Dinámica de la evolución de las formas de Blastocystis spp. en un medio de cultivo simple / Dynamics of the evolution of the forms of Blastocystis spp. in a simple culture medium

Resumen Blastocystis spp. es un parásito muy frecuente en materia fecal humana, pero la naturaleza polimórfica y el número de Blastocystis en la muestra pueden complicar su detección por microscopía. El objetivo del trabajo fue describir la dinámica de los morfotipos de Blastocystis a corto plazo en un medio de cultivo simple y determinar su aplicabilidad para utilizarlo como complemento del análisis coproparasitológico y para estudios morfológicos, bioquímicos y moleculares del parásito. Se sembraron 10 muestras de materia fecal con Blastocystis en un medio Pavlova adaptado, se examinaron diariamente por examen microscópico durante 6 días y se registraron las formas y el recuento. El desarrollo fue regular y abundante y las formas fueron de tamaños variables y claramente identificables. El cultivo ensayado puede ser útil para la detección de Blastocystis cuando existan dudas diagnósticas por microscopía, para estudios de sensibilidad y especificidad diagnóstica o cuando se requiera aumentar la carga para realizar otros estudios.

Abstract Blastocystis spp. is a very frequent parasite in human fecal matter, but the polymorphic nature and the number of Blastocystis in a sample can complicate its detection by microscopy. The objective of the present work was to describe the dynamics of Blastocystis morphotypes in the short term in a simple culture medium and to determine its applicability to use it as a complement to coproparasitological analysis and for morphological, biochemical and molecular studies of the parasite. Ten stool samples with Blastocystis were cultured in an adapted Pavlova medium and examined during 6 days by microscopy to record the forms and the count. The development was regular and abundant and the shapes were of variable sizes and clearly identifiable. The tested culture could be used for the detection of Blastocystis when microscopic diagnosis is dubious, for studies of diagnostic sensitivity and specificity or when it is necessary to increase the load to perform other studies.

Resumo Blastocystis spp. é um parasita muito frequente nas fezes humanas, mas a natureza polimórfica e o número de Blastocystis na amostra podem complicar a sua detecção através do microscópio. O objetivo do trabalho foi descrever a dinâmica dos morfotipos de Blastocystis no curto prazo em um meio de cultura simples e determinar sua aplicabilidade para ser utilizado como complemento da análise coproparasitológica e para estudos morfológicos, bioquímicos e moleculares do parasita. Foram semeadas dez amostras de fezes com Blastocystis em um meio Pavlova adaptado e examinadas diariamente através de exame microscópico durante 6 dias, registrando as formas e fazendo recontagem. O desenvolvimento foi regular e abundante e as formas foram de tamanhos variáveis e claramente identificáveis. A cultura testada pode ser útil para a detecção de Blastocystis quando houver dúvidas diagnósticas por microscopia; para estudos de sensibilidade e especificidade diagnóstica ou quando for necessário aumentar a carga para a realização de outros estudos.

Interactions between a pathogenic Blastocystis subtype and gut microbiota: in vitro and in vivo studies.

BACKGROUND: Blastocystis is a common gut eukaryote detected in humans and animals. It has been associated with gastrointestinal disease in the past although recent metagenomic studies also suggest that it is a member of normal microbiota. This study investigates interactions between pathogenic human isolates belonging to Blastocystis subtype 7 (ST7) and bacterial representatives of the gut microbiota. RESULTS: Generally, Blastocystis ST7 exerts a positive effect on the viability of representative gut bacteria except on Bifidobacterium longum. Gene expression analysis and flow cytometry indicate that the bacterium may be undergoing oxidative stress in the presence of Blastocystis. In vitro assays demonstrate that Blastocystis-induced host responses are able to decrease Bifidobacterium counts. Mice infected with Blastocystis also reveal a decrease in beneficial bacteria Bifidobacterium and Lactobacillus. CONCLUSIONS: This study shows that particular isolates of Blastocystis ST7 cause changes in microbiota populations and potentially lead to an imbalance of the gut microbiota. This study suggests that certain isolates of Blastocystis exert their pathogenic effects through disruption of the gut microbiota and provides a counterpoint to the increasing reports indicating the commensal nature of this ubiquitous parasite.

Resistance towards metronidazole in Blastocystis sp.: A pathogenic consequence.

