Encysted Stages Research Articles

Novel Insights into the Wattle and Daub Model of Entamoeba Cyst Wall Formation and the Importance of Actin Cytoskeleton.

The "Wattle and Daub" model of cyst wall formation in Entamoeba invadens has been used to explain encystment in Entamoeba histolytica, the causal agent of amoebiasis, and this process could be a potential target for new antiamoebic drugs. In this study, we studied the morphological stages of chitin wall formation in E. invadens in more detail using fluorescent chitin-binding dyes and the immunolocalization of cyst wall proteins. It was found that chitin deposition was mainly initiated on the cell surface at a specific point or at different points at the same time. The cystic wall grew outward and gradually covered the entire surface of the cyst over time, following the model of Wattle and Daub. The onset of chitin deposition was guided by the localization of chitin synthase 1 to the plasma membrane, occurring on the basis of the Jacob lectin in the cell membrane. During encystation, F-actin was reorganized into the cortical region within the early stages of encystation and remained intact until the completion of the chitin wall. The disruption of actin polymerization in the cortical region inhibited proper wall formation, producing wall-less cysts or cysts with defective chitin walls, indicating the importance of the cortical actin cytoskeleton for proper cyst wall formation.

NLRP3 Plays a Key Role in Antihelminth Immunity in the Enteral and Parenteral Stages of Trichinella spiralis-Infected Mice.

Trichinellosis is an important foodborne zoonosis, and no effective treatments are yet available. Nod-like receptor (NLR) plays a critical role in the host response against nematodes. Therefore, we aimed to explore the role of the NLRP3 inflammasome (NLRP3) during the adult, migrating, and encysted stages of Trichinella spiralis infection. The mice were treated with the specific NLRP3 inhibitor MCC950 after inoculation with T. spiralis. Then, the role that NLRP3 plays during T. spiralis infection of mice was evaluated using enzyme-linked immunosorbent assay (ELISA), Western blotting, flow cytometry, histopathological evaluation, bone marrow-derived macrophage (BMDM) stimulation, and immunofluorescence. The in vivo results showed that NLRP3 enhanced the Th1 immune response in the adult and migrating stages and weakened the Th2 immune response in the encysted stage. NLRP3 promoted the release of proinflammatory factors (interferon gamma [IFN-γ]) and suppressed the release of anti-inflammatory factors (interleukin 4 [IL-4]). Pathological changes were also improved in the absence of NLRP3 in mice during T. spiralis infection. Importantly, a significant reduction in adult worm burden and muscle larvae burden at 7 and 35 days postinfection was observed in mice treated with the specific NLRP3 inhibitor MCC950. In vitro, we first demonstrated that NLRP3 in macrophages can be activated by T. spiralis proteins and promotes IL-1β and IL-18 release. This study revealed that NLRP3 is involved in the host response to T. spiralis infection and that targeted inhibition of NLRP3 enhanced the Th2 response and accelerated T. spiralis expulsion. These findings may help in the development of protocols for controlling trichinellosis.

Phycotoxins and marine annelids – A global review

Several species of microalgae can produce potent phycotoxins that negatively affect aquatic organisms and their consumers following different exposure routes, as well as toxicokinetic (TK) and toxicodynamic (TD) processes. Benthic organisms are especially vulnerable as they are exposed to both benthic and planktonic species causative of harmful algal blooms (HABs). While benthic algae can come into direct contact with annelids during substrate remobilization, planktonic cells can settle to the bottom mostly during senescence and/or encystment stages, and in shallow and calm waters. We performed a systematic, qualitative review of the literature on the phycotoxin TK and TD processes in marine annelids, summarizing the most relevant findings and general trends. Besides, by using innovative analytical/statistical approaches, we were able to detect patterns and gaps in the current literature, thus pointing to future research directions. We retrieved and analyzed studies involving diarrhetic shellfish toxins (DSTs), paralytic shellfish toxins (PSTs), brevetoxins (PbTXs), domoic acid (DA), as well as palytoxin and its congeners, the ovatoxins (treated together as PLTXs). It is worth mentioning that studies evaluating other phycotoxins (e.g., ciguatoxins, yessotoxins) were not found in the literature. The absence of data on PbTXs, PSTs and DA is the largest gap hampering TK assessment in annelids, although some relevant information on TD is already available. Whereas lethal effects from DSTs have not been reported, more potent toxins like PbTXs, PSTs, DA and those grouped as PLTX-like compounds can cause mortality and/or marked decrease in annelid abundance. In addition, phycotoxins have been linked to sublethal effects on annelid cells. Although very sparse, field and laboratory studies offer strong evidence that annelids may be reliable indicators of toxin exposure and their negative effects during both early and later stages of HABs in marine environments. Besides quickly responding to these compounds at both organismic and suborganismic levels, annelids are easily found in areas affected by HABs. The use of annelids in future investigations evaluating the action mechanisms of toxic microalgae on marine invertebrates should be thus encouraged. In this case, the choice for widely dispersed and numerically dominant species of annelids would strengthen the validation and extrapolation of results from risk assessments in areas affected by HABs worldwide.

