Dispersal and invasive stages of Urospora eugregarines (Apicomplexa) from brown bodies of a polychaete host.

Urosporid eugregarines (Apicomplexa: Urosporidae) are unicellular eukaryotic parasites inhabiting the coelom or the intestine of marine invertebrates such as annelids, molluscs, nemerteans, and echinoderms. Despite the availability of published morphological and phylogenetical analyses of coelomic gregarines, their long-term survival in the host body cavity and dispersal routes into the marine environment remain unclear. Here, we focus on Urospora gametocysts and oocysts with sporozoites, which were found viable inside the so-called brown bodies floating in the body cavity of the polychaete Travisia forbesii. Brown bodies form as a result of host defence where coelomocytes encapsulate dead host cells and foreign objects including potential pathogens. We hypothesise the long-term persistence of Urospora eugregarines in brown bodies through evasion of the host immunity and outline possible pathways for their egress into the marine environment, applicable as dispersal routes for other parasites as well. Unique features revealed by detailed ultrastructural analysis of detected eugregarine stages include asynchronous sporogony, a massive sporozoite secretion apparatus, as well as the presence of free (possibly autoinfective) sporozoites within the gametocyst. The assignment to the genus Urospora and the complete identity with U. ovalis and U. travisiae were confirmed by analysing 18S rDNA sequences obtained from isolated gametocysts. The 18S rDNA phylogeny confirmed the affiliation of Urosporidae to Lecudinoidea and the grouping of all Urospora sequences with Difficilina from nemerteans and environmental sequences from the Artic region. We also enriched the Apicomplexa set by partial 28S rDNA sequences of two Urospora species enabling more complex phylogenetic analyses prospectively.

Eudiplozoon nipponicum: morphofunctional adaptations of diplozoid monogeneans for confronting their host.

BACKGROUND: Monogeneans, in general, show a range of unique adaptations to a parasitic lifestyle, making this group enormously diverse. Due to their unique biological properties, diplozoid monogeneans represent an attractive model group for various investigations on diverse biological interactions. However, despite numerous studies, there are still gaps in our knowledge of diplozoid biology and morphofunctional adaptations. RESULTS: In this study, we provide a comprehensive microscopic analysis of systems/structures involved in niche searching, sensing and self-protection against the host environment, and excretory/secretory processes in Eudiplozoon nipponicum. Freeze-etching enabled us to detect syncytium organisational features not visible by TEM alone, such as the presence of a membrane subjacent to the apical plasma membrane (separated by a dense protein layer) and a lack of basal plasma membrane. We located several types of secretory/excretory vesicles and bodies, including those attached to the superficial membranes of the tegument. Giant unicellular glands were seen accumulating predominantly in the apical forebody and hindbody haptor region. Muscle layer organisation differed from that generally described, with the outer circular and inner longitudinal muscles being basket-like interwoven by diagonal muscles with additional perpendicular muscles anchored to the tegument. Abundant muscles within the tegumentary ridges were detected, which presumably assist in fixing the parasite between the gill lamellae. Freeze-etching, alongside transmission electron and confocal microscopy with tubulin labelling, enabled visualisation of the protonephridia and nervous system, including the peripheral network and receptor innervation. Three types of receptor were identified: 1) uniciliated sensory endings with a subtle (or missing) tegumentary rim, 2) obviously raised uniciliated receptors with a prominent tegumentary rim (packed with massive innervation and muscles) and 3) non-ciliated papillae (restricted to the hindbody lateral region). CONCLUSIONS: This study points to specific morphofunctional adaptations that have evolved in diplozoid monogeneans to confront their fish host. We clearly demonstrate that the combination of different microscopic techniques is beneficial and can reveal hidden differences, even in much-studied model organisms such as E. nipponicum.

Olanzapine exposure diminishes perfusion and decreases volume of sensorimotor cortex in rats.

BACKGROUND: Olanzapine is a frequently used atypical antipsychotic drug known to exert structural brain alterations in animals. This study investigated whether chronic olanzapine exposure alters regional blood brain perfusion assessed by Arterial Spin Labelling (ASL) magnetic resonance imaging (MRI) in a validated model of olanzapine-induced metabolic disturbances. An effect of acute olanzapine exposure on brain perfusion was also assessed for comparison. METHODS: Adult Sprague-Dawley female rats were treated by intramuscular depot olanzapine injections (100 mg/kg every 14 days) or vehicle for 8 weeks. ASL scanning was performed on a 9.4 T Bruker BioSpec 94/30USR scanner under isoflurane anesthesia. Serum samples were used to assay leptin and TNF-α level while brains were sliced for histology. Another group received only one non-depot intraperitoneal dose of olanzapine (7 mg/kg) during MRI scanning, thus exposing its acute effect on brain perfusion. RESULTS: Both acute and chronic dosing of olanzapine resulted in decreased perfusion in the sensorimotor cortex, while no effect was observed in the piriform cortex or hippocampus. Furthermore, in the chronically treated group decreased cortex volume was observed. Chronic olanzapine dosing led to increased body weight, adipose tissue mass and leptin level, confirming its expected metabolic effects. CONCLUSION: This study demonstrates region-specific decreases in blood perfusion associated with olanzapine exposure present already after the first dose. These findings extend our understanding of olanzapine-induced functional and structural brain changes.

