Protozoa In Water Research Articles

Guarding the health of individuals: Effects of bacterial toxins in drinking water

Contamination of drinking water is one of the major concern’s that affect the health of the community due to bacterial toxins. This research paper closely focuses on the impact of these toxins in human health, and the available ways of tracking and controlling their distribution in water resources. This paper will therefore consider a literature review that will help in the generation of epidemiological data from different studies done on bacterial toxins in drinking water. The figures and levels of pathogenic bacteria, viruses, and protozoa in water make their presence or absence affect the lives of people all over the world. Waterborne pathogens can affect humanity through drinking water, fresh produce and raw foods, processed foods, or water for recreation. This increased chemical exposure has led to drinking water becoming a large source of chemical exposure despite drinking water treatment research mainly concentrating on microbial contaminants because of the large burden of diseases caused and high childhood mortality among the victims. Consequently, use of water polluted with heavy metals and bacterial toxins leads to high morbidity and mortality coefficient in various regions of the world. The general objective of this paper is to focus on bacterial toxins’ contamination of drinking water globally and evaluate the health risks that are likely to be occasioned by the intake of the said water. It will also focus on the link between exposure limits and the latent effects on health and the primary organs that are affected.

Molecular characterization of waterborne protozoa in surface water and sediment in Brazil: a taxonomic survey of ciliated protozoa and their correlation with Giardia duodenalis and Cryptosporidium spp.

The detection of Giardia duodenalis and Cryptosporidium spp. was performed, along with the identification of the ciliated protozoa biodiversity, to evaluate the correlation between these protozoa in freshwater quality monitoring. Water and sediment samples from two sites in the Atibaia River (Campinas, São Paulo, Brazil) were collected monthly for 2years (n = 96). Pathogenic protozoa in water and sediment were detected by using immunomagnetic separation, followed by visualization by immunofluorescence assay (IFA). All positive aliquots in IFA were subjected to DNA extraction and subsequently nested PCR. Qualitative (in vivo observation and silver impregnation) and quantitative (in vivo enumeration) analyses were performed for the ciliated protozoa. Giardia cysts were detected in 62.5% of the surface water samples and Cryptosporidium spp. in 25.0%. In the sediment, cysts were detected in 35.4% samples and oocysts in 16.6%. A total of 57 samples positive for Giardia cysts were subjected to sequencing, 40 of which were harboring G. duodenalis (24 were characterized as sub-assemblage AII). For ciliated protozoa, 73 taxa belonging to 53 genera were identified over the period of the study. These results revealed a high degree of contamination by waterborne protozoa in the main water source which supplies drinking water for more than one million people in Campinas (São Paulo), highlighting the need for continuous monitoring of this catchment site. In addition, the present study provides important data regarding the sources of the water body degradation, i.e., fecal contamination of human origin, in addition to the survey of the ciliated protozoa.

Near dissolved organic matter microfiltration (NDOM MF) coupled with UVC LED disinfection to maximize the efficiency of water treatment for the removal of Giardia and Cryptosporidium

Microfiltration (MF) membranes with a mean pore size same as or smaller than 0.45 µm have been typically used to separate pathogenic protozoa in water since materials larger than 0.45 µm are considered particulates. However, 0.45 µm is too small to separate protozoa which are 4–6 µm (Cryptosporidium oocyst) or 8–15 µm (Giardia cyst) in size. In this study, we optimized the mean pore size of MF membranes to maximize the producibility and guarantee a high removal rate simultaneously and proposed the membrane filtration using an MF membrane with an optimum mean pore size larger than but close to dissolved organic matter (DOM), which is called near DOM MF (NDOM MF). According to the MF test using polystyrene surrogate beads with diameters of 3 and 8 µm, an MF membrane with a 0.8 µm mean pore size was the best in that it showed 52% to 146% higher water fluxes than a 0.45 µm MF membrane while maintaining the removal rate at 3–4 log. It was also the case for a low-temperature MF test, revealing the NDOM MF is highly effective regardless of temperature changes. Lastly, we tried to find the possibility of combining the NDOM MF with disinfection by an ultraviolet light emitting diode (UVC LED) to further guarantee the high quality of treated water while providing high process efficiency.

