Process Of Encystation Research Articles

Stress Response in Entamoeba histolytica Is Associated with Robust Processing of tRNA to tRNA Halves.

tRNA-derived fragments have been reported in many different organisms and have diverse cellular roles, such as regulating gene expression, inhibiting protein translation, silencing transposable elements, and modulating cell proliferation. In particular, tRNA halves, a class of tRNA fragments produced by the cleavage of tRNAs in the anti-codon loop, have been widely reported to accumulate under stress and regulate translation in cells. Here, we report the presence of tRNA-derived fragments in Entamoeba, with tRNA halves being the most abundant. We further established that tRNA halves accumulate in the parasites upon different stress stimuli such as oxidative stress, heat shock, and serum starvation. We also observed differential expression of tRNA halves during developmental changes of trophozoite-to-cyst conversion, with various tRNA halves accumulating during early encystation. In contrast to other systems, the stress response does not appear to be mediated by a few specific tRNA halves, as multiple tRNAs appear to be processed during the various stresses. Furthermore, we identified some tRNA-derived fragments associated with Entamoeba Argonaute proteins, EhAgo2-2 and EhAgo2-3, which have a preference for different tRNA-derived fragment species. Finally, we show that tRNA halves are packaged inside extracellular vesicles secreted by amoebas. The ubiquitous presence of tRNA-derived fragments, their association with the Argonaute proteins, and the accumulation of tRNA halves during multiple different stresses, including encystation, suggest a nuanced level of gene expression regulation mediated by different tRNA-derived fragments in Entamoeba. IMPORTANCE In the present study, we report for the first time the presence of tRNA-derived fragments in Entamoeba. tRNA-derived fragments were identified by bioinformatics analyses of small-RNA sequencing data sets from the parasites and also confirmed experimentally. We found that tRNA halves accumulated in parasites exposed to environmental stress or during the developmental process of encystation. We also found that shorter tRNA-derived fragments are bound to Entamoeba Argonaute proteins, indicating that they may have a potential role in the Argonaute-mediated RNA-interference pathway, which mediates robust gene silencing in Entamoeba. We noticed that in response to heat shock, the protein translation levels were elevated in the parasites. This effect was reversed in the presence of an analog of leucine, which also reduced the levels of the tRNA halves in the stressed cells. Our results suggest that tRNA-derived fragments in Entamoeba have a possible role in regulating gene expression during environmental stress.

Investigating how clathrin adaptor complex AP-2 participates in Giardia lamblia encystation

The protozoan parasite Giardia lamblia acquires cholesterol from the environment since it is unable to synthesise cholesterol de novo and this is vital for trophozoite growth. Conversely, the lack of cholesterol was described as an essential event to trigger encystation, the differentiation of trophozoites to mature cysts. During the G. lamblia cell cycle, cholesterol is acquired as a free molecule as well as through receptor-mediated endocytosis (RME) of lipoproteins. In this work, we describe the involvement of RME in the cell differentiation process of G. lamblia. We found that a reduction in the expression of the medium subunit (Glµ2) of the giardial adaptin protein GlAP2 impaired RME, triggering the process of encystation in growing cells. Contrary to expectations, decreasing Glµ2 expression produced a cohort of trophozoites that yielded significantly less mature cysts when cells were induced to encyst. Analysis of the subcellular localization of Glµ2 and the cyst wall protein 1 (CWP1) during encystation was later performed, to dissect the process. Our results showed, on one hand, that blocking RME by inhibiting Glµ2 expression, and probably cholesterol entry, is sufficient to induce cell differentiation but not to complete the process of encystation. On the other hand, we observed that GlAP2 is necessary to accomplish the final steps of encystation by sorting CWP1 to the plasma membrane for cyst wall formation. The understanding of the mechanisms involved in cyst formation should provide novel insights into the control of giardiasis, an endemic worldwide neglected disease.

Novel life cycle stages of Colpodella sp. (Apicomplexa) identified using Sam-Yellowe's trichrome stains and confocal and electron microscopy.

