had three distinct layers and included cellulose. The outer wall was coated with numerous bacteria. The orange-red speckled eyespot was observed all tomont developmental stages and in the dinospore of A. ocellatum. Tomonts proliferation required successive nuclear division, the formation of new cyst walls, and cytoplasmic segregation. The cytoplasm comprises mainly the matrix, organelles, and inclusions. The matrix was grainy and evenly distributed. In addition to organelles, including mitochondria with tubular cristae, Golgi apparatus, and endoplasmic reticulum, the cytoplasm had starch grains and lipid droplets as inclusions. The A. ocellatum cells lacked chloroplasts. This study provides the first ultrastructural view of the cytoplasmic structure of the free-living stages of A. ocellatum.

Protists show diverse lifestyles and fulfill important ecological roles as primary producers, predators, symbionts, and parasites. The degradation of dead microbial biomass, instead, is mainly attributed to bacteria and fungi, while necrophagy by protists remains poorly recognized. Here, we assessed the food range specificity and feeding behavior of the algivorous flagellate Orciraptor agilis (Viridiraptoridae, Cercozoa) with a large-scale feeding experiment. We demonstrate that this species is a broad-range necrophage, which feeds on a variety of eukaryotic and prokaryotic algae, but fails to grow on the tested fungi. Furthermore, our microscopic observations reveal an unexpected flexibility of O. agilis in handling food items of different structures and biochemistry, demonstrating that sophisticated feeding strategies in protists do not necessarily indicate narrow food ranges.

Human babesiosis is a malaria-like, tick-borne infectious disease with a global distribution. Babesiosis is caused by intraerythrocytic, apicomplexan parasites of the genus Babesia. In the United States, human babesiosis is caused by Babesia microti and Babesia duncani. Current treatment for babesiosis includes either the combination of atovaquone and azithromycin or the combination of clindamycin and quinine. However, the side effects of these agents and the resistance posed by these parasites call for alternative approaches for treating human babesiosis. Proteases play several roles in the context of parasitic lifestyle and regulate basic biological processes including cell death, cell progression, and cell migration. Using the SYBR Green-1 assay, we screened a protease inhibitor library that consisted of 160 compounds against B. duncani in vitro and identified 13 preliminary hits. Dose response assays of hit compounds against B. duncani and B. microti under in vitro conditions identified five effective inhibitors against parasite growth. Of these compounds, we chose ixazomib, a proteasome inhibitor as a potential drug for animal studies based on its lower IC and a higher therapeutic index in comparison with other compounds. Our results suggest that Babesia proteasome may be an important drug target and that developing this class of drugs may be important to combat human babesiosis.

Pirsoniales is a stramenopile order composed of marine parasitoids of diatoms with unique life cycle. Until recently, a single genus, Pirsonia, uniting six species, was known. The recent identification of new free-living eukaryotrophic Pirsoniales Pirsonia chemainus, Feodosia pseudopoda, and Koktebelia satura changed our understanding of this group as exclusively parasitic. However, their cell ultrastructure and feeding preferences were not fully studied due to the death of the cultures. In this study, we re-isolated some of these Pirsoniales and established six new strains exhibiting predatory behavior, including a first freshwater representative. This allowed us to describe five new genera and species, as well as to emend the diagnosis of the order Pirsoniales. The 18S rRNA gene phylogenetic analysis revealed the position of new strains within Pirsoniales and their relationships with parasitoid relatives and environmental sequence lineages. Feeding experiments on novel Pirsoniales strains using diverse algal prey showed that they were not able to form trophosomes and auxosomes. The ability of cell aggregation in Pirsoniales was observed for the first time. One of the studied strains contained intracellular gammaproteobacteria distantly related to Coxiella. Ultrastructural analyses revealed a more complex cytoskeleton structure in Pirsoniales than previously thought and supported the monophyly of Bigyromonadea and Pseudofungi.