Waterborne transmission of protozoan parasites: a review of water resources in Iran – an update 2020

Waterborne protozoan parasitic diseases are considered a global disease. With no comprehensive review for the country, we evaluated only the available studies (n = 27) related to the waterborne transmission of protozoan parasites in Iran. The most commonly reported protozoan parasites were Acanthamoeba spp., Naegleria spp., Vahlkampfiid spp., Cryptosporidium spp., Giardia spp., Hartmannella spp., Saccamoeba spp., Entamoeba spp., Blastocystis spp., and other free-living amoeba. Immunofluorescence assay (IFA), polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), differential interference contrast (DIC), microscopic detection, and enumer-ation methods are the most reported methods to identify protozoan parasites in water samples. The literature showed untreated water, recreational water, and contaminated water supply sources by animal feces as the main routes of waterborne parasites transmission. No study reported stan-dard protocols to identify protozoan parasites. In addition, the investigation of protozoan parasites in water resource has not been performed all around the country. Iran, as a developing country, use surface water to supply drinking water. Many recreational rivers and springs can be potentially contaminated with parasitic protozoa and thus reliable surveillance systems need to be established. Furthermore, quick and applicable methods to survey the parasitic protozoa in water are necessary. © 2021 Desalination Publications.

Relationships between chemical and microbial faecal source tracking markers in urban river water and sediments during and post-discharge of human sewage

This study explores the relationships between faecal source tracking (FST) markers (quantitative Polymerase Chain Reaction (qPCR) markers and steroids), microbial indicators, the faecal ageing ratio of atypical colonies/total coliforms (AC/TC) and potential human pathogens (Giardia, Cryptosporidium and Campylobacter). Faecal source PCR markers tested were GenBac3, HumM3, HumBac (HF183-Bac708R); Bifidobacterium adolescentis, wildfowl and canine-associated markers. Sediment and water samples from the Avon River were collected during and post-discharge of untreated human sewage inputs, following a series of earthquakes, which severely damaged the Christchurch sewerage system.Significant, positive Spearman Ranks (rs) correlations were observed between human-associated qPCR markers and steroid FST markers and Escherichia coli and F-specific RNA bacteriophage (rs 0.57 to 0.84, p < 0.001) in water samples. These human source indicative FST markers demonstrated that they were also effective predictors of potentially pathogenic protozoa in water (rs 0.43–0.74, p ≤ 0.002), but correlated less well with Campylobacter. Human-associated qPCR and steroid markers showed significant, substantial agreement between the two FST methods (Cohen's kappa, 0.78, p = 0.023), suggesting that water managers could be confident in the results using either method under these contamination conditions.Low levels of fluorescent whitening agents (FWA) (mean 0.06 μg/L, range 0.01–0.40 μg/L) were observed in water throughout the study, but steroids and FWA appeared to be retained in river sediments, months after continuous sewage discharges had ceased. No relationship was observed between chemical FST markers in sediments and the overlying water, and few correlations in sediment between chemical FST markers and target microorganisms. The low values observed for the faecal ageing ratio, AC/TC in water, were significantly, negatively correlated with increasing pathogen detection. This study provides support for the use of the AC/TC ratio, and qPCR and steroid FST markers as indicators of health risks associated with the discharge of raw human sewage into a freshwater system.

Prevalence, characteristics and correlates of enteric pathogenic protozoa in drinking water sources in Molyko and Bomaka, Cameroon: a cross-sectional study.

BackgroundAccess to potable water remains a major challenge particularly in resource-limited settings. Although the potential contaminants of water are varied, enteric pathogenic protozoa are known to cause waterborne diseases greatly. This study aimed at investigating the prevalence, characteristics and correlates of enteric pathogenic protozoa in drinking water sources in Buea, Cameroon.MethodsA cross-sectional study was conducted using 155 water samples collected from various drinking sources (boreholes, springs, taps and wells). Each sample was subjected to physicochemical examinations (pH, turbidity, odour and sliminess) and parasitological analysis (wet mount, modified Ziehl-Neelsen stain) to determine the presence of enteric pathogenic protozoa. A data collection tool was used to note characteristics of collected samples and the data was analysed using EPI-INFO Version 3.5.3.ResultsThe overall prevalence of enteric pathogenic protozoa in water sources was 62.6 %. Eight species of enteric protozoa were observed with Cryptosporidium parvum being the most predominant (45.8 %). Spring water was the most contaminated source with enteric protozoa (85.7 %) while pipe borne water had all eight species of protozoa identified. A pH of 6 was the only significant factor associated with the prevalence of these pathogens in water sources.ConclusionThe prevalence of enteric protozoa in water sources in Molyko and Bomaka is high, spring water is the most contaminated water source and Cryptosporidium parvum is the most common protozoa contaminating water. A water pH of 6 is associated to the prevalence of protozoa. Community members need to be educated to treat water before drinking to avoid infection by enteric protozoa in water and further studies with larger samples of water need to be conducted to find other correlates of the presence of protozoa in water.