Colpodella spp. are free-living flagellates closely related to the apicomplexans. Human infections by Colpodella sp. have been reported. A biflagellated trophozoite and cyst stage comprise the known life cycle stages of Colpodella sp. However, the process of encystation and excystation within the life cycle is unclear. Life cycle stages initiating human infections are unknown. We performed a detailed investigation of the life cycle of Colpodella sp. (ATCC 50594) in culture using Sam-Yellowe's trichrome stains and differential interference contrast (DIC) for light microscopy and fluorescence microscopy of Congo red-stained cells and investigated ultrastructure using transmission electron microscopy (TEM). We report previously undocumented stages of Colpodella sp. Asymmetric and asynchronous division was detected inside cysts by trichrome staining and by TEM. Odd-numbered juveniles and cysts containing more than four juvenile trophozoites were identified. Live imaging of active cultures captured the excystation and egress of juvenile trophozoites and confirmed the presence of multinucleate cysts. The ultrastructure of the multinucleate cyst is reminiscent of apicomplexan schizonts. Insights gained from the life cycle stages observed in culture allowed the construction of the life cycle of Colpodella sp. Knowledge of the life cycle will aid biochemical and molecular characterization of Colpodella sp. and help identify stages in human infections.

Advances in Entamoeba histolytica Biology Through Transcriptomic Analysis.

A large number of transcriptome-level studies in Entamoeba histolytica, the protozoan parasite that causes amoebiasis, have investigated gene expression patterns to help understand the pathology and biology of the organism. They have compared virulent and avirulent strains in lab culture and after tissue invasion, cells grown under different stress conditions, response to anti-amoebic drug treatments, and gene expression changes during the process of encystation. These studies have revealed interesting molecules/pathways that will help increase our mechanistic understanding of differentially expressed genes during growth perturbations and tissue invasion. Some of the important insights obtained from transcriptome studies include the observations that regulation of carbohydrate metabolism may be an important determinant for tissue invasion, while the novel up-regulated genes during encystation include phospholipase D, and meiotic genes, suggesting the possibility of meiosis during the process. Classification of genes according to expression levels showed that amongst the highly transcribed genes in cultured E. histolytica trophozoites were some virulence factors, raising the question of the role of these factors in normal parasite growth. Promoter motifs associated with differential gene expression and regulation were identified. Some of these motifs associated with high gene expression were located downstream of start codon, and were required for efficient transcription. The listing of E. histolytica genes according to transcript expression levels will help us determine the scale of post-transcriptional regulation, and the possible roles of predicted promoter motifs. The small RNA transcriptome is a valuable resource for detailed structural and functional analysis of these molecules and their regulatory roles. These studies provide new drug targets and enhance our understanding of gene regulation in E. histolytica.

Two potential evolutionary origins of the fruiting bodies of the dictyostelid slime moulds.

Dictyostelium discoideum and the other dictyostelid slime moulds ('social amoebae') are popular model organisms best known for their demonstration of sorocarpic development. In this process, many cells aggregate to form a multicellular unit that ultimately becomes a fruiting body bearing asexual spores. Several other unrelated microorganisms undergo comparable processes, and in some it is evident that their multicellular development evolved from the differentiation process of encystation. While it has been argued that the dictyostelid fruiting body had similar origins, it has also been proposed that dictyostelid sorocarpy evolved from the unicellular fruiting process found in other amoebozoan slime moulds. This paper reviews the developmental biology of the dictyostelids and other relevant organisms and reassesses the two hypotheses on the evolutionary origins of dictyostelid development. Recent advances in phylogeny, genetics, and genomics and transcriptomics indicate that further research is necessary to determine whether or not the fruiting bodies of the dictyostelids and their closest relatives, the myxomycetes and protosporangids, are homologous.

Second harmonic generation microscopy imaging of cellulose transportation during the encystment process of Acanthamoeba castellanii.

