Journal of Signal Processing

ABSTRACTMobilid ciliates are a morphologically distinct group of protists that form a wide range of symbiotic relationships with aquatic animals and includes three subgroups: Trichodinidae, Urceolariidae, and Polycyclidae. Trichodinids are best known for infecting fishes, whereas urceolariids infect diverse marine invertebrates. Polycyclidae was established for mobilid ciliates infecting sea cucumbers; however, molecular data have been unavailable for this group. In this study, we discovered and characterized two novel mobilid species, one infecting two species of sea cucumbers (Eupentacta quinquesemita and Cucumaria miniata) and one infecting brachiopods or lamp shells (Terebratalia transversa) collected from the Northeast Pacific Ocean. These new mobilid species were characterized at the morphological level using light microscopy (LM) and scanning electron microscopy (SEM). We also inferred the molecular phylogenetic positions of these species using small subunit (SSU) rDNA sequences. Based on combined morphological and molecular data, we demonstrate that the two new species belong to Urceolaria, U. clepsydra n. sp. and U. bratalia n. sp., and support synonymization of Polycycla with Urceolaria. By providing the first molecular data from new species of mobilids infecting sea cucumbers and brachiopods, we expand the host range and improve our knowledge of this diverse but poorly understood group of symbionts.

ABSTRACTGoniomonads are commonly found heterotrophic biflagellates in both marine and freshwater environments. Despite the high genetic diversity inferred from 18S rDNA data, many goniomonad species remain undescribed. In this study, we established a total of 21 marine goniomonad culture strains, and from these, describe three new species by using 18S rDNA phylogeny, light microscopy, and electron microscopy. Molecular sequence analyses suggest the presence of several distinct sub‐lineages within the marine goniomonad clade. Two of these are Goniomonas ulleungensis sp. nov. and G. lingua sp. nov., which are similar in size, flagellar length, appendage, and orientation and have a tongue‐like protrusion at the anterior. The two species can be differentiated by the periplast plate pattern with G. ulleungensis displaying one additional plate on the right side. G. duplex sp. nov. differed from these two species by having two unequal flagella with the longer one trailing posteriorly and having the opposite cell orientation when skidding. Comparative analyses of five marine goniomonad species showed that genetically distinct goniomonad groups can be delineated by morphological data as well, and of several morphological features that are of taxonomic utility, the periplast plate pattern, observable by SEM, is particularly informative in goniomonad taxonomy.

AbstractThe class Labyrinthulomycetes constitutes a multitude of species found ubiquitously in the environment, and includes pathogens of corals, hard clams, turfgrasses, and seagrasses. Labyrinthula zosterae, the causative agent of seagrass wasting disease, has been associated with declines in seagrass coverage since the 1930s. However, pathogenic and nonpathogenic Labyrinthula spp. have been isolated from seagrass tissue. These isolates are difficult to distinguish morphologically, and the diversity of isolates where seagrass wasting disease is present is often unknown. This study aimed to increase knowledge on the pathogenicity and phylogeny of Labyrinthula spp. in Washington and Oregon, USA where a high prevalence of seagrass wasting disease has been associated with eelgrass, Zostera marina, declines. We tested the pathogenicity of 14 Labyrinthula isolates and compared partial 18S rRNA gene sequences of 12 isolates to sequences from around the world through the NCBI database. We found that pathogenic isolates could be identified as Labyrinthula zosterae, while nonpathogenic isolates did not form a clade with any previously identified SSU ribotypes. These results add to the growing data on Labyrinthula and seagrass wasting disease and can improve our understanding of pathogen evolution and spread in the future.

AbstractThe genus Pelomyxa includes 15 species of anaerobic Archamoebae with remarkable diverse nucleoplasm morphology. Nuclear structures, like chromatin and nucleoli, of several members of the genus was previously identified only based on their ultrastructural similarity to typical structures of somatic cells of higher eukaryotes. Here, we explored an easy‐to‐use, one‐step intravital staining method with DAPI and pyronin to distinguish between DNA and RNA structures in nuclei of unfixed cells of Pelomyxa belevskii and P. stagnalis significantly varying by nuclear organization. Our method can be used for rapid diagnosis of DNA and RNA‐containing structures in species with complex nuclear organization.