The Parasitology Unbound Collective: Commission Report

The Parasitology Unbound Collective:Commission Report David Hollingshead (bio) OPENING REMARKS This interdisciplinary commission was formed to assess and respond to the sudden collapse of the Parasitology Unbound Collective, a research initiative supported by the United States Department of Agriculture (USDA) in partnership with the National Health Association (NHA). The Collective was conceived as a comprehensive three-year program to study the effects of the common brain parasite Toxoplasma gondii on human behavior and psychology. However, the operation was forced to shut down less than two years into its projected timeline for reasons that are still under investigation. The following report was written in affiliation with the A/N/I/M/A/C/I/E/S/ROOM ("A Room for Sciences Studies in Action") at Alberta Regional University. It compiles a record of the events leading up to and including the incident at the Collective's primary testing facility in Beltsville, Maryland, on May 20, 2019, and the contemporaneous disappearance of lead researcher Charles Wilson along with seven (7) live cats infected with toxoplasmosis. This record contains primary documentation, beginning with Dr. Wilson's NHA grant application, and includes correspondences between NHA officials and Wilson, as well as excerpts from private journals and other miscellaneous [End Page 715] documents recovered from the Beltsville facility. In order to secure its publication in ASAP/Journal, the commission has taken the opportunity to provide relevant contextualizing documents, including discursive paratextual notes and additional secondary scholarship, when necessary. The paratextual notes were written by two ARU graduate students who were hired as summer researchers. ________ Dr. Charles V. Wilson Assistant Professor of Parasitology NHA Grant Application November 9th, 2016 Reassessing the behavioral effects of Toxoplasma gondii in humans through integrated "in-house" testing and a revised personality trait taxonomy Introduc on1 Toxoplasma gondii (T. gondii) is one of the most common parasites on the planet. It is a "highly successful Apicomplexan protozoan capable of infecting any warm-blooded animal worldwide" (Khan & Grigg, 2017). Estimates suggest that nearly one-third of the world's human population is chronically infected (Dubey, 2010). Beyond its ubiquity, T. gondii is notable for its peculiar life cycle, which consists of three stages: a non-transmissible stage of rapid multiplication (tachyzoites); a transmissible, encysted stage of slow multiplication (bradyzoite); and a sexual stage that can only occur in the intestine of the feline species (Frenkel, 1973; Frenkel et al., 1970). In this last stage, the infected cat will shed T. gondii oocysts with its feces, which can then be reingested by another animal, thus beginning [End Page 716] the cycle again. T. gondii oocysts are extraordinarily hardy and can endure a wide range of "environmental, physical and chemical insults," including laboratory grade disinfectants and municipal water treatment (Fritz et al., 2012). In order for the parasite to successfully reproduce, its final (or "definitive") host must belong to the feline family. All other hosts, including humans, are designated "intermediate," meaning that they are reproductive "dead ends" for the parasite. Humans are most commonly infected with T. gondii through their contact with domestic cats, but meat contamination and exposure to oocysts through soil and water are also common vectors of infection. Seroprevalence rates in humans diverge widely from country to country, and studies suggest that region-specific differences in agricultural practices, foodways traditions, and cultures of pet-keeping may be determinative factors (Opsteegh et al., 2016; Scallan et al., 2011). For most of its known existence, Toxoplasma gondii and its associate disease, toxoplasmosis, were not considered serious public health risks.2 In humans, certain flu-like symptoms, as well as an increased chance of meningitis and encephalitis in the severely immunocompromised, had been noted upon infection. Serious physiological symptoms during the parasite's latent stage, however, were [End Page 717] believed nonexistent (Sugden et al., 2016). In the 1970s, researchers began to observe that rodents—one of the most common intermediate host-reservoirs of T. gondii—infected with the parasite exhibited significant behavioral changes that rendered them far more susceptible to feline predation. Most notably, the rats ceased to display their species' innate aversion to feline urine. Experiments conducted to test the effects of T. gondii on rats' perception of predation...