Motility and cytoskeletal organisation in the archigregarine Selenidium pygospionis (Apicomplexa): observations on native and experimentally affected parasites.

Representatives of Apicomplexa perform various kinds of movements that are linked to the different stages of their life cycle. Ancestral apicomplexan lineages, including gregarines, represent organisms suitable for research into the evolution and diversification of motility within the group. The vermiform trophozoites and gamonts of the archigregarine Selenidium pygospionis perform a very active type of bending motility. Experimental assays and subsequent light, electron, and confocal microscopic analyses demonstrated the fundamental role of the cytoskeletal proteins actin and tubulin in S. pygospionis motility and allowed us to compare the mechanism of its movement to the gliding machinery (the so-called glideosome concept) described in apicomplexan zoites. Actin-modifying drugs caused a reduction in the movement speed (cytochalasin D) or stopped the motility of archigregarines completely (jasplakinolide). Microtubule-disrupting drugs (oryzalin and colchicine) had an even more noticeable effect on archigregarine motility. The fading and disappearance of microtubules were documented in ultrathin sections, along with the formation of α-tubulin clusters visible after the immunofluorescent labelling of drug-treated archigregarines. The obtained data indicate that subpellicular microtubules most likely constitute the main motor structure involved in S. pygospionis bending motility, while actin has rather a supportive function.

Application of Advanced Microscopic Methods to Study the Interaction of Carboxylated Fluorescent Nanodiamonds with Membrane Structures in THP-1 Cells: Activation of Inflammasome NLRP3 as the Result of Lysosome Destabilization.

Nanodiamonds (ND), especially fluorescent NDs, represent potentially applicable drug and probe carriers for in vitro/in vivo applications. The main purpose of this study was to relate physical-chemical properties of carboxylated NDs to their intracellular distribution and impact on membranes and cell immunity-activation of inflammasome in the in vitro THP-1 cell line model. Dynamic light scattering, nanoparticle tracking analysis, and microscopic methods were used to characterize ND particles and their intracellular distribution. Fluorescent NDs penetrated the cell membranes by both macropinocytosis and mechanical cutting through cell membranes. We proved accumulation of fluorescent NDs in lysosomes. In this case, lysosomes were destabilized and cathepsin B was released into the cytoplasm and triggered pathways leading to activation of inflammasome NLRP3, as detected in THP-1 cells. Activation of inflammasome by NDs represents an important event that could underlie the described toxicological effects in vivo induced by NDs. According to our knowledge, this is the first in vitro study demonstrating direct activation of inflammasome by NDs. These findings are important for understanding the mechanism(s) of action of ND complexes and explain the ambiguity of the existing toxicological data.

Cellulose fibrils formation and organisation of cytoskeleton during encystment are essential for Acanthamoeba cyst wall architecture.

Acanthamoebae success as human pathogens is largely due to the highly resistant cysts which represent a crucial problem in treatment of Acanthamoeba infections. Hence, the study of cyst wall composition and encystment play an important role in finding new therapeutic strategies. For the first time, we detected high activity of cytoskeletal elements - microtubular networks and filamentous actin, in late phases of encystment. Cellulose fibrils - the main components of endocyst were demonstrated in inter-cystic space, and finally in the ectocyst, hereby proving the presence of cellulose in both layers of the cyst wall. We detected clustering of intramembranous particles (IMPs) and their density alterations in cytoplasmic membrane during encystment. We propose a hypothesis that in the phase of endocyst formation, the IMP clusters represent cellulose microfibril terminal complexes involved in cellulose synthesis that after cyst wall completion are reduced. Cyst wall impermeability, due largely to a complex polysaccharide (glycans, mainly cellulose) has been shown to be responsible for Acanthamoeba biocide resistance and cellulose biosynthesis pathway is suggested to be a potential target in treatment of Acanthamoeba infections. Disruption of this pathway would affect the synthesis of cyst wall and reduce considerably the resistance to chemotherapeutic agents.

Effect of jasplakinolide and cytochalasin D on cortical elements involved in the gliding motility of the eugregarine Gregarina garnhami (Apicomplexa).