The planktonic dinoflagellate genus Centrodinium has been understudied, with the type species C. elongatum remaining undocumented since the original description. Here, we report C. elongatum isolated from Mazatlán, Mexican Pacific. In the chains, the posterior daughter cell with an incomplete apical horn shows the morphology of C. elongatum, while the anterior daughter cell with complete epitheca corresponds to C. pulchrum. For the first time, a species of Centrodinium sensu stricto (highly laterally flattened species with horns) was cultured. An unarmored life stage, known as Murrayella ovalis, derived from the spheroplast after ecdysis. In the rDNA molecular phylogenies, C. elongatum (=C. pulchrum) nested as basal to morphologically similar species (C. eminens and C. intermedium) and as a sister group of a former Murrayella species, C. punctatum. C. elongatum differs from C. eminens and C. intermedium in the chain formation, second apical (2') plate not being divided, horns with coarse poroid ornamentation, and missing prominent distal spinules. The taxonomy of Centrodinium sensu stricto is revised, with a discussion in the identities of C. complanatum, C. eminens, and C. maximum. The name C. deflexum is restored as a senior synonym of C. intermedium and C. ovale.

Metamonads are a large and exclusively anaerobic group of protists. Additionally, they are one of the three clades proposed to ancestrally possess an "excavate" cell morphology, with a conspicuous ventral groove accompanied by a posterior flagellum with a vane. Here, we cultivate and characterize four anaerobic bacterivorous flagellates from hypersaline and alkaline soda lake environments, which represent a novel clade. Small subunit ribosomal RNA (SSU rRNA) gene phylogenies support recent phylogenomic analyses in placing them as the sister of barthelonids, a group that is itself sister to or deeply branching within Fornicata (Metamonada). The new isolates have a distinctive morphology: the hunchbacked cell body is traversed by a narrow ventral groove ending in a large opening to a conspicuous recurrent cytopharynx. The right margin of the groove is defined by a thin "lip." The posterior flagellum bears a wide ventral-facing vane. The narrow ventral groove and elongate cytopharynx are shared with barthelonids. We describe one isolate as Skoliomonas litria, gen. et sp. nov. Further investigation of their mitochondrial-related organelles (MROs) and detailed ultrastructural studies would be important to understanding the adaptation to anaerobic conditions in Metamonads-especially fornicates-as well as the evolution of the "excavate" cell architecture.

Glugea sp. found infecting the liver of the teleost fish Carangoides bajad from the Red Sea, Egypt, is described based on light microscopy and ultrastructural characteristics combined with phylogenetic analyses. This microsporidium forms whitish xenomas up to ~4 mm in size. Xenomas display numerous parasitophorous vacuoles totally filled by mature spores, no other life cycle stages were observed. Mature spores ellipsoidal and measuring 6.3 × 4.0 μm in size. The polaroplast appears composed of two distinct regions: an electron-dense vesicular region and a densely packed lamellar region. The polar tubule forms approximately 24-27 coils arranged in three layers encircling the posterior vacuole. The small subunit (SSU) rRNA gene and its ITS region were sequenced and showed the highest similarity of 99.4% to other Glugea spp. Bayesian inference and maximum likelihood analyses place the novel isolate within the Glugea clade, more specifically within a subclade that predominantly grouped species described from fish inhabiting the Arabian Gulf or Red Sea. The results validate the parasite's classification in the Glugea genus. Nevertheless, until more detailed ultrastructural and molecular data are obtained, the identification of the current Glugea species is hampered by the absence of some developmental stages and the high degree of genetic similarity.

The Diphyllatea (CRuMs) are heterotrophic protists currently divided into three distinct clades (Diphy I-III). Diphy I are biflagellates in the genus Diphylleia, whereas Diphy II and III represent cryptic clades comprising Collodictyon-type quadriflagellates that were recently distinguished based on rRNA gene phylogenies. Here, we isolated Diphyllatea from freshwater crater lakes on two South Pacific islands and generated high-quality transcriptomes from species representing each clade, including the first transcriptomic data from Diphy III. Phylogenomic analyses support the separation of Diphy II and III, while transcriptome completeness highlights the utility of these data for future studies. Lastly, we discuss the biogeography and ecology of Diphyllatea on these remote islands.

When mechanical stimulation was applied to free swimming Paramecium, forward swimming velocity transiently increased due to activation of the posterior mechanosensory channels. The behavior response, known as "escape response," requires membrane hyperpolarization and the activation of K-channel type adenylate cyclases. Our hypothesis is that this escape response also involves activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. HCN channels are activated by hyperpolarization and are modulated by cyclic nucleotides such as cAMP and cGMP. They play a critical role in many excitable cells in higher animals. If HCN channels act in Paramecium, this should help to enhance and prolong hyperpolarization, thereby increasing the swimming speed of Paramecium. This study used RNAi to examine the role of the HCN channel 1 in the escape responses by generating hcn1-gene knockdown cells (hcn1-KD). These cells showed reduced mechanically-stimulated escape responses and a lack of cGMP-dependent increases in swimming speed. Electrophysiological experiments demonstrated reduced hyperpolarization upon injection of large negative currents in hcn1-KD cells. This is consistent with a decrease in HCN1 channel activity and changes in the escape response. These findings suggest that HCN1 channels are K channels that regulate the escape response of Paramecium by amplifying the hyperpolarizations elicited by posterior mechanical stimulation.