Antifreeze Water-Rich Dormant Cysts of the Terrestrial Ciliate Colpoda cucullus Nag-1 at −65 ℃: Possible Involvement of Ultra-Antifreeze Polysaccharides

We found that the water-rich (osmolality below 0.052 Osm/l) wet resting cysts of the soil ciliate Colpoda cucullus Nag-1 were tolerant to extremely low temperature (−65℃). When cell fluid obtained from the resting cysts was cooled at −65℃, small particles of ice crystals did not grow into large ice crystals. At −65℃, the cysts shrank due to an outflow of water, because a vapor pressure difference was produced between the cell interior and freezing surrounding medium. The osmolality of these shrunk cells was estimated 0.55 Osm/l, and the freezing point depression of the shrunk cell fluid was estimated to be 1.02℃. Hence, the antifreeze ability of wet cysts at −65℃can not be explained by freezing point depression due to elevation of cytoplasmic osmolality. The cytoplasm of resting cysts was vividly stained red with periodic acid-Schiff (PAS) and stained purple with toluidine blue. On the other hand, the excystment-induced cysts were not stained with PAS, and exhibited a loss of the antifreeze activity. PAS staining of SDSPAGE gel obtained from encysting Colpoda cells showed that a large amount of PAS-positive macromolecules accumulated as the encystment stage progressed. These results suggest that antifreeze polysaccharides may be involved in the antifreeze activity of C. cucullus Nag-1 dormant forms.

Broad North Atlantic distribution of a meiobenthic annelid – against all odds

DNA barcoding and population genetic studies have revealed an unforeseen hidden diversity of cryptic species among microscopic marine benthos, otherwise exhibiting highly similar and simple morphologies. This has led to a paradigm shift, rejecting cosmopolitism of marine meiofauna until genetically proven and challenging the “Everything is Everywhere, but the environment selects” hypothesis that claims ubiquitous distribution of microscopic organisms. With phylogenetic and species delimitation analyses of worldwide genetic samples of the meiofaunal family Dinophilidae (Annelida) we here resolve three genera within the family and showcase an exceptionally broad, boreal, North Atlantic distribution of a single microscopic marine species with no obvious means of dispersal besides vicariance. With its endobenthic lifestyle, small size, limited migratory powers and lack of pelagic larvae, the broad distribution of Dinophilus vorticoides seems to constitute a “meiofaunal paradox”. This species feasts in the biofilm among sand grains, but also on macroalgae and ice within which it can likely survive long-distance rafting dispersal due to its varying lifecycle stages; eggs encapsulated in cocoons and dormant encystment stages. Though often neglected and possibly underestimated among marine microscopic species, dormancy may be a highly significant factor for explaining wide distribution patterns and a key to solving this meiofaunal paradox.

Microscopy and phylogeny of Pyramimonas tatianae sp. nov. (Pyramimonadales, Chlorophyta), a scaly quadriflagellate from Golden Horn Bay (eastern Russia) and formal description of Pyramimonadophyceae classis nova