Since apicomplexans represent exclusively parasitic unicellular organisms with medical and economic impacts, the principles of their motility have been studied intensively. By contrast, the movement in apicomplexan basal groups, such as gregarines, remains to be elucidated. The present study focuses on Gregarina garnhami parasitising the digestive tract of the locust Schistocerca gregaria, and investigates the involvement of cytoskeletal elements (the ectoplasmic network and myonemes) and the secretion of mucosubstances during eugregarine gliding motility. Combined microscopic analyses were used to verify the role of actin filaments and membranes' organisation in G. garnhami motility. A freeze-etching analysis of membranes revealed the size, density, and arrangement of intramembranous particles along with the distribution and size of pores and ducts. Experimental assays using actin-modifying drugs (jasplakinolide, cytochalasin D) confirmed that actin most likely plays a role in cell motility, principally in its filamentous form (=F-actin). Myonemes, localised in the border between the ectoplasm and endoplasm, correspond to the concentric bundles of F-actin. Microscopic analyses confirmed that changes in gamonts motility corresponding to the changes in the organisation and density of myonemes and the ectoplasmic network in drug-treated cells, suggesting that these structures might serve as contractile elements facilitating gliding motility in G. garnhami.

Motility in blastogregarines (Apicomplexa): Native and drug-induced organisation of Siedleckia nematoides cytoskeletal elements.

Recent studies on motility of Apicomplexa concur with the so-called glideosome concept applied for apicomplexan zoites, describing a unique mechanism of substrate-dependent gliding motility facilitated by a conserved form of actomyosin motor and subpellicular microtubules. In contrast, the gregarines and blastogregarines exhibit different modes and mechanisms of motility, correlating with diverse modifications of their cortex. This study focuses on the motility and cytoskeleton of the blastogregarine Siedleckia nematoides Caullery et Mesnil, 1898 parasitising the polychaete Scoloplos cf. armiger (Müller, 1776). The blastogregarine moves independently on a solid substrate without any signs of gliding motility; the motility in a liquid environment (in both the attached and detached forms) rather resembles a sequence of pendular, twisting, undulation, and sometimes spasmodic movements. Despite the presence of key glideosome components such as pellicle consisting of the plasma membrane and the inner membrane complex, actin, myosin, subpellicular microtubules, micronemes and glycocalyx layer, the motility mechanism of S. nematoides differs from the glideosome machinery. Nevertheless, experimental assays using cytoskeletal probes proved that the polymerised forms of actin and tubulin play an essential role in the S. nematoides movement. Similar to Selenidium archigregarines, the subpellicular microtubules organised in several layers seem to be the leading motor structures in blastogregarine motility. The majority of the detected actin was stabilised in a polymerised form and appeared to be located beneath the inner membrane complex. The experimental data suggest the subpellicular microtubules to be associated with filamentous structures (= cross-linking protein complexes), presumably of actin nature.

The enigma of eugregarine epicytic folds: where gliding motility originates?

BACKGROUND: In the past decades, many studies focused on the cell motility of apicomplexan invasive stages as they represent a potential target for chemotherapeutic intervention. Gregarines (Conoidasida, Gregarinasina) are a heterogeneous group that parasitize invertebrates and urochordates, and are thought to be an early branching lineage of Apicomplexa. As characteristic of apicomplexan zoites, gregarines are covered by a complicated pellicle, consisting of the plasma membrane and the closely apposed inner membrane complex, which is associated with a number of cytoskeletal elements. The cell cortex of eugregarines, the epicyte, is more complicated than that of other apicomplexans, as it forms various superficial structures. RESULTS: The epicyte of the eugregarines, Gregarina cuneata, G. polymorpha and G. steini, analysed in the present study is organised in longitudinal folds covering the entire cell. In mature trophozoites and gamonts, each epicytic fold exhibits similar ectoplasmic structures and is built up from the plasma membrane, inner membrane complex, 12-nm filaments, rippled dense structures and basal lamina. In addition, rib-like myonemes and an ectoplasmic network are frequently observed. Under experimental conditions, eugregarines showed varied speeds and paths of simple linear gliding. In all three species, actin and myosin were associated with the pellicle, and this actomyosin complex appeared to be restricted to the lateral parts of the epicytic folds. Treatment of living gamonts with jasplakinolide and cytochalasin D confirmed that actin actively participates in gregarine gliding. Contributions to gliding of specific subcellular components are discussed. CONCLUSIONS: Cell motility in gregarines and other apicomplexans share features in common, i.e. a three-layered pellicle, an actomyosin complex, and the polymerisation of actin during gliding. Although the general architecture and supramolecular organisation of the pellicle is not correlated with gliding rates of eugregarines, an increase in cytoplasmic mucus concentration is correlated. Furthermore, our data suggest that gregarines utilize several mechanisms of cell motility and that this is influenced by environmental conditions.