Entamoeba nuttalli is genetically the closest to Entamoeba histolytica, the causative agent of human amebiasis. E. nuttalli is found in Macaca species, exhibiting no symptoms while potentially virulent. Using comparative genomics of Entamoeba species, we identified a gene encoding an E. nuttalli-specific protein containing 42 repeats of an octapeptide (PTORS). In the present study, we analyzed the genes in E. nuttalli strains derived from various geographic locations and host species. Sequence analysis of genomic DNA from four strains indicated 43, 44, and 48 repeat types in addition to 42 repeats and remarkable genetic diversity in the repeat region, although all nucleotide substitutions were synonymous. In contrast, the sequences of the N-terminal side region and C-terminus were identical among the strains. Monoclonal antibodies prepared against recombinant PTORS were reactive to the repeat regions but not to the N-terminal side regions. Polyclonal antibodies did not react with the N-terminal region, demonstrating that the repeat region had higher antigenicity. Analysis using synthetic peptides revealed that the two repeats of the octapeptide functioned as epitopes. Immunofluorescence microscopy using monoclonal antibodies demonstrated the surface localization of PTORS. These results suggest that the repeat region of PTORS plays an important role in host-parasite interactions.

Gregarine apicomplexans are ubiquitous endosymbionts of invertebrate hosts. Despite their ecological and evolutionary importance, inferences about the phylogenetic relationships of major gregarine groups, such as the Lecudinidae and Urosporidae, have been hindered by vague taxonomic definitions and limited molecular and morphological data. In this study, we investigated five gregarine species collected from four families of polychaete hosts (Nereididae, Oenonidae, Hesionidae, and Phyllodocidae) using light microscopy (LM) and scanning electron microscopy (SEM). We also generated small subunit ribosomal DNA sequences from these species and conducted molecular phylogenetic analyses to elucidate the evolutionary relationships within the Lecudinoidea. Our results include new molecular and morphological data for two previously described species (Lecudina cf. platynereidis and Lecudina cf. arabellae), the discovery of a new species of Lecudina (L. oxydromus n. sp.), and the discovery of two novel species, namely Amplectina cordis n. gen. et. n. sp. and Sphinctocystis inclina n. sp. These two species exhibited unique shapes and movements, resembling those of urosporids but with a phylogenetic affinity to lecudinids, blurring the border between lecudinids and urosporids. Our study emphasizes the need for further investigations into this highly diverse group, which has achieved great success across multiple animal phyla with diverse shapes and movements.

Coccidiosis is one of the most prevalent diseases found in local rabbits (Oryctolagus cuniculus), which is caused by the Eimeria. The study aimed to more reliably identify Eimeria species (Eimeria magna) infecting Local Rabbits in Alkarg City, Saudi Arabia, based the method on the molecular properties and morphological and molecular biological techniques. Sub-spheroidal oocysts measuring 21-27 × 12-16 (24 × 14.4) μm (20 n) and with a length/width (L/W) ratio of 0.9-1.1 (1.0) were identified by microscopic analysis of a fecal sample. Oocysts feature a bi-layered wall that is 1.0-1.2 (1.1) μm thick. About two-thirds of the wall's thickness is made up of a smooth outer layer. A polar granule is present, but neither a micropyle nor an oocyst residuum is present. The ovoidal sporozoites measure 15-18 × 8-11 (16.5 × 9.5) μm, have an L/W ratio of 1.6-1.8 (1.7), and take up around 21% of the oocyst's total surface. The mean size of the sub-Stieda body is 1.4 × 2.3 μm, while the average size of the Stieda body is 0.9 × 1.8 μm. The para-Stieda body is lacking. Sporocyst residuum appears membrane-bound and has an uneven form made up of several granules. With two refractile bodies below the striations and pronounced striations at the more pointed end, sporozoites are vermiform, measuring an average of 11.6 × 4.0 μm. The results of the sequencing for the 18S rDNA gene confirmed the species of Eimeria parasites found in the host (rabbits). The current parasite species is closely related to the previously described and deposited E. magna and deeply embedded in the genus Eimeria (family Eimeriidae). According to the findings, single oocyst molecular identification of Eimeria may be accomplished through consistent use of the morphological and molecular results. It is possible to draw the conclusion that the current research supplies relevant facts that help assess the potential infection and future control measures against rabbit coccidiosis to reduce the financial losses that can be incurred by the rabbit industry in Saudi Arabia.

