European journal of protistology最新文献

Tetrahymena thermophila is an alternative organism for recombinant protein production. However, the production efficiency in T. thermophila is quite low mainly due to the rich cysteine proteases. In this study, we studied whether supplementation of the E-64 inhibitor to T. thermophila cultures increases the recombinant protein production efficiency without any toxic side effects. Our study showed that supplementation of E-64 had no lethal effects on T. thermophila cells in flask culture at 30 °C and 38 °C. In vitro protease activity analysis using secretome as protease enzyme source from E-64-supplemented cell cultures showed a reduced protein substrate degradation using bovine serum albumin, rituximab, and milk lactoglobulin proteins. E-64 also prevented proteolysis of the recombinantly produced and secreted TtmCherry2-sfGFP fusion protein at some level. This reduced inhibitory effect of E-64 could be due to genetic compensation of the inhibited proteases. As a result, the 5 µM concentration of E-64 was found to be a non-toxic protease inhibitory supplement to improve extracellular recombinant protein production efficiency in T. thermophila. This study suggests that the use of E-64 may increase the efficiency of extracellular recombinant protein production by continuously reducing extracellular cysteine protease activity during cultivation.

Acanthamoeba castellanii, a free-living amoeba, can be pathogenic to humans causing a corneal infection named Acanthamoeba keratitis (AK). The mannose-binding protein (MBP) is well established as the major factor related to Acanthamoeba pathogenesis. However, additional factors that participate in the adhesion process and protect trophozoites from cytolytic effects caused by host immune responses remain unknown. Ectonucleotidases, including 3′-nucleotidase/nuclease (3′-NT/NU), a bifunctional enzyme that was recently reported in A. castellanii, are frequently related to the establishment of parasitic infections. We verified that trophozoites can hydrolyze 3′-AMP, and this activity is similar to that observed in other protists. The addition of 3′-AMP increases the adhesion of trophozoites to LLC-MK2 epithelial cells, and this stimulation is completely reversed by DTT, an inhibitor of ecto-3′-nucleotidase activity. Lesions in corneal cells caused by AK infection may elevate the extracellular level of 3′-AMP. We believe that ecto-3′-nucleotidase activity can modulate the host immune response, thus facilitating the establishment of parasitic infection. This activity results from the generation of extracellular adenosine, which can bind to purinergic receptors present in host immune cells. Positive feedback may occur in this cascade of events once the ecto-3′-nucleotidase activity of trophozoites is increased by the adhesion of trophozoites to LLC-MK2 cells.

Gregarines are symbiotic protists that are found in a broad spectrum of invertebrates, including insects, crustaceans, and annelids. Among these the globally distributed amphipod Gammarus pulex is one of the earliest recognized hosts for aquatic gregarines and is prevalent among macroinvertebrates in freshwater environments. In this study, samples of G. pulex were collected in the Water of Leith river, Scotland, UK. Gregarines were identified using light and scanning electron microscopy as well as standard molecular techniques. We identified three septate eugregarine symbionts—Heliospora longissima, Cephaloidophora gammari, and the here newly characterized Cephaloidophora conus n. sp. (formerly Cephaloidophora sp.) associated with Gammarus pulex in the Water of Leith. Prevalences for identified gregarine species were calculated and seasonal dynamics of gregarine infections/colonization were analyzed. Prevalences were highest in autumn and spring reaching almost 50 %. While the two Cephaloidophora species showed similar colonization patterns, the prevalence of Heliospora showed an opposite trend. Identifying gregarine infection/colonization patterns is one step towards better understanding the gregarine–host relationship, as well as possible impacts of the gregarines on their hosts.

The frequently encountered macroscopic slime molds of the genus Ceratiomyxa have long been recognized by mycologists and protistologists for hundreds of years. These organisms are amoebozoan amoebae that live and grow inside and on the surface of decaying wood. When conditions are favorable, they form subaerial sporulating structures called fruiting bodies which take on a variety of forms. These forms are typically some arrangement of column and/or branches, but one is uniquely poroid, forming folds instead. Originally, this poroid morphology was designated as its own species. However, it was not always clear what significance fruiting body morphology held in determining species. Currently, Ceratiomyxa fruticulosa var. porioides, the poroid form, is considered a taxonomic variety of Ceratiomyxa fruticulosa based on morphological designation alone. Despite its long history of observation and study, the genus Ceratiomyxa has been paid little molecular attention to alleviate these morphological issues. We have obtained the first transcriptomes of the taxon C. fruticulosa var. porioides and found single gene phylogenetic and multigene phylogenomic support to separate it from C. fruticulosa. This provides molecular evidence that fruiting body morphology does correspond to species level diversity. Therefore, we formally raise Ceratiomyxa porioides to species level.

Many terrestrial microbes have evolved cell behaviors that help them rise above their substrate, often to facilitate dispersal. One example of these behaviors is found in the amoebae of Sappinia pedata, which actively lift most of their cell mass above the substrate, known as standing. This standing behavior was first described in S. pedata in the 1890s from horse dung isolates but never molecularly characterized from dung. Our study expands this understanding, revealing the first molecularly confirmed S. pedata from herbivore dung in Mississippi, USA, and describing a new species, Sappinia dangeardi n. sp., with larger trophozoite cells. Additionally, we isolated another standing amoeba, Thecamoeba homeri n. sp., from soil, exhibiting a previously unreported “doughnut shape” transient behavior. In S. dangeardi n. sp., we discovered that standing is likely triggered by substrate drying, and that actin filaments actively localize in the “stalk” to support the standing cells, as observed through confocal microscopy. While the purpose of standing behaviors has not been investigated, we hypothesize it is energetically expensive and therefore a significant evolutionary strategy in these organisms. Overall, this study emphasizes behavioral adaptations to terrestrial environments within Amoebozoa, stressing the importance of diverse laboratory conditions that replicate natural habitats.