Acanthamoeba are single-celled protists able to survive in many different environments, but are most commonly found in soil and freshwater sources. Due to their free-living nature, Acanthamoeba often face a variety of precarious, environmentally stressful conditions, which for many microbes means death. Acanthamoeba are able to cope with extreme conditions by means of a biphasic life-cycle resulting in a round, resistant double-walled dormant cyst state when conditions are adverse, and a dividing, metabolically active free-living trophozoite state when conditions are favorable. The purpose of this study is to observe the transformation from a trophozoite to a mature cyst stage, specifically analyzing the change in morphology associated with the process of encystation. In order to directly observe the production of cellulose, the main component of the interior cell wall that gives an Acanthamoeba cyst its durability, cells were subjected to a non-nutrient medium with added salt concentrations, which has been shown in preliminary trials to be an encystation-inducing stressor. During the process of encystment, trophozoites were imaged frequently using second harmonic generation microscopy to identify cellulose forming in the cell walls of the cysts. Through this method, possible locations of intracellular cellulose while it is being transported to the cell wall can be observed, as well as the dynamic morphology of the cell caused by the build-up of cellulose, resulting in round, mature cysts. Calcofluor white, a cellulose-specific stain commonly used for identifying cell walls in plants and algae, was also implemented to achieve the same goals.

Coordinated Changes in Gene Expression Throughout Encystation of Giardia intestinalis.

Differentiation into infectious cysts through the process of encystation is crucial for transmission and survival of the intestinal protozoan parasite Giardia intestinalis. Hitherto the majority of studies have focused on the early events, leaving late encystation poorly defined. In order to further study encystation, focusing on the later events, we developed a new encystation protocol that generates a higher yield of mature cysts compared to standard methods. Transcriptome changes during the entire differentiation from trophozoites to cysts were thereafter studied using RNA sequencing (RNA-seq). A high level of periodicity was observed for up- and down-regulated genes, both at the level of the entire transcriptome and putative regulators. This suggests the trajectory of differentiation to be coordinated through developmentally linked gene regulatory activities. Our study identifies a core of 13 genes that are consistently up-regulated during initial encystation. Of these, two constitute previously uncharacterized proteins that we were able to localize to a new type of encystation-specific vesicles. Interestingly, the largest transcriptional changes were seen in the late phase of encystation with the majority of the highly up-regulated genes encoding hypothetical proteins. Several of these were epitope-tagged and localized to further characterize these previously unknown genetic components of encystation and possibly excystation. Finally, we also detected a switch of variant specific surface proteins (VSPs) in the late phase of encystation. This occurred at the same time as nuclear division and DNA replication, suggesting a potential link between the processes.

Eukaryotic cell encystation and cancer cell dormancy: is a greater devil veiled in the details of a lesser evil?

Cancer cell dormancy is the main cause of cancer recurrence and failure of therapy as dormant cells evade not only the anticancer drugs but also the host immune system. These dormant cells veil themselves from detection by imaging and/or using biomarkers, which imposes an additional problem in targeting such cells. A similar form of hibernation process known as encystation is studied in detail for pathogenic unicellular eukaryotic microorganisms. By examination using microarray gene expression profiles, immunocytochemistry tools, and siRNAs during the process of encystation, understanding the covert features of cancer cell dormancy as proposed could be possible. This knowledge can be extended to dormant cancer cells to uncover the mechanisms that underlie this ghost, yet dangerous state of human cancers. We propose a strategy to induce dormancy and exit this state by application of knowledge gained from the encystation induction and retrieval processes in pathogenic eukaryotic microorganisms. Given that early detection and characterization of dormant malignant tumor cells is important as a general strategy to monitor and prevent the development of overt metastatic disease, this homology may enable the design of therapies that could either awake the dormant cell from dormancy to make it available for therapies or prolong such a phase to make cancer appear as a chronic disease.

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”.

Mechanisms of adaptation in the intestinal parasite Giardia lamblia

Giardia lamblia, a parasite of humans, is a major source of waterborne diarrhoeal disease. Giardia is also an excellent system to study basic biochemical processes because it is a single-celled eukaryote with a small genome and its entire life cycle can be replicated in vitro. Giardia trophozoites undergo fundamental changes to survive outside the intestine of their host by differentiating into infective cysts. Encystation entails the synthesis, processing, transport, secretion and extracellular assembly of cyst wall components. To survive within the intestine, Giardia undergoes antigenic variation, a process by which the parasite continuously switches its major surface molecules, allowing the parasite to evade the host's immune response and produce chronic and recurrent infections. The objective of the present chapter is to provide a better understanding of the molecular mechanisms involved in adaptation and differentiation in Giardia, with a particular focus on the process of encystation and antigenic variation of this interesting micro-organism.