AbstractEuglenids are flagellates with diverse modes of nutrition, including the photosynthetic Euglenophyceae, which acquired plastids via secondary endosymbiosis with green algae, and a diverse assemblage of predators of bacteria and other microeukaryotes. Most heterotrophic euglenids have never been cultivated, so their morphology remains poorly characterized and limited to only a few studies. “Ploeotids” are a paraphyletic group representing much of the diversity of heterotrophic euglenids and are characterized by their feeding apparatus and a rigid pellicle of 10–12 longitudinally arranged strips. Ploeotid‐like euglenids gave rise to the Spirocuta, a large clade of heterotrophic and photosynthetic euglenids defined by a flexible pellicle of helically arranged strips. Using single‐cell approaches, we report the first ultrastructural characterization of Olkasia polycarbonata, a ploeotid that is consistently positioned as the sister lineage to the Spirocuta in multigene phylogenetic analyses. O. polycarbonata shares several morphological characteristics with members of Spirocuta, such as prominent swellings on the paraxonemal rods and a robust feeding apparatus consisting of rods and vanes. These morphological traits are consistent with the phylogenetic position of O. polycarbonata and demonstrate an increase in cytoskeletal complexity that occurred prior to the key strip duplication event in the most recent common ancestor of Spirocuta.

AbstractThe globally distributed ciliate Balanion planctonicum is a primary consumer of phytoplankton spring blooms. Due to its small size (~20 μm), identification and quantification by molecular tools is preferable as an alternative to the laborious counting of specimen in quantitative protargol stains. However, previous sequencing of the 18S rDNA V9 region of B. planctonicum from Lake Zurich (Switzerland) and subsequent quantification by fluorescence in situ hybridization yielded significantly lower cell numbers than using morphotype counting. This raised the question of whether B. planctonicum shows a cryptic diversity or whether it is just a ‘complex species’ with intra‐clonal polymorphisms. Over three years, we established numerous monoclonal cultures, and long‐read sequencing of rDNA operons revealed four distinct dominant haplotypes (BpHs 1–4). The gene sequences of BpHs 1 and 3 differed by 6% and did not share intra‐clonal polymorphisms, providing evidence for two distinct clades. Furthermore, phylogenetic analyses corroborate the sister relationship between Balanion and Askenasia (plus Hexasterias and Radiosperma). Morphologically, the two Balanion clades are nearly indistinguishable with small differences in macronucleus size and in the cell length to width ratio. CARD‐FISH analyses indicated that the diversity of B. planctonicum is even more extensive with still unidentified clades.

AbstractKinetoplastids are a large and diverse protist group, spanning ecologically important free‐living forms to medically important parasites. The taxon Allobodonidae holds an unresolved position within kinetoplastids, and the sole described species, Allobodo chlorophagus, is uncultivated, being a necrotroph/parasite of macroalgae. Here we describe Allobodo yubaba sp. nov. and Novijibodo darinka gen. nov. et sp. nov., both free‐living bacterivores isolated into monoeukaryotic cultures. Electron microscopy shows that both A. yubaba and N. darinka have a microtubular prism in the feeding apparatus (absent in A. chlorophagus), and an ovoid eukinetoplast, rather than pan‐kDNA as in A. chlorophagus. Phylogenetic analyses of SSU rDNA sequences robustly place A. yubaba as the sister to A. chlorophagus, while N. darinka branches separately within Allobodonidae, as a sister group of undescribed freshwater isolates. We view Allobodonidae as containing at least four genus‐level clades: Allobodo (A. chlorophagus and A. yubaba n. sp.), an undescribed fresh‐water clade, an undescribed marine clade, and now Novijibodo—with N. darinka as its sole known member. Electron microscopy also revealed a rod‐shaped gram‐negative bacterial cytoplasmic endosymbiont in our N. darinka isolate. The availability of these species in monoeukaryotic culture should facilitate future research, including resolving the position of Allobodonidae using phylogenomic approaches.