Periphytic protists including ciliates are the primary components of microbial communities in which they play a vital role in the progression of food webs by moving resources from lower to higher trophic levels. However, the toxic effects of veterinary antibiotics on periphytic protists across four seasons are minimally understood. Therefore, in this study, a 1-year survey was conducted with the antibiotic nitrofurazone (NFZ) applied at concentrations of 0.0, 1.5, 3.0, 6.0, and 12.0 mg/L. Samples of protist communities were collected using microscope glass slides during four seasons in the coastal waters of the Yellow Sea, Qingdao, northern China. The abundance of protists dropped with an increase in NFZ concentrations, and almost all species were dead at a concentration of 12.0 mg/L. The 12 h-LC₅₀ values of NFZ for the protist biota were similar among the four seasons, despite significant seasonal variability in the community structure. The present results suggest that the periphytic protist biota may be used as a biomarker for assessing the ecotoxicity of NFZ in marine environments regardless of the year season.

Gregarines are the most biodiverse group of apicomplexan parasites. This group specializes on invertebrate hosts (e.g., ascidians, crustaceans, and polychaetes). Marine gregarines are of particular interest because they are considered to be the earliest evolving apicomplexan lineage, having subsequently speciated (and radiated) through virtually all existing animal groups. Still, mechanisms governing the broad (global) distribution and speciation patterns of apicomplexans are not well understood. The present study examines Pacific lecudinids, one of the most species-rich and diverse groups of marine gregarines. Here, marine polychaetes were collected from intertidal zones. Single trophozoite cells were isolated for light and electron microscopy, as well as molecular phylogenetic analyses using the partial 18S rRNA gene. The cytochrome c oxidase subunit 1 gene was used to confirm morphology-based host identification. This study introduces Undularius glycerae n. gen., n. sp. and Lecudina kitase n. sp. (Hokkaido, Japan), as well as Difficilina fasoliformis n. sp. (California, USA). Occurrences of Lecudina cf. longissima and Lecudina cf. tuzetae (California, USA) are also reported. Phylogenetic analysis revealed a close relationship between L. pellucida, L. tuzetae, and L. kitase n. sp. Additionally, clustering among North Atlantic and Pacific L. tuzetae formed a species complex, likely influenced by biogeography.

When the ciliate Spirostomum ambiguum is transected into two pieces, both fragments regenerate and proliferate. In the anterior fragments, which have lost their contractile vacuoles due to transection, new contractile vacuoles were formed at their posterior ends in a few minutes. When the cells were cut into three pieces, new contractile vacuoles were formed in the anterior and middle fragments, both at their posterior ends. Thus, the anterior-posterior axis of S. ambiguum was maintained after transection. Morphological repair, including the formation of the contractile vacuole, was also observed when only the anteriormost portion was transected to cut out a small fragment that did not contain part of the macronucleus. Scanning electron microscopy was performed to observe changes in the shape of the cleavage surface of S. ambiguum during the wound healing process. Within minutes after cutting, the cut surface was covered with a cilia-free membrane, preventing leakage of cytoplasmic contents. The surface of the cut area then rounded with time and was covered with cilia, completing the repair of the cut area in about one day.

Osmoregulation is the homeostatic mechanism essential for the survival of organisms in hypoosmotic and hyperosmotic conditions. In freshwater or soil dwelling protists this is frequently achieved through the action of an osmoregulatory organelle, the contractile vacuole. This endomembrane organelle responds to the osmotic challenges and compensates by collecting and expelling the excess water to maintain the cellular osmolarity. As compared with other endomembrane organelles, this organelle is underappreciated and under-studied. Here we review the reported presence or absence of contractile vacuoles across eukaryotic diversity, as well as the observed variability in the structure, function, and molecular machinery of this organelle. Our findings highlight the challenges and opportunities for constructing cellular and evolutionary models for this intriguing organelle.

In Euplotes, protein pheromones regulate cell reproduction and mating by binding cells in autocrine or heterologous fashion, respectively. Pheromone binding sites (receptors) are identified with membrane-bound pheromone isoforms determined by the same genes specifying the soluble forms, establishing a structural equivalence in each cell type between the two twin proteins. Based on this equivalence, autocrine and heterologous pheromone/receptor interactions were investigated analyzing how native molecules of pheromones Er-1 and Er-13, distinctive of mating compatible E. raikovi cell types, associate into crystals. Er-1 and Er-13 crystals are equally formed by molecules that associate cooperatively into oligomeric chains rigorously taking a mutually opposite orientation, and each burying two interfaces. A minor interface is pheromone-specific, while a major one is common in Er-1 and Er-13 crystals. A close structural inspection of this interface suggests that it may be used by Er-1 and Er-13 to associate into heterodimers, yet inapt to further associate into higher complexes. Pheromone-molecule homo-oligomerization into chains accounts for clustering and internalization of autocrine pheromone/receptor complexes in growing cells, while the heterodimer unsuitability to oligomerize may explain why heterologous pheromone/receptor complexes fail clustering and internalization. Remaining on the cell surface, they are credited with a key role in cell–cell mating adhesion.