Blastocsytis sp. is a protozoan parasite that has been linked to common gastrointestinal illnesses. Metronidazole, the first line therapy, was reported to show frequent inefficacy. Previously, Blastocystis sp. isolated from different population showed varying metronidazole resistance. However, the effect of metronidazole treatment on pathogenic potentials of Blastocystis sp. isolated from different populations, which is known to have different gut environment, is unclear. This study investigates the in vitro effect of metronidazole on the pathogenic potentials of Blastocystis sp. isolated from urban and orang asli individuals. Blastocystis sp. ST 3 isolated from symptomatic and asymptomatic individuals were treated with a range of metronidazole concentration. The parasites' growth characteristics, apoptotic rate, specific protease activity and the ability to proliferate cancer cells were analyzed upon treatment with 0.001 mg/l metronidazole. The study demonstrates that Blastocystis sp. isolates showed increase in the parasite numbers especially the amoebic forms (only in urban isolates) after treating with metronidazole at the concentration of 0.001 mg/ml. High number of cells in post-treated isolates coincided with increase of apoptosis. There was a significant increase in cysteine protease of Blastocystis sp. isolates upon treatment despite the initial predominance of serine protease in asymptomatic isolates. Metronidazole resistant Blastocystis sp. also showed significant increase in cancer cell proliferation. Resistance to metronidazole did not show significant different influence on the pathogenicity between Blastocystis sp. isolated from urban and orang asli individual. However, an increase in parasite numbers, higher amoebic forms, cysteine protease and ability to proliferate cancer cells implicates a pathogenic role. The study provides evidence for the first time, the effect of metronidazole towards enhancing pathogenic potentials in Blastocystis sp. when isolated from different gut environment. This necessitates the need for reassessment of metronidazole treatment modalities.

Establishing a protocol for water sample processing for the detection of Blastocystis sp.

This study was aimed at establishing a protocol for water sample processing for the detection of Blastocystis sp. using distilled water spiked with Blastocystis sp. cysts. The study established a protocol involving eight technical aspects, namely, storage temperature, storage duration, minimum water sample volume, optimum relative centrifugal force, centrifugation duration, minimum number of cyst for inoculation in Jones' medium and turn-around-time for the detection of vacuolar forms of Blastocystis sp. Results showed a minimum of 1.0 L water sample should be collected and processed on the same day. Otherwise, it should be stored at 4 °C and processed within 3 days. Water sample should be centrifuged at 1400×g for 10 min. For the isolation of Blastocystis sp. cysts, parasite pellet could be layered on top of Ficoll-Paque™ PLUS, centrifuged at 1400×g for 20 min and washed twice using 0.9% saline with centrifugation at 1400×g for 10 min. A minimum of 1 × 105 cysts could then be inoculated in Jones' medium supplement with 10% horse serum, incubated at 37 °C and examined for any presence of vacuolar forms of Blastocystis sp. after 3 days of inoculation. A protocol for water sample processing for the detection of Blastocystis sp. has successfully been established. The protocol was validated using 106 various water samples. This protocol will be very useful in determining the extent of Blastocystis sp. contamination in water sources in order to identify the seriousness of contamination.

The Human Gut Colonizer Blastocystis Respires Using Complex II and Alternative Oxidase to Buffer Transient Oxygen Fluctuations in the Gut.

Blastocystis is the most common eukaryotic microbe in the human gut. It is linked to irritable bowel syndrome (IBS), but its role in disease has been contested considering its widespread nature. This organism is well-adapted to its anoxic niche and lacks typical eukaryotic features, such as a cytochrome-driven mitochondrial electron transport. Although generally considered a strict or obligate anaerobe, its genome encodes an alternative oxidase. Alternative oxidases are energetically wasteful enzymes as they are non-protonmotive and energy is liberated in heat, but they are considered to be involved in oxidative stress protective mechanisms. Our results demonstrate that the Blastocystis cells themselves respire oxygen via this alternative oxidase thereby casting doubt on its strict anaerobic nature. Inhibition experiments using alternative oxidase and Complex II specific inhibitors clearly demonstrate their role in cellular respiration. We postulate that the alternative oxidase in Blastocystis is used to buffer transient oxygen fluctuations in the gut and that it likely is a common colonizer of the human gut and not causally involved in IBS. Additionally the alternative oxidase could act as a protective mechanism in a dysbiotic gut and thereby explain the absence of Blastocystis in established IBS environments.