Detection of protozoa in water samples by formalin/ether concentration method

Methods to detect protozoa in water samples are expensive and laborious. We evaluated the formalin/ether concentration method to detect Giardia sp., Cryptosporidium sp. and Toxoplasma in water. In order to test the properties of the method, we spiked water samples with different amounts of each protozoa (0, 10 and 50 cysts or oocysts) in a volume of 10 L of water. Immunofluorescence assay was used for detection of Giardia and Cryptosporidium. Toxoplasma oocysts were identified by morphology. The mean percent of recovery in 10 repetitions of the entire method, in 10 samples spiked with ten parasites and read by three different observers, were for Cryptosporidium 71.3 ± 12, for Giardia 63 ± 10 and for Toxoplasma 91.6 ± 9 and the relative standard deviation of the method was of 17.5, 17.2 and 9.8, respectively. Intraobserver variation as measured by intraclass correlation coefficient, was fair for Toxoplasma, moderate for Cryptosporidium and almost perfect for Giardia. The method was then applied in 77 samples of raw and drinkable water in three different plant of water treatment. Cryptosporidium was found in 28 of 77 samples (36%) and Giardia in 31 of 77 samples (40%). Theses results identified significant differences in treatment process to reduce the presence of Giardia and Cryptosporidium. In conclusion, the formalin ether method to concentrate protozoa in water is a new alternative for low resources countries, where is urgently need to monitor and follow the presence of theses protozoa in drinkable water.

Spatial distribution and enteroparasite contamination in peridomiciliar soil and water in the Apucaraninha Indigenous Land, southern Brazil.

The prevalence and distribution of soil and water samples contaminated with enteroparasites of humans and animals with zoonotic potential (EHAZP) in Apucaraninha Indigenous Land (AIL), southern Brazil, was evaluated. An environmental survey was conducted to evaluate the presence of parasitic forms in peridomiciliary soil and associated variables. Soil samples were collected from 40/293 domiciles (10 domiciles per season), from November 2010 to June 2011, and evaluated by modified methods of Faust et al. and Lutz. Analyses of water from seven consumption sites were also performed. The overall prevalence of soil samples contaminated by EHAZP was 23.8%. The most prevalent parasitic forms were cyst of Entamoeba spp. and eggs of Ascaris spp. The highest prevalence of contaminated soil samples was observed in winter (31%). The probability map obtained with geostatistical analyses showed an average of 47% soil contamination at a distance of approximately 140m. The parasitological analysis of water did not detect Giardia spp. or Cryptosporidium spp. and showed that all collection points were within the standards of the Brazilian law. However, the microbiological analysis showed the presence of Escherichia coli in 6/7 sampled points. Despite the low level of contamination by EHAZP in peridomiciliar soil and the absence of pathogenic protozoa in water, the AIL soil and water (due to the presence of fecal coliforms) are potential sources of infection for the population, indicating the need for improvements in sanitation and water treatment, in addition periodic treatment of the population with antiparasitic.

Prevalence of protozoa species in drinking and environmental water sources in Sudan.