Dinoflagellates are an abundant and diverse group of protists representing a wealth of unique biology and ecology. While many dinoflagellates are photosynthetic or mixotrophic, many taxa are heterotrophs, often with complex feeding strategies. Compared to their photosynthetic counterparts, heterotrophic dinoflagellates remain understudied, as they are difficult to culture. One exception, a long-cultured isolate originally classified as Amphidinium but recently reclassified as Oxytoxum, has been the subject of a number of feeding, growth, and chemosensory studies. This lineage was recently determined to be closely related to Prorocentrum using phylogenetics of ribosomal RNA gene sequences, but the exact nature of this relationship remains unresolved. Using transcriptomes sequenced from culture and three single cells from the environment, we produce a robust phylogeny of 242 genes, revealing Oxytoxum is likely sister to the Prorocentrum clade, rather than nested within it. Molecular investigations uncover evidence of a reduced, nonphotosynthetic plastid and proteorhodopsin, a photoactive proton pump acquired horizontally from bacteria. We describe the ultrastructure of O. lohmannii, including densely packed trichocysts, and a new type of mucocyst. We observe that O. lohmannii feeds preferentially on cryptophytes using myzocytosis, but can also feed on various phytoflagellates using conventional phagocytosis. O. lohmannii is amenable to culture, providing an opportunity to better study heterotrophic dinoflagellate biology and feeding ecology.

An astonishing range of morphologies and life strategies has arisen across the vast diversity of protists, allowing them to thrive in most environments. In model protists, like Tetrahymena, Dictyostelium, or Trypanosoma, life cycles involving multiple life stages with different morphologies have been well characterized. In contrast, knowledge of the life cycles of free-living protists, which primarily consist of uncultivated environmental lineages, remains largely fragmentary. Various life stages and lineage-specific cellular innovations have been observed in the field for uncultivated protists, but such innovations generally lack functional characterization and have unknown physiological and ecological roles. In the actual state of knowledge, evidence of sexual processes is confirmed for 20% of free-living protist lineages. Nevertheless, at the onset of eukaryotic diversification, common molecular trends emerged to promote genetic recombination, establishing sex as an inherent feature of protists. Here, we review protist life cycles from the viewpoint of life cycle transitions and genetics across major eukaryotic lineages. We focus on the scarcely observed sexual cycle of free-living protists, summarizing evidence for its existence and describing key genes governing its progression, as well as, current methods for studying the genetics of sexual cycles in both cultivable and uncultivated protist groups.

Microsporidia comprise a large phylum of single-cell and obligate intracellular parasites that can infect a wide range of invertebrate and vertebrate hosts including humans. These fungal-related parasites are characterized by a highly reduced genome, a strong energy dependence on their host, but also by their unique invasion organelle known as the polar tube which is coiled within the resistant spore. Upon appropriate environmental stimulation, the long hollow polar tube (ranging from 50 to 500 μm in length) is extruded at ultra-fast speeds (300 μm/s) from the spore acting as a harpoon-like organelle to transport and deliver the infectious material or sporoplasm into the host cell. To date, seven polar tube proteins (PTPs) with distinct localizations along the extruded polar tube have been described. For example, the specific location of PTP4 and PTP7 at the tip of the polar tube supports their role in interacting with cellular receptor(s). This chapter provides a brief overview on the current understanding of polar tube structure and dynamics of extrusion, primarily through recent advancements in cryo-tomography and 3D reconstruction. It also explores the various mechanisms used for host cell invasion. Finally, recent studies on the structure and maturation of sporoplasm and its moving through the tube are discussed.