A Novel ARID/Bright-like Protein Involved in Transcriptional Activation of Cyst Wall Protein 1 Gene in Giardia lamblia

The capability of protozoan parasite Giardia lamblia to encyst is critical for survival outside the host and its transmission. AT-rich interaction domain (ARID) or Bright homologs constitute a large family of transcription factors in higher eukaryotes that regulate cell proliferation, development, and differentiation. We asked whether Giardia has ARID-like genes and whether they influence gene expression during Giardia encystation. Blast searches of the Giardia genome data base identified two genes with putative ARID/Bright domains (gARID1 and 2). Epitope-tagged gARID1 was found to localize to nuclei. Recombinant gARID1 specifically bound to the encystation-induced cyst wall protein (cwp) gene promoters. Mutation analysis revealed that AT-rich initiators were required for binding of gARID1 to the cwp promoters. gARID1 contains several key residues for DNA binding, and its binding sequences are similar to those of the known ARID family proteins. The gARID1 binding sequences were positive cis-acting elements of the cwp1 promoter during both vegetative growth and encystation. We also found that gARID1 transactivated the cwp1 promoter through its binding sequences in vivo. Our results suggest that the ARID family has been conserved during evolution and that gARID1 is an important transactivator in regulation of the Giardia cwp1 gene, which is key to Giardia differentiation into cysts.

Oral Immunization of BALB/c Mice with Giardia duodenalis Recombinant Cyst Wall Protein Inhibits Shedding of Cysts

The process of encystation is a key step in the Giardia duodenalis life cycle that allows this intestinal protozoan to survive between hosts during person-to-person, animal-to-person, waterborne, or food-borne transmission. The release of cysts from infected persons and animals is the main contributing factor to contamination of the environment. Genes coding for cyst wall proteins (CWPs), which could be used for developing a transmission-blocking vaccine, have been cloned. Since the immunogenicity of recombinant Giardia CWP is unknown, we have investigated the immunogenicity of recombinant CWP2 (rCWP2) and its efficacy in interfering with the phenomenon of encystation taking place in the small bowels of BALB/c mice vaccinated with the recombinant protein. Here we report that the immunization of BALB/c mice with rCWP2 stimulated the immune system in a manner comparable to that for a live infection with Giardia muris cysts. Fecal and serum anti-rCWP2 immunoglobulin A (IgA) antibodies were detected in the immunized mice. In addition, anti-rCWP2 IgG1 and IgG2a antibodies were detected in the serum. mRNAs coding for Th1 and Th2 types of cytokines were detected in spleen and Peyer's patch cells from immunized mice. When the vaccinated mice were challenged with live cysts, the animals shed fewer cysts. We conclude that rCWP2 is a possible candidate antigen for the development of a transmission-blocking vaccine.

Tubulovesicular elements in Blastocystis hominis from the caecum of experimentally-infected rats

By transmission electron microscopy (TEM), tubulovesicular elements were seen in Blastocystis hominis obtained from the caecum of experimentally-infected rats. These appeared to arise from the peripheral cytoplasm and were rounded, oval or elongate in sections. It is suggested that these elements form a network for transfer of nutrients to the periphery during the process of encystation.

Studies on the rate of selective uptake of amino acids by Trichinella larvae in vivo

Groups of C57BL/6J mice, orally infected with 300 larvae each of Trichinella spiralis or T. pseudospiralis were injected with [3H]-alanine, tyrosine, tryptophan or glycine. The incorporation of isotope labelled amino acids into larval proteins was measured at 2, 6, and 12 months post-infection. It was shown that there is a significant increase in the in vivo uptake of isotope labelled amino acids with time by the larvae of T. spiralis and T. pseudospiralis. The level of uptake was highest for tyrosine followed by tryptophan, alanine and then glycine, for both species. The in vivo uptake of amino acids by T. pseudospiralis larvae was always higher than T. spiralis or the host at 6 and 12 months post-infection. At 2 months post-infection, T. spiralis uptake of these amino acids was higher, except for tyrosine. This may be related to the special needs of these larvae during the process of encystation. The higher metabolic requirements of T. pseudospiralis may be related to the higher energy needs of these non encapsulated, highly motile and mobile muscle larvae.