ABSTRACT Nearly two decades ago a scaly quadriflagellate culture was established from a sample collected in Golden Horn Bay, eastern Russia. Here we present a comparative analysis of pheno- and genotypic characters and show that the isolate did not match any existing species of Pyramimonas and is therefore described as P. tatianae sp. nov. The species was probably identified as P. aff. cordata in previous studies on material from the same area. Based on an ultrastructural account of the cell and its external body scales it was found to belong to the subgenus Vestigifera. This was supported by a phylogenetic analysis using the chloroplast-encoded rbcL gene. The cell dimensions of P. tatianae were 6–7 µm long and 5–6 µm wide and it thus represented one of the smaller species of the genus. The cup-shaped chloroplast was divided into four lobes reaching from the middle to the anterior part of the cell. A single posterior eyespot was observed adjacent to an excentric pyrenoid. The ultrastructure of the body and flagellar scales were illustrated from material prepared for whole mounts and thin sections. The new species was compared with P. cordata and P. mitra, two other vestigiferans with which it shares some morphological features. The sequence divergence between P. tatianae and the most closely related species, P. mitra, was 2.5%. The phylogeny of Vestigifera revealed two lineages, one comprising cold-water species and the cosmopolitan P. orientalis, and the other species from temperate-subtropical waters. The tree topology suggested a southbound dispersal route. This was further supported by all antarctic Pyramimonas species having encystment stages as part of their life cycle. It was therefore probable that their ancestors also were capable of producing cysts allowing transportation over great distances. A formal description of the class Pyramimonadophyceae comprising both extant and extinct species was also provided.

Galactose as novel target against Acanthamoeba cysts.

Galactose as novel target against Acanthamoeba cysts.

Black-spot syndrome in Caribbean fishes linked to trematode parasite infection (Scaphanocephalus expansus)

Despite the evidence that diseases have increased in marine taxa, parasites remain underrepresented in studies of marine ecology. Recently, observations of black-spot syndrome (BSS) in Caribbean fishes, especially ocean surgeonfishes, Acanthurus tractus (Poey, 1860), have been reported, although its cause(s) has remained conjectural. We investigated the etiology of BSS and whether the pathology was functionally and consistently associated with particular infections. By examining the patterns of BSS among reef fishes in Bonaire, we show that observed dermal spots are the encysted stages (metacercariae) of the heterophyid trematode Scaphanocephalus expansus (Creplin, 1842). Metacercariae were detected within ten species of fishes, with loads ranging from 1–315 per fish. These represent the first published accounts of the genus Scaphanocephalus in the Caribbean and are all new host records. Molecular analysis of sequences identified the infection as a heterophyid, while morphological examinations and comparisons with archival specimens confirmed the species identity as S. expansus. Field-based estimates of BSS by scuba divers and subsequent video analysis correlated positively with parasite counts from necropsy. No such correlation was observed between BSS and metacercariae of a second trematode genus, Bucephalus. The consistent link between host burdens of S. expansus and BSS highlights the potential for low-cost and low-impact assessments in fish populations, including the use of images from citizen scientists and public databases. Taken together, these results help establish the foundation for future investigations into the pathology, geographic distribution, life cycle, and ecological consequences of BSS.

A model for the dynamics of the parasitic stages of equine cyathostomins

A model was developed to reproduce the dynamics of the parasitic stages of equine cyathostomins. Based on a detailed review of published literature, a deterministic simulation model was constructed using the escalator boxcar-train approach, which allows for fully-overlapping cohorts of worms and approximately normally distributed variations in age/size classes. Key biological features include a declining establishment of ingested infective stage larvae as horses age. Development rates are constant for all the parasitic stages except the encysted early third stage larvae, for which development rates are variable to reflect the sometimes extended arrestment of this stage. For these, development is slowed in the presence of adult worms in the intestinal lumen, and when ingestion of infective larvae on herbage is high or extended. In the absence of anthelmintic treatments, the life span of adult worms is approximately 12 months, and the presence of an established adult worm burden largely blocks the transition of luminal fourth stage larvae to the adult stage, resulting in mortality of the larvae. This inhibition is removed by effective anthelmintic treatment allowing the rapid replacement of adult worms from the pool of mucosal stages.Within the model, the rate and seasonality at which infective stage larvae are ingested strongly influences the dynamics of the pre-adult stages. While the adult worm burden remains relatively stable within a year, due to the negative feedback they have on developing stages, the numbers and proportions of larval stages relative to the total worm burden increase with the numbers of infective larvae ingested. Further, within the model, the seasonal rise and fall of encysted stages is largely driven by the seasonal pattern of infective larvae on pasture. Because of this, the model reproduces the contrasting seasonal patterns of mucosal larvae, typical of temperate and tropical environments, using only the appropriate seasonality of larvae on pasture. Thus, the model reproduces output typical of different climatic regions and suggests that observed patterns of arrested development may simply reflect the numbers and seasonality of free-living stages on pasture as determined by different management practices and weather patterns.