Evaluating rodent experimental models for studies of Blastocystis ST1.

Blastocystis is a common inhabitant of the human gut, colonizing at least one billion people at a prevalence ranging from <10% to 100% in healthy human populations globally. The majority of carriers remain asymptomatic, suggesting that Blastocystis is largely a commensal, though Blastocystis has also been implicated in disease in some people. However, there are no in vivo model systems in which to experimentally test the impact of Blastocystis on mammalian hosts and the gut ecosystem and determine which factors underlie these variable clinical outcomes. We evaluated a rat model for sustaining of a human-derived Blastocystis ST1 and assess colonization success and longevity. Because of the broad host range of Blastocystis, we compared the rat with three other rodent species to establish the reproducibility of our method. Blastocystis was introduced by esophageal gavage and colonization success evaluated by Blastocystis culture. Culture was also used to determine that all animals were negative prior to colonization and negative controls remain Blastocystis-free. In this study, Blastocystis ST1 established in 100% of the outbred rats (Rattus norvegicus) and gerbils (Meriones unguiculatus) challenged. Rats were colonized asymptomatically for more than one year, but Blastocystis ST1 was not transmitted between rats. Mus musculus strain CD1 and Mastomys coucha were not susceptible to Blastocystis ST1. Thus, rats appear to be a suitable in vivo model for studies of Blastocystis ST1, as do gerbils though testing was less extensive. This work lays the foundation for experimental work on the role of Blastocystis in health and disease.

Viability Screen of LOPAC1280 Reveals Phosphorylation Inhibitor Auranofin as a Potent Inhibitor of Blastocystis Subtype 1, 4, and 7 Isolates.

Blastocystis is an enteric parasite with extensive global prevalence. Studies have linked infection with this protist with a variety of gastrointestinal disorders, including irritable bowel syndrome. Due to the polymorphic nature of Blastocystis, studies on the parasite could be complicated, as results can be easily misinterpreted. Metronidazole is the commonly prescribed drug for Blastocystis infection, although there have been increasing reports of drug resistance. Hence, there is a need to identify alternative drugs to eliminate Blastocystis infection. In this study, LOPAC1280 was screened and drugs that can decrease the viability of three Blastocystis isolates in cultures were identified. Using apoptosis assay and imaging flow cytometry, phenotypic changes in Blastocystis cells after treatment were also analyzed to obtain insights into the possible mechanism of action of these drugs. Three drugs-diphenyleneiodonium chloride, auranofin, and BIX 01294 trihydrochloride hydrate-were effective against all three isolates tested. Repurposing of these drugs for Blastocystis treatment could be a way of combating metronidazole resistance relatively quickly and at a lower cost.

Presence of Parasites in Environmental Waters in Samsun and Its Districts.

OBJECTIVE: The aim of this study was to detect the presence of parasites in environmental waters in Samsun and its districts. METHODS: At the center of Samsun, 13 stations were determined. The research was performed between March 2012 and February 2013, and every month, water samples were collected on the dates stated. The samples were stained with Kinyoun acid-fast, modified trichrome, and trichrome dyes after examining with the direct bond. The preparations were evaluated in terms of parasitologic under a light microscope. RESULTS: Totally, 180 of 228 water samples analyzed were from streams; of these, 48 were drinking water samples. The following were found: 142 Giardia spp., 132 Cryptosporidium spp., 56 Cyclospora spp., 38 microsporidia, 47 Blastocystis spp., 38 Entamoeba coli cysts, 18 Dientamoeba, 9 Chilomastix, 9 Strongyloides spp., and 6 hookworms. CONCLUSION: The widespread use of animal husbandry and agriculture in the region and the use of stream surroundings as a grazing area increase the presence of some determined protozoa during a certain period. Parasitological studies in humans and animals in the region should be conducted, and control programs should be applied.

Blastocystis: Isolation, Xenic Cultivation, and Cryopreservation.