Microsporidia are prolific producers of effector molecules, encompassing both proteins and nonproteinaceous effectors, such as toxins, small RNAs, and small peptides. These secreted effectors play a pivotal role in the pathogenicity of microsporidia, enabling them to subvert the host's innate immunity and co-opt metabolic pathways to fuel their own growth and proliferation. However, the genomes of microsporidia, despite falling within the size range of bacteria, exhibit significant reductions in both structural and physiological features, thereby affecting the repertoire of secretory effectors to varying extents. This review focuses on recent advances in understanding how microsporidia modulate host cells through the secretion of effectors, highlighting current challenges and proposed solutions in deciphering the complexities of microsporidial secretory effectors.

Methionine aminopeptidases (MetAPs) have emerged as a target for medicinal chemists in the quest for novel therapeutic agents for treating cancer, obesity, and other disorders. Methionine aminopeptidase is a metalloenzyme with two structurally distinct forms in humans, MetAP-1 and MetAP-2. The MetAP2 inhibitor fumagillin, which was used as an amebicide in the 1950s, has been used for the successful treatment of microsporidiosis in humans; however, it is no longer commercially available. Despite significant efforts and investments by many pharmaceutical companies, no new MetAP inhibitors have been approved for the clinic. Several lead compounds have been designed and synthesized by researchers as potential inhibitors of MetAP and evaluated for their potential activity in a wide range of diseases. MetAP inhibitors such as fumagillin, TNP-470, beloranib, and reversible inhibitors and their analogs guide new prospects for MetAP inhibitor development in the ongoing quest for new pharmacological indications. This perspective provides insights into recent advances related to MetAP, as a potential therapeutic target in drug discovery, bioactive small molecule MetAP2 inhibitors, and data on the role of MetAP-2 as a therapeutic target for microsporidiosis.

Microsporidia are intracellular parasites that all possess a unique infection apparatus involving a polar tube. Upon contact with a host cell, this tube forms the conduit through which the parasite enters the host. Infecting mostly animals, microsporidian species can be transmitted vertically or horizontally, and exert various effects on their hosts: infections range from being relatively benign to lethal. Microsporidian genomes possess highly divergent sequences and are often substantially reduced in size. Their divergent sequences and unique morphology created early challenges to our understanding of their phylogenetic position within the tree of eukaryotes. Over the last couple of decades, advances in both sequencing technology and phylogenetic methodology supported a clear relationship between microsporidia and fungi. However, the specifics of this relationship were muddied by the lack of known microsporidian relatives. With increased taxon discovery and the morphological and molecular characterization of microsporidia-like taxa, rozellids and aphelids, a better resolved picture is emerging. Here we review the history of microsporidian taxonomy and current status of genomics of microsporidia and their nearest relatives, with an aim to understand their morphological and metabolic differences, along with their evolutionary relationships.

Microsporidia are opportunistic fungal-like pathogens that cause microsporidiosis, which results in significant economic losses and threatens public health. Infection of domesticated silkworms by the microsporidium Nosema bombycis causes pébrine disease, for which this species of microsporidia has received much attention. Research has been conducted extensively on this microsporidium over the past few decades to better understand its infection, transmission, host-parasite interaction, and detection. Several tools exist to study this species including the complete genome sequence of N. bombycis. In addition to the understanding of N. bombycis being important for the silkworm industry, this species has become a model organism for studying microsporidia. Research on biology of N. bombycis will contribute to the development of knowledge regarding microsporidia and potential antimicrosporidia drugs. Furthermore, this will provide insight into the molecular evolution and functioning of other fungal pathogens.

Microsporidia, a group of unicellular eukaryotic parasites, rely intensely on secretory effectors for successful invasion and proliferation within host cells. This review focuses on the identification, characterization, and functional roles of effectors, including secretory proteins and microRNAs. The adhesion proteins like the Ricin-B-lectin facilitate initial invasion, which binds to the host cell surface. Once inside, microsporidia deploy a range of effectors to modulate host immune responses, such as serpin proteins, and redirect host cell metabolism to meet the parasite's nutritional needs through hexokinase. Some effectors such as microRNAs, alter the host gene expression to create a more favorable intracellular parasitic environment. In conclusion, the secretory effectors of microsporidia play a pivotal role spanning from host cell invasion to intracellular establishment. In the future, more effectors secreted by microsporidia will be studied, which will not only help to elucidate the molecular mechanism of pathogenic manipulation of the host but also help to provide the potential targets for anti-parasitic treatments.