Transcriptome Analysis Reveals the Molecular Mechanism of Resting Cyst Formation in Colpoda aspera.

Resting cyst formation is a remarkable survival strategy used by ciliates in response to the adverse environmental conditions. However, the mechanisms underlying encystment are poorly understood. Here, the genetic basis of encystment in Colpoda aspera was examined through RNA sequencing to identify transcriptome-wide changes in gene expression between vegetative and encystment stages. After de novo assembly, 49,543 transcripts were identified. Gene annotation and pathway mapping analysis revealed marked changes in biosynthesis, energy metabolism, and autophagy pathways during cyst formation. In addition, some differentially regulated genes were predicted to function in the interconnected cAMP, AMPK, mTOR, and PI3K/AKT signaling pathways, potentially forming a regulatory network for encystment. The present study conducted a large-scale assessment of Colpoda aspera genomic resources and provides new insight into the molecular mechanisms underlying cyst formation.

Mitochondria fightToxoplasmafor fat

Cell Biology![Figure][1] Toxoplasma parasites (blue), shown here in their encysted stage within liver tissue, compete with mitochondria for lipids. CREDIT: MOREDUN SCIENTIFIC LTD./SCIENCE SOURCE Mitochondria provide platforms for innate immunity. The intracellular parasite Toxoplasma gondii

Resting Cysts of the Pigmented Ciliate Blepharisma sinuosum Sawaya, 1940 (Ciliophora: Heterotrichea)

The morphology of Blepharisma sinuosum resting cysts and the dynamics of pigmentation at different stages of encystment are presented for the first time. Cyst morphometrics are similar to other Blepharisma species, with three-wall layers, bacteria surrounding the ectocyst, a conical plug, and wrinkly surface toward the plug in mature stages. The vegetative moniliform macronucleus changes to a horseshoe shape, and at early stages, the cystic cytoplasm is homogeneously pigmented, comprising a contractile vacuole; later, pigments polarize toward the plug, decorate the cortical layer, and become brownish. This work reinforces the potential role of pigment dynamics on cyst biology.

Surgical Management of Pyothorax: A Series of 172 Cases

Introduction: The pyothorax or thoracic empyema is defined by the presence between the two pleural layers of a purulent liquid, or a non-purulent liquid, but with bacteriological or biochemical characteristics testifying to a microbial invasion. The use of early medical treatment prevents the passage to pleural encystment, whose management remains surgical. Material and Methods: Our study was performed retrospectively, in the department of thoracic surgery of CHU Hassan II of Fez, between 2010 and 2016, involving 172 patients operated for pyothorax. Results: Among the 172 patients included in our study, there were 110 men and 67 women. The average age was 32.11 years old. The clinical picture was dominated by pleural syndrome in 56% of cases, signs of tuberculous impregnation in 44.1% of cases. Preoperative preparation with thoracic drainage, bi-antibiotic therapy and respiratory physiotherapy was performed in 90% of cases. The attack was right in 55.8% of cases and left in 44.1%. A chest CT scan performed in all patients showed pachypleuritis in all cases. The tuberculous pyothorax accounted for 54% of cases, those by intra pleural rupture of a pulmonary hydatid cyst in 10.4%, para-pneumonic origin in 5.2% and post-traumatic in 4.06%. The origin was undetermined in 26.16%. A conservative posterolateral thoracotomy was performed in all our patients. The pulmonary release was done through the extrapleural plane in 94% of cases, and pleuropulmonary decortication performed in all cases. Atypical resection was associated in 4.3% of cases and peri-cytectomy in 13.9% of cases. Operative follow-up was simple in 79.6% of cases. The main postoperative complications were a prolonged aerial leak in 16.7%, atelectasis in 4.5%, a wall infection in 6.5%, a hemothorax refrained in 3.2% of cases. The average follow-up was 2.5 years. Conclusion: Tuberculosis remains the most common etiology of pyothorax in our setting. Early management plays an important role in reducing the morbidity and mortality of this pathology. However, surgery remains the only effective treatment at the pleural encystment stage.