Blastocystis is an intestinal parasite that is very easily isolated in culture from fresh stool samples. In fact, the parasite grows so readily in culture that short-term in vitro culture is sometimes used as a diagnostic tool in the absence of DNA-based methods. While axenizing Blastocystis cultures remains a significant challenge, the parasite can be propagated for several months in the presence of metabolically active bacteria (xenic culture). Hence, culture can be used for maintaining live Blastocystis strain libraries. This enables the production of a stable resource of reference material, which for instance can be used for DNA-based assays and research. Blastocystis isolates can also be cryopreserved with a view to reestablishing them in culture. Here, we provide protocols for xenic in vitro culture and cryopreservation of Blastocystis. © 2016 by John Wiley & Sons, Inc.

Colonization with the enteric protozoa Blastocystis is associated with increased diversity of human gut bacterial microbiota.

Alterations in the composition of commensal bacterial populations, a phenomenon known as dysbiosis, are linked to multiple gastrointestinal disorders, such as inflammatory bowel disease and irritable bowel syndrome, or to infections by diverse enteric pathogens. Blastocystis is one of the most common single-celled eukaryotes detected in human faecal samples. However, the clinical significance of this widespread colonization remains unclear, and its pathogenic potential is controversial. To address the issue of Blastocystis pathogenicity, we investigated the impact of colonization by this protist on the composition of the human gut microbiota. For that purpose, we conducted a cross-sectional study including 48 Blastocystis-colonized patients and 48 Blastocystis-free subjects and performed an Ion Torrent 16S rDNA gene sequencing to decipher the Blastocystis-associated gut microbiota. Here, we report a higher bacterial diversity in faecal microbiota of Blastocystis colonized patients, a higher abundance of Clostridia as well as a lower abundance of Enterobacteriaceae. Our results contribute to suggesting that Blastocystis colonization is usually associated with a healthy gut microbiota, rather than with gut dysbiosis generally observed in metabolic or infectious inflammatory diseases of the lower gastrointestinal tract.

[Morphological identification of blastocystis].

The authors have attempted to systematize the currently known specific morphological features of the composition of Blastocystis spp. existing in different forms and to present this material as a reference table, by understanding the need for further data clarification. In addition, the paper describes observations of variations in the forms of human blastocysts. In particular, it depicts the species of multinucleated cysts, the identification of which may cause difficulties in diagnosing and differentiating these forms with some representative species of the genus Entamoeba.

Blastocystis tropism in the pig intestine.

Blastocystis has been reported in pig feces but the sites of development in the gastrointestinal tract are unknown. The present study was undertaken to determine predilection sites of Blastocystis in 11 naturally infected pigs examined at 20 weeks of age. At necropsy, feces and contents of the duodenum, jejunum, ileum, and cecum were examined by immunofluorescence (IFA) microscopy and PCR and tissues from these sites as well as the proximal and distal colon were processed for histology from pigs 1 to 5. Feces were examined by IFA microscopy, and segments from the jejunum and ileum were processed for histology from pigs 6 to 11. Multiple sections were cut from each tissue segment, and each was stained with the following: hematoxylin and eosin, polyclonal rabbit antibody to Blastocystis, and ParaFlor B monoclonal antibody to Blastocystis. Blastocystis was detected in feces of all 11 pigs by IFA microscopy and determined by PCR and gene sequencing to be subtype 5 for pigs 1-5. Blastocystis was also detected in the lumen contents removed from the cecum of pigs 1-5 examined by IFA microscopy and in the cecum of pigs 4 and 5 by PCR. Blastocystis was also observed in tissue sections from the jejunum of 7 of the 11 pigs, in the proximal and distal colon of pigs 1-5, and in the cecum of 4 of these 5 pigs but was not detected in the duodenum or ileum of any pigs. In tissue sections, Blastocystis was found primarily in the lumen usually associated with digested food debris, sometimes in close proximity or appearing to adhere to the epithelium, but no stages were found to penetrate the epithelium or the lamina propria.

Establishing mono-eukaryotic Histomonas meleagridis cultures from in vivo infection contaminated with Tetratrichomonas gallinarum and Blastocystis spp.