Protozoa are eukaryotic cells distributed worldwide in nature and are receiving increasing attention as reservoirs and potential vectors for the transmission of pathogenic bacteria. In the environment, on the other hand, many genera of the protozoa are human and animal pathogens. Only limited information is available on these organisms in developing countries and so far no information on their presence is available from Sudan. It is necessary to establish a molecular identification of species of the protozoa from drinking and environmental water. 600 water samples were collected from five states (Gadarif, Khartoum, Kordofan, Juba, and Wad Madani) in Sudan and analysed by polymerase chain reaction (PCR) and sequencing. 57 out of 600 water samples were PCR positive for protozoa. 38 out of the 57 positive samples were identified by sequencing to contain 66 protozoa species including 19 (28.8%) amoebae, 17 (25.7%) Apicomplexa, 25 (37.9%) ciliates, and 5 (7.6%) flagellates. This study utilized molecular methods identified species belonging to all phyla of protozoa and presented a fast and accurate molecular detection and identification of pathogenic as well as free-living protozoa in water uncovering hazards facing public health.

Vectorial role of Acanthamoeba in Legionella propagation in water for human use

Legionella spp. is the causative agent of Legionnaires' disease and is transmitted through aerosols emanating from man-made water systems. Legionella resistance to water treatments has been related to its association with environmental amoebae such as Acanthamoeba. Due to the high presence of this protozoon in Spain and the high rate of notification of Legionnaires' disease of this country, the aims of this work were to study the coexistence of these bacteria and protozoa in water as well as their interaction. The usefulness of Acanthamoeba co-culture for the isolation of environmental Legionella was also studied. For this purpose, 70 water samples were collected in 2011 from three Drinking Water Treatment Plants, three Wastewater Treatment Plants and five Natural Pools in Spain. Acanthamoeba was found by PCR in 87.1% (61/70) samples and, by culture in 85.7% (60/70) samples. Legionella was detected by PCR in 58.6% (41/70) of water samples, in 5.7% (4/70) by agar culture and 75.7% (53/70) by Acanthamoeba co-culture. From the 54 Acanthamoeba water isolates, Legionella was detected in 43 of them independently of Acanthamoeba's genotype (T3, T4 and T11). Legionella feeleii, Legionella birminghamiensis, Legionella gresilensis/berliardensis, Legionella fairfieldensis, Legionella drozanski and Legionella falloni were identified. In conclusion, our results showed that environmental Acanthamoeba is infected by Legionella to a high percentage, and due to its ubiquity, high resistance and its pathogenic potential per se, new methods for its elimination should be studied. Also, the high effectivity of Acanthamoeba co-culture for Legionella detection has been shown.

Detection of &lt;i&gt;Cryptosporidium parvum&lt;/i&gt; and &lt;i&gt;Giardia lamblia&lt;/i&gt; in Water Supply by Quantitative Real-Time PCR

Cryptosporidium parvum and Giardia lamblia are common pathogenic protozoa in water, which pose high risk to drinking water supply. In the present study, detection of C. parvum and G. lamblia was performed by quantitative real-time PCR (RT-PCR). Pairs of PCR primers were evaluated for the detection specificity to pathogenic C. parvum and G. lamblia. The recovery of the RT-PCR detection procedure was examined and high recovery rates (i.e., more than 45％ for C. parvum and more than 50％ for G. lamblia ) were achieved. The RT-PCR method was used to detect C. parvum and G. lamblia in a secondary water supply. The results indicated the potential application of the quantitative RT- PCR method in detection of C. parvum and G. lamblia in water supply.

Antiprotozoal activity of 2, 3-dihydroxybenzoic acid isolated from the fruit extracts ofFlacourtia inermisRoxb.

An attempt has been made to isolate, purify and characterize an antiprotozoal compound from the fruit extract of Flacourtia inermis. Chromatographic technique was used for the isolation and purification of the active principle and the structural elucidation of the compound was done by spectroscopic analysis. Antiprotozoal activity was tested by microscopic count method using fresh water protozoa and rectal ciliates of frog. The study revealed that the purified compound belongs to the phenolic acid class and identified as 2, 3-dihydroxybenzoic acid. Antiprotozoal assay showed that the purified compound at a concentration of 2.5 mg/ml was lethal to the tested organisms. The study can be concluded that the fruit of Flacourtia inermis possesses 2, 3-dihydroxybenzoic acid, which is a powerful antiprotozoal agent against fresh water protozoa and rectal ciliates of frog.