A Puf RNA-binding protein encoding gene PlM90 regulates the sexual and asexual life stages of the litchi downy blight pathogen Peronophythora litchii

Sexual and asexual reproduction are two key processes in the pathogenic cycle of many filamentous pathogens. However in Peronophythora litchii, the causal pathogen for the litchi downy blight disease, critical regulator(s) of sexual or asexual differentiation has not been elucidated. In this study, we cloned a gene named PlM90 from P. litchii, which encodes a putative Puf RNA-binding protein. We found that PlM90 was highly expressed during asexual development, and much higher than that during sexual development, while relatively lower during cyst germination and plant infection. By polyethylene glycol (PEG)-mediated protoplast transformation, we generated three PlM90-silenced transformants and found a severely impaired ability in sexual spore production and a delay in stages of zoospore release and encystment. However, the pathogenicity of P. litchii was not affected by PlM90-silencing. Therefore we conclude that PlM90 specifically regulates the sexual and asexual differentiation of P. litchii.

Local and systemic inflammatory and immunologic reactions to cyathostomin larvicidal therapy in horses

Encysted cyathostomin larvae are ubiquitous in grazing horses. Arrested development occurs in this population and can lead to an accumulation of encysted larvae. Large numbers of tissue larvae place the horse at risk for developing larval cyathostominosis. This disease complex is caused by mass emergence of these larvae and is characterized by a generalized acute typhlocolitis and manifests itself as a profuse protein-losing watery diarrhea with a reported case-fatality rate of about 50%. Two anthelmintic formulations have a label claim for larvicidal therapy of these encysted stages; moxidectin and a five-day regimen of fenbendazole. There is limited knowledge about inflammatory and immunologic reactions to larvicidal therapy. This study was designed to evaluate blood acute phase reactants as well as gene expression of pro-inflammatory cytokines, both locally in the large intestinal walls and systemically. Further, mucosal tissue samples were evaluated histopathologically as well as analyzed for gene expression of pro- and anti-inflammatory cytokines, cluster of differentiation (CD) cell surface proteins, and select transcription factors. Eighteen juvenile horses with naturally acquired cyathostomin infections were randomly assigned to three treatment groups; one group served as untreated controls (Group 1), one received a five-day regimen of fenbendazole (10mg/kg) (Group 2), and one group received moxidectin (0.4mg/kg) (Group 3). Horses were treated on day 0 and euthanatized on days 18–20. Serum and whole blood samples were collected on days 0, 5, and 18. All horses underwent necropsy with collection of tissue samples from the ventral colon and cecum. Acute phase reactants measured included serum amyloid A, iron and fibrinogen, and the cytokines evaluated included interferon γ, tumor necrosis factor α, transforming growth factor (TGF)-β, and interleukins 1β, 4, 5, 6, and 10. Transcription factors evaluated were FoxP3, GATA3 and tBet, and CD markers included CD163, CD3z, CD4, CD40, and CD8b. Histopathology revealed an inflammatory reaction with higher levels of lymphocytes, T cells, B cells, eosinophils and fibrous tissue in the moxidectin-treated group compared to controls or horses treated with fenbendazole. No apparent systemic reactions were observed. Expression of IL-5 and TGF-β in intestinal tissues was significantly lower in Group 3 compared to Group 1. This study revealed a subtle inflammatory reaction to moxidectin, which is unlikely to cause clinical issues.

Dracomyxa pallida gen. et sp. nov.: A New Giant Freshwater Foraminifer, with Remarks on the Taxon Reticulomyxidae (emend.)

Reticulomyxids are organotrophic freshwater foraminifers typically placed within the radiation of early monothalamous Foraminifera. Though ubiquitous, reticulomyxids are rarely reported, which may be due both to their concealed life style and to inappropriate isolation techniques. Their taxonomic position is largely based on developmental stages of the life cycle. Here, we describe a new freshwater species, Dracomyxa pallida gen. nov. et sp. nov., isolated from submersed plant material. It has two possible life cycles including small cells, large plasmodia and encysted stages. Both types of development occur simultaneously in cultures derived from a single cell as well as in crude cultures. We amplified and sequenced a short sequence fragment of the 18S rRNA gene of the new isolate. Comparative phylogenetic analysis and molecular characterization indicate that it is a new reticulomyxid species, with Reticulomyxa filosa Nauss, Haplomyxa saranae Dellinger and Wobo gigas Hülsmann as the closest relatives. Unique features such as tripodal pediculated cysts and the lack of cleansing process justify the erection of a new genus.