SUMMARY The necessity to easily establish Histomonas meleagridis cultures has been underlined extensively by many researchers in order to gain more insights in the biology of H. meleagridis. In addition the occurrence of different protozoa in the caeca of birds impedes, however, the isolation and propagation of H. meleagridis from field outbreaks. Therefore, in a kinetic study using transmission electron microscopy the deleterious effects of adventitious protozoa including Tetratrichomonas gallinarum and Blastocystis spp. on cultured H. meleagridis were examined. To overcome this issue, an easy and successful approach to establish the mono-eukaryotic H. meleagridis culture free of other host's protozoa is proposed. At 10 days post infection, liver lesions of H. meleagridis-infected birds were isolated and inoculated into culture media pre-incubated with caecal bacteria. After 48 h of incubation, presence of H. meleagridis in the cultures was confirmed through morphological evaluation. Additionally, TEM examination and analysis by PCR amplification of the small subunit rRNA gene could exclude the co-cultivation of T. gallinarum and Blastocystis spp. Furthermore, after successful propagation and maintenance of the cultured H. meleagridis, its pathogenicity was affirmed in an infection experiment in turkeys.

[Blastocystis spp. culturing as a method for diagnosing the parasite].

The technique for isolating blastocysts in the culture was assessed, by examining the fresh specimens from 196 patients with gastrointestinal tract (GIT) disorders and from 56 persons with an uncertain protozoological diagnosis. A set of techniques, such as triple examination of native smears, parallel inoculation of the Pavlova medium, and microscopy of obtained permanent specimens as may be required, contributes to the timely identification of protozoa, including blastocysts, in persons with GIT disorder. The efficiency of the technique for isolating blastocysts on Pavlova's medium and the native smear test averaged 34.8% and 49.4%, respectively. Among those known to be infected, the rate of parasite development in vitro was not greater than 52.9%. The culture showed amoeboids generally with the clinical manifestation of infection. The culture-based diagnostic method cannot be changed for routine microscopy techniques in detecting blastocysts, although it is essential to investigate the morphofunctional and phylogenetic properties of the parasite. The pleomorphism of cultured blastocysts with variability of growth properties was shown as a manifestation of phenotypic characteristics when isolating the parasite.

Amoebic forms of Blastocystis spp. - evidence for a pathogenic role.

BACKGROUND: Blastocystis spp. are one of the most prevalent parasites isolated from patients suffering from diarrhea, flatulence, constipation and vomiting. It's pathogenicity and pathophysiology remains controversial to date. Protease activity and amoebic forms have been reported previously in symptomatic isolates but there has been no conclusive evidence provided to correlate the protease activity and any specific life cycle stage of the parasite thus far. METHODS: Symptomatic isolates with amoebic form were tested for protease activity and compared with symptomatic and asymptomatic isolates without amoebic form for 10 days culture period. RESULTS: The present study demonstrates an elevated protease activity in cultures having a higher percentage of amoebic forms seen in symptomatic isolates. The growth curve demonstrated a significantly (p < 0.05) higher average number of parasite counts in asymptomatic compared to symptomatic isolates. Symptomatic isolates showed amoebic forms with percentages ranging from 5% to 17%. Elevated protease activity was demonstrated in isolates that had higher percentages of amoebic forms with intense bands at higher molecular weight proteases (60 - 100 kDa). As days of culture proceeded, the protease quantification also showed a steady increase. CONCLUSION: This study elucidates a correlation between protease activity and percentage of amoebic forms. The finding implies that these forms could play a role in exacerbation of intestinal symptoms during Blastocystis spp. infection.

Comparison of microscopy, culture, and conventional polymerase chain reaction for detection of blastocystis sp. in clinical stool samples.

We tested 513 stool samples from patients in Sydney, Australia for Blastocystis by using five diagnostic techniques: microscopy of a permanently stained smear using a modified iron-hematoxylin stain, two xenic culture systems (a modified Boeck and Drbohlav's medium and tryptone, yeast extract, glucose, methionine-9 medium), and two published conventional polymerase chain reaction methods specific for the small subunit ribosomal DNA. Ninety-eight (19%) samples were positive for Blastocystis in one or more of the diagnostic techniques. The PCR 2 method was the most sensitive at detecting Blastocystis with a sensitivity of 94%, and the least sensitive was microscopy of the permanent stain (48%). Subtype 3 was the most predominant subtype (present in 43% of samples assigned to this group). This study highlights the low sensitivity of microscopy when used as the sole diagnostic modality for detection of Blastocystis sp.

A rapid, high-throughput viability assay for Blastocystis spp. reveals metronidazole resistance and extensive subtype-dependent variations in drug susceptibilities.