Environmental Monitoring of Opportunistic Protozoa in Rivers and Lakes in the Neotropics Based on Yearly Monitoring

The goal of this study was to standardise and use parasitological and molecular techniques in the analysis and seasonal monitoring of opportunistic protozoa in water from fluvial systems for human usage in the municipality of Goiânia, the capital of the state of Goias, in the mid- western region of Brazil. We focused on Cryptosporidium sp. Cyclospora cayetanensis, Isopora belli, Microsporidia and Giardia lamblia. Water samples were collected monthly from February 2006 to January 2007 and concentrated using vacuum filtration and a positively charged membrane. Sev- eral methods were used to identify the different protozoa of interest. To detect coccidia (Cryptosporidium sp., Isospora belli, and Cyclospora cayetanensis), we used a Kinyoun hot- staining method and a modified Ziehl-Neelsen technique. Enteral microsporidia were detected by a hot-chromotrope technique while a MERIFLUOR ® Cryptosporidium kit was used to confirm the presence of Cryptosporidium sp. Finally, we used PCR to detect Cryptosporidium parvum/hominis. Water is of vital importance to living beings; however, due to anthropic action, several microorganisms are disseminated into aquatic environments. Among them are opportunistic protozoa that infect mainly immunodepressed and immuno- suppressed individuals, children and elderly people. These protozoa pose a significant health hazard. Nevertheless, the presence of these pathogens is underestimated because they are not considered during routine environmental monitor- ing. In our study, we were able to observe the presence of Giardia cysts, Cryptosporidium sp. and Cryptosporidium parvum oocysts in the bodies of water monitored in this re- search.

Incorporating method recovery uncertainties in stochastic estimates of raw water protozoan concentrations for QMRA

The impact of incorporating recovery data on protozoan concentration estimates was investigated for Cryptosporidium and Giardia using a large dataset (n=99) of [oo]cyst assay results with paired recovery estimates. Stochastic [oo]cyst concentration was estimated using three approaches: I-no availability/consideration of recovery, II-limited recovery data, where sample recovery was considered as an independent random variable, and III-every [oo]cyst assay result was adjusted for a concurrently derived recovery estimate. Critically, Approach I underestimated [oo]cyst concentrations by about 100% compared to Approaches II and III, which were similar. The impact of dataset size on statistical uncertainty about the concentration estimate for Approach II was investigated; little improvement in parameter uncertainty was achieved beyond n=20. It is suggested that recovery data be incorporated into source water concentration estimates, especially when used to infer health risks to consumers, so as not to underestimate the risk. Where none is available, conservatively low recoveries should be assumed. When designing monitoring programmes, recovery data should be collected as a pair with [oo]cyst count data for an initial period at least, so that site-specific relationships between those parameters may be ascertained and incorporated into source water concentration estimates.

Interaction between Mycobacterium avium subsp. paratuberculosis and environmental protozoa

BackgroundInteractions between Mycobacterium avium subsp. paratuberculosis (Map) and free-living protozoa in water are likely to occur in nature. The potential impact of ingestion of Map by two naturally occurring Acanthamoeba spp. on this pathogen's survival and chlorine resistance was investigated.ResultsBetween 4.6 and 9.1% of spiked populations of three Map strains (NCTC 8578, B2 and ATCC 19698), which had been added at a multiplicity of infection of 10:1, were ingested by Acanthamoeba castellanii CCAP 1501/1B and A. polyphaga CCAP 1501/3B during co-culture for 3 h at 25°C. Map cells were observed to be present within the vacuoles of the amoebae by acid-fast staining. During extended co-culture of Map NCTC 8578 at 25°C for 24 d with both A. castellanii and A. polyphaga Map numbers did not change significantly during the first 7 days of incubation, however a 1–1.5 log10 increase in Map numbers was observed between days 7 and 24 within both Acanthamoeba spp. Ingested Map cells were shown to be more resistant to chlorine inactivation than free Map. Exposure to 2 μg/ml chlorine for 30 min resulted in a log10 reduction of 0.94 in ingested Map but a log10 reduction of 1.73 in free Map (p < 0.001).ConclusionThis study demonstrated that ingestion of Map by and survival and multiplication of Map within Acanthamoeba spp. is possible, and that Map cells ingested by amoebae are more resistant to inactivation by chlorine than free Map cells. These findings have implications with respect to the efficacy of chlorination applied to