Recognition of Giardia lamblia cyst-specific antigens by monoclonal antibodies.

Immunization of BALB/c mice with a sonicated extract of in vitro-generated Giardia lamblia cysts produced six cyst-specific monoclonal antibodies (MoAbs). Two MoAbs (8C5.C11 and 5A4.G6), which recognize proteinaceous cyst antigens, were selected for further study. In indirect immunofluorescence (IFA), MoAb 8C5.C11 reacted with encystation-specific vesicles in trophozoites beginning 3 h after the induction of encystation in vitro. This MoAb also recognized cysts which began to appear at 12 h. In contrast, MoAb 5A4.G6 stained only cyst walls. In Western blots, both MoAbs also reacted with cyst antigens, but not trophozoite antigens. MoAb 8C5.C11 first recognized cyst antigen from 3 h encysting cultures, reacting with 26, 28, 42 and 46 kD bands. MoAb 5A4.G6 reacted with a 38 kD band, beginning with 12 h encysting cultures. When added to G. lamblia encysting cultures before the appearance of cysts (0 to 9 h) and in the presence of a source of complement, MoAb 8C5.C11 caused a significant reduction in the numbers of water-resistant cysts produced in vitro compared to the control. MoAb 5A4.G6 did not affect in vitro encystation. These findings confirm the heterogeneity of cyst antigens, and also indicate that the process of encystation in vitro can be interrupted by antibodies and complement.

Giardia lamblia: Absence of Cyst Antigens and Reduced Secretory Vesicle Formation and Bile Salt Uptake in an Encystation-Deficient Subline

Encystation of Giardia lamblia entails the appearance of a number of new antigens, as well as formation of a novel class of large encystation-specific secretory vesicles (ESV) that transport stage-specific proteins to the nascent cyst wall. The monoclonal antibody GCSA-1, which was raised against purified cyst walls, recognizes protein species of ~26-46 kDa that are regulated by exposure to bile (plus lactic acid) and alkaline pH, the factors that induce encystation. The GCSA-1 epitope is maximally expressed after ~14 hr of encystation and localizes to the interior, but not the membrane of the ESV as shown by frozen section immunoelectron microscopy. To further understand the process of encystation, we compared two sublines of strain WB that differ in their ability to encyst in vitro. Water-resistant cysts were not detected in subline A6 under conditions in which subline C6 formed ~2 × 105 cysts/ml. Moreover, subline A6 did not form ESV efficiently or detectably express antigens recognized by mAb GCSA-1 or by polyclonal anti-cyst sera. Finally, uptake of the bile salt taurocholate by A6 was reduced 4- to 20-fold, compared with that of C6, although transport by both strains was sodium-dependent and regulated by bile salt starvation. The decrease in bile salt uptake by A6 may be related to its defect in encystation.

Differentiation of pathogenic amoebae: encystation and excystation ofAcanthamoeba culbertsoni — A model

Differentiation into dormant cysts and vegetative trophozoites is an inherent character intimately associated with the life cycle and infectivity of pathogenic amoebae. In the case of human intestinal amoebiasis encystation and excystation are of immediate relevance to the process of transmission of the disease from healthy carriers to susceptible individuals. Using a pathogenic free living amoebaAcanthamoeba culbertsoni as a model, considerable progress has been achieved in understanding the mechanism and control of the process of differentiation. The turnover of the regulatory molecule cyclic 3: ’5′ adenosine monophosphate is responsible for triggering the process of encystation. Amoebae bind effector molecules such as biogenic amines to a membrane localized receptor which itself resembles the β-adrenergic receptor of mammalian organisms. The activation of adenylate cyclase or inhibition of cyclic AMP phosphodiesterase maintain the dynamic intracellular cyclic AMP. The cytosol fraction of amoebae has a cyclic AMP binding protein. During encystation, enzymes needed for synthesis of cellulose and glycoproteins are induced. Control is exercised at transcriptional level and the process is subject to catabolic repression.