AbstractAll insect trypanosomatids of the subfamily Strigomonadinae harbor a proteobacterial symbiont in their cytoplasm and unique ultrastructural cell organization. Here, we report an unexpected finding within the Strigomonadinae subfamily: the identification of a new species lacking bacterial symbiont, represented by two isolates obtained from Calliphoridae flies in Brazil and Uganda. This species is hereby designated as Kentomonas inusitatus n. sp. Molecular investigations targeting symbiont DNA, cell proliferation, and ultrastructural analyses agreed with the absence of bacterial symbionts in cultured flagellates. PCR‐screening specifically targeting symbiont DNA corroborated the absence of symbionts in K. inusitatus present in the intestine of the respective host flies. K. inusitatus exhibited forms varying in size and shape. While displaying overall ultrastructural features of the Strigomonadinae, the novel species showed mitochondrial branches juxtaposed to the plasma membrane in locations both without and notable, with subpellicular microtubules. The discovery of the first Strigomonadinae species naturally lacking a symbiont and closely related to K. sorsogonicus, suggests a unique evolutionary history for the genus Kentomonas. Our findings provide novel insights into the complex relationships between trypanosomatids and their symbionts.

AbstractWhile a longstanding method for identifying dinoflagellates, morphology alone can be inaccurate because of convergent evolution of truly different species toward a particular cellular shape. The dinoflagellate genus Togula is a case in point in that its type species Togula britannica was previously assigned to the genus Amphidinium based on morphology but was recently recognized as an independent genus following phylogenetic characterization. Chemotaxonomy can be a useful tool to complement the characterization of dinoflagellates by phylogeny. To this point, some, but not all, species of Amphidinium, such as Amphidinium carterae, have been observed to produce the rare 4α‐methyl‐substituted, Δ8(14)‐nuclear‐unsaturated major sterol 4α‐methyl‐5α‐ergosta‐8(14),24(28)‐dien‐3β‐ol (amphisterol) that has historically been considered a potential chemotaxonomic biomarker for the genus as it is found in very few other dinoflagellate genera. To date, no isolates within the genus Togula have had their sterols characterized; our objective was thus to provide a first examination of the sterols of an isolate of Togula to compare to the sterols of Amphidinium. To this end, we have characterized the sterols of Togula jolla, one of the few members of Togula available for study, to demonstrate the production of amphisterol, among other Δ8(14)‐nuclear unsaturated sterols, as its major sterol.

AbstractThecate amoebae play important roles in terrestrial and aquatic ecosystems. This study introduces a novel thecofilosean amoeba from Arctic and Antarctic sea sediments. Phylogenetic analysis based on the 18S rDNA sequence places it in the family Chlamydophryidae (order Tectofilosida, class Thecofilosea). However, the novel organism exhibits a significant genetic divergence and distinct morphology from its closest relatives, prompting us to erect the novel genus Katarium with its type species Katarium polorum. K. polorum is a consumer of diatoms and prokaryotes, indicating an important role in nutrient cycling in the polar marine food webs.

AbstractAs chloroplast‐stealing or “kleptoplastidic” lineages become more reliant on stolen machinery, they also tend to become more specialized on the prey from which they acquire this machinery. For example, the ciliate Mesodinium rubrum obtains > 95% of its carbon from photosynthesis, and specializes on plastids from the Teleaulax clade of cryptophytes. However, M. rubrum is sometimes observed in nature containing plastids from other cryptophyte species. Here, we report on substantial ingestion of the blue‐green cryptophyte Hemiselmis pacifica by M. rubrum, leading to organelle retention and transient increases in M. rubrum's growth rate. However, microscopy data suggest that H. pacifica organelles do not experience the same rearrangement and integration as Teleaulax amphioxeia's. We measured M. rubrum's functional response, quantified the magnitude and duration of growth benefits, and estimated kleptoplastid photosynthetic rates. Our results suggest that a lack of discrimination between H. pacifica and the preferred prey T. amphioxeia (perhaps due to similarities in cryptophyte size and swimming behavior) may result in H. pacifica ingestion Thus, while blue‐green cryptophytes may represent a negligible prey source in natural environments, they may help M. rubrum survive when Teleaulax are unavailable. Furthermore, these results represent a useful tool for manipulating M. rubrum's cell biology and photophysiology.

AbstractAmyloodinium 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.

AbstractHuman 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 IC50 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.

AbstractProtists 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.