Ribosomal RNA and protein transcripts persist in the cysts of Entamoeba invadens

In most organisms rDNA transcription ceases under conditions of growth stress. However, we have earlier shown that pre-rRNA accumulates during encystation in Entamoeba invadens. We labeled newly-synthesized rRNA during encystation, with [methyl-3H] methionine in the presence of chitinase to enable uptake of isotope. Incorporation rate reduced after 24h, and then increased to reach levels comparable with normal cells. The label was rapidly chased to the ribosomal pellet in dividing cells, while at late stages of encystation the ratio of counts going to the pellet dropped 3-fold. The transcript levels of selected ribosomal protein genes also went down initially but went up again at later stages of encystation. This suggested that rRNA and ribosomal protein transcription may be coordinately regulated. Our data shows that encysting E. invadens cells accumulate transcripts of both the RNA and protein components of the ribosome, which may ensure rapid synthesis of new ribosomes when growth resumes.

The Proteome Landscape of Giardia lamblia Encystation

Giardia lamblia is an intestinal protozoan parasite required to survive in the environment in order to be transmitted to a new host. To ensure parasite survival, flagellated trophozoites colonizing the small intestine differentiate into non-motile environmentally-resistant cysts which are then shed in the environment. This cell differentiation process called encystation is characterized by significant morphological remodeling which includes secretion of large amounts of cyst wall material. Although much is known about the transcriptional regulation of encystation and the synthesis and trafficking of cyst wall material, the investigation of global changes in protein content and abundance during G. lamblia encystation is still unaddressed.In this study, we report on the quantitative analysis of the G. lamblia proteome during encystation using tandem mass spectrometry. Quantification of more than 1000 proteins revealed major changes in protein abundance in early, mid and late encystation, notably in constitutive secretory protein trafficking. Early stages of encystation were marked by a striking decrease of endoplasmic reticulum-targeted variant-specific surface proteins and significant increases in cytoskeleton regulatory components, NEK protein kinases and proteins involved in protein folding and glycolysis. This was in stark contrast to cells in the later stages of encystation which presented a surprisingly similar proteome composition to non-encysting trophozoites. Altogether these data constitute the first quantitative atlas of the Giardia proteome covering the whole process of encystation and point towards an important role for post-transcriptional control of gene expression in Giardia differentiation. Furthermore, our data provide a valuable resource for the community-based annotation effort of the G. lamblia genome, where almost 70% of all predicted gene models remains “hypothetical”.

Anthelmintic resistance in equine parasites—Current evidence and knowledge gaps

Anthelmintic resistance is becoming increasingly prevalent among equine nematode parasites. The first reports documenting resistance were published in the 1960s, just a short time after introduction of the first modern anthelmintics phenothiazine and thiabendazole. Several factors are known to influence development of resistance, but evidence specific to equine parasites is limited. Most current knowledge and applications have been extrapolated from research with trichostrongylid parasites of sheep. The number of cyathostomin species co-infecting horses adds to the complexity of investigating drug resistance but, given their apparent limited biological diversity, viewing these in a unispecific context remains a pragmatic approach. Factors affecting resistance development in cyathostomins include parasite seasonality, life span and fecundity, host immunity, and the existence of encysted stages. Further, parasite refugia have been shown to play a vital role in resistance development in other parasites, and likely is also important in equine parasites. Specific genetic factors for drug resistance and possible modes of inheritance have been identified for trichostrongylid nematodes, but it is widely accepted that several more remain undiscovered. Current evidence with equine and ruminant parasites suggests that fitness is not significantly compromised in drug resistant strains. Attempts to develop in vitro and molecular assays for diagnosing anthelmintic resistance in equine nematodes have had only limited success, standardized guidelines are sorely needed for performing the fecal egg count reduction test in horse populations. Taken together, this review illustrates the complexity of understanding anthelmintic resistance in equine nematodes, and emphasizes the need for further research.