Entamoeba invadens and E. histolytica: Separation and purification of precysts and cysts by centrifugation on discontinuous density gradients of percoll

The different cell forms in the life cycle of Entamoeba invadens (trophozoites, precysts, and cysts) were rapidly and quantitatively separated on density step gradients of polyvinylpyrolidone-coated colloidal silica particles (Percoll). With this method, the gradual process of encystation by E. invadens trophozoites could be monitored. Percoll gradients were also efficient in separating trophozoites of Entamoeba histolytica and bacteria. After purification on Percoll, trophozoites display no evidence of damage when examined by light microscopy and no loss in viability as judged by their ability to multiply.

Ultrastructure of the Metacercarial Cyst of Posthodiplostomum minimum (Maccallum, 1921)

The 2-layered cyst consists of an outer fibrous cellular capsule composed of endothelial cells and an inner noncellular primary cyst wall of homogeneous and moderately electron-dense texture. An osmiophilic interface zone separates these layers. Adjacent to the primary cyst wall is a cyst cavity filled with flocculent material and membrane-bound vesicles. These vesicles are found in close proximity to the cyst wall and resemble similar vesicles in teguminal extrusions of the metacercaria, suggesting that cystogenous materials are formed by the parasite, released into the cyst cavity, and eventually deposited onto the cyst wall. The endothelial layers of the outer fibrous capsule contain numerous intracellular microfilaments that may function in providing additional tensile strength, protecting the metacercaria during ingestion by the definitive host. Micropinocytotic vesicles and extracellular deposits of collagen are characteristic of loose fibroblasts which surround the periphery of the cyst. Necrotic areas are designated the interface zone. It is suggested that they may be attributed to mechanical compression or isolation from oxygen and important nutrients. The metacercariae of the trematode Posthodiplostomum minimum utilize numerous species of freshwater fish as second intermediate hosts, particularly sunfish of the family Centrarchidae. The cercariae penetrate epidermal tissues and enter the general systemic circulation. The larval trematode forms a thin cyst wall which appears as a frail baglike structure loosely covering the metacercariae after reaching those visceral organs which are usually associated with a plentiful blood supply, e.g., liver, heart, or kidney. The process of encystation has been studied by electron microscopy in certain families of digenetic trematodes in order to elucidate the structure, function, host response, and relative contributions of the host and parasite during elaboration of the cyst wall. Electron microscopy of Fasciola hepatica metacercariae which openly encyst in an external environment has revealed that cyst components form a fourlayer wall which is solely contributed by cystogenous gland secretions from the parasite. The quinone-tanned, keratinlike proteinaceous layers are rapidly deposited, providing protection against desiccation and fungal and bacterial injury (Dixon and Mercer, 1964, 1967; Mercer and Dixon, 1967). The cyst of Notocotylus attenuatus has been described by Southgate (1971) as three-layered, originating in a similar manner from parenchymal gland Received for publication 1 May 1973. 67 cell secretions. Cyst wall ultrastructure within a second intermediate host has been described in one psilostomatid (Macy et al., 1968) and in two microphallid species (Strong and Cable, 1972; Stanier et al., 1968). These cysts, protected internally by their hosts from the environment, complete differentiation after periods of up to 4 weeks in which as many as four layers may be present. There is evidence in various heterophyid species of Ascocotyle that the cyst wall is derived from tegumental extrusions which form one or more layers. Host involvement is limited to peripheral infiltration of fibroblasts with extracellular reticular collagen (Lumsden, 1968; Stein and Lumsden, 1971, 1972). This paper reports on the ultrastructure of Posthodiplostomum minimum metacercarial cyst wall organization as an initial step in describing the process of encystation in strigeids. MATERIALS AND METHODS Metacercarial cysts were obtained from naturally infected sunfish (Lepomis sp.) which were seined from ponds in the vicinity of Marion, Ohio, and the Bonnet Carre' Spillway, Norco, Louisiana. Fish were maintained in aquaria and fed a diet of commercial fish food, mealworms, and earthworms. Cysts were excised from liver parenchyma and processed for electron microscopy. Cysts were fixed for 6 hr (1 to 4 C) in 3% glutaraldehyde buffered at pH 7.3 in 0.12 mI monobasic sodium phosphate-NaOH, containing 1% CaCl2 and 3% sucrose. These were washed overnight in cold phosphate buffer, postfixed for 1/, hr with 1% phosphate-buffered OsO4, and This content downloaded from 207.46.13.15 on Fri, 26 Aug 2016 05:32:13 UTC All use subject to http://about.jstor.org/terms 68 THE JOURNAL OF PARASITOLOGY, VOL. 60, NO. 1, FEBRUARY 1974 washed in several rinses of veronal acetate buffer at pH 5.3. Tissue was stained en bloc in 1% uranyl acetate (in veronal acetate buffer, pH 5.3) for 1 hr, and dehydrated with ethanol before gradual embedment in low viscosity epoxy resin mixture (Spurr, 1969). Sections were cut on glass knives with a Sorvall MT-2 ultramicrotome, collected on uncoated copper grids, and stained with 1% aqueous uranyl acetate and with lead citrate. Electron micrographs were made with a Siemens Elmiskop IA operating at an accelerating voltage of 80 kv. Cysts were cut open during fixation allowing access of the fixatives and embedding media into both inner and outer surfaces of the cyst wall.