Blastocystis is an emerging protistan parasite of controversial pathogenesis. Although metronidazole (Mz) is standard therapy for Blastocystis infections, there have been accumulating reports of treatment failure, suggesting the existence of drug-resistant isolates. Furthermore, very little is known about Blastocystis susceptibility to standard antimicrobials. In the present study, we established resazurin and XTT viability microassays for Blastocystis spp. belonging to subtypes 4 and 7, both of which have been suggested to represent pathogenic zoonotic subtypes. The optimized resazurin assay was used to screen a total of 19 compounds against both subtypes. Interestingly, subtype 7 parasites were resistant to Mz, a 1-position-substituted 5-nitroimidazole (5-NI), while subtype 4 parasites were sensitive. Some cross-resistance was observed to tinidazole, another 1-position 5-NI. Conversely, subtype 4 parasites were resistant to emetine, while subtype 7 parasites were sensitive. Position 2 5-NIs were effective against both subtypes, as were ornidazole, nitazoxanide, furazolidone, mefloquine, quinicrine, quinine, cotrimoxazole (trimethoprim-sulfamethoxazole), and iodoacetamide. Both subtypes were resistant to chloroquine, doxycycline, paromomycin, ampicillin, and pyrimethamine. This is the first study to report extensive variations in drug sensitivities among two clinically important subtypes. Our study highlights the need to reevaluate established treatment regimens for Blastocystis infections and offers clear new treatment options for Mz treatment failures.

Autophagy is involved in starvation response and cell death in Blastocystis.

Previous studies have demonstrated that colony forms of Blastocystis undergo cell death with numerous membrane-bound vesicles containing organelles located within the central vacuole, resembling morphological features of autophagy. In this study, we investigated whether Blastocystis underwent autophagy upon amino acid starvation and rapamycin treatment. Concurrently, we provide new insight into a possible function of the central vacuole. The use of the autophagy marker monodansylcadaverine, and the autophagy inhibitors 3-methyladenine and wortmannin, showed the existence of autophagy in amino-acid-starved and rapamycin-treated Blastocystis. Confocal microscopy and transmission electron microscopy studies also showed morphological changes that were suggestive of autophagy. The unusually large size of the autophagic compartments within the parasite central vacuole was found to be unique in Blastocystis. In addition, autophagy was found to be triggered when cells were exposed to the cytotoxic antibody mAb 1D5, and autophagy was intensified in the presence of the caspase inhibitor zVAD.fmk. Taken together, our results suggest that the core machinery for autophagy is conserved in Blastocystis, and that it plays an important role in the starvation response and cell death of the parasite.

[The morphological study of the cysts Pelomyxa palustris Greeff, 1874].

Pelomyxa palustris Greeff, 1874, is the only species of pelomixoid amoebas with the rest cysts in its life cycle. The morphology of the P. palustris has been studied by the light and electronic microscopy. Encystation of P. palustris under climatic conditions of North-West of Russia occurs within August-September. Rest cysts have a complex, trilaminar wall. Two inner lamina are the dense endocyst and the laminated mesocyst, thickness of each layer runs up to 0.6-0.7 microm. Thickness of the electron-dense ectocyst usually does not exceed 0.1-0.2 microm. The encystated cell of P. palustris has the unique structure. About 60 % of the cell volume are occupied by a huge vacuole placed in the center and filled up with the prokaryotic cytobionts. Different vacuoles, small vesicles of various nature, autophagosomes and lipid drops could be found inside that huge vacuole. The amoebae cytoplasm occupies the space in between endocyst's inner surface and the central vacuole. No any inclusions, prokaryotic cytobionts and most of cell organelles are absent in the cytoplasm. There are 4 large nuclei filled with relatively homogeneous karyoplasm lying in the cytoplasm. Nuclear envelope forms a lot of long tubular channels, running through the cytoplasm and lining the membrane of the central vacuole. Encysted pelomixoid stay in this state up until the beginning of excystation. Excystation of P. palustris in the studied region occurs in spring, during the latter half of April and the beginning of May. Cysts undergo complex morphofunctional changes, related to the reorganization of the wall and formation of young multinucleate amoebas. Only one wall lamina of the 3 initial ones is left up to the moment of excystation. The central vacuole endures ruination and its content penetrates into the cytoplasm. Pelomixoid nuclei divide twice. Prokaryotic cytobionts are localized in cytoplasm and in the perinuclear area. Young multinuclear species of P. palustris coming out of the cysts do not differ in their structure from the adult forms.