Studies on Entamoeba histolytica

1. The oxidation-reduction potential in cultures of E. histolytica has been studied. It was found that a close relation exists between the potential in the culture and the growth, encystation, and excystation of E. histolytica. The small differences observed in different cultures indicate the reproducibility of the potentials and the reliability of the potential condition in explaining these biological activities of the amoebae in culture.2. The best growth of trophozoites was observed at potentials between −350 and −425mV., less at potentials between −275 and −350mV., still less at between −200 and −275mV. At potentials between −150 and −200mV. a bare increase in number of trophozoites was noticed. Between potentials of −50 and −150mV. no increase of growth was observed. Trophozoites were found dead in 12 hr. or more at potentials between −50 and +50mV., and died and disintegrated in a couple of hours between potentials of +100 and +200mV.3. The life span of a culture of E. histolytica was also found to be related to the potential condition. The longer the potential in the culture remains below −150mV., the longer the culture remains positive.4. Excystation of E. histolytica, no matter in what media, took place only at potentials of −115mV. or lower.5. Mass encystation of E. histolytica was induced in cultures in which a ‘sharp rise of potential’ occurred after a greatly increased rate of multiplication of trophozoites after 22–24 hr. of incubation. Maturation of cysts occurred during this period of ‘sharp rise of potentials’. If the potential went down shortly after its sharp rise, the cysts failed to become mature and disappeared in about 12 hr.6. Trophozoites and cysts observed in cultures with very low potentials (below −300mV.) and a rapid multiplication were definitely smaller than those in cultures having higher potentials and trophozoites multiplied slowly.7. In cultures of amoebae in which mass encystation failed to occur, or mass encystation occurred but cysts failed to become mature, some success was observed in a few trials by artificially creating the ‘sharp rise of potential’ by oxygenation.8. The pH values noticed in cultures at times of encystation ranged from 6.4 to 6.8. This pH range was a little lower than that observed previously. Mass encystation failed to occur at the change in potential if the pH was 7.2 or higher.9. It is felt that the process of encystation requires, at least, two extrinsic and one intrinsic factors. The first two are the potential change from more favourable to less favourable conditions and the pH. The last one is the tendency to encyst possessed by the amoebae.10. The low potential condition in a culture of E. histolytica was found chiefly provided by the activity of the accompanying bacteria. It is, therefore, felt that the indispensability of a bacterial flora for cultivation of amoebae may be explained, partly if not entirely, by the potential relation. To verify this statement, it is hoped to propagate E. histolytica in cultures in which a strongly reducing condition is provided without the presence of bacteria.The author wishes to acknowledge the kindness of Dr David Weinman of this department in making many invaluable corrections of this manuscript, and he is very grateful to Mrs Elizabeth B. Keller and Mrs Wanda Reed for their help in preparing the graphs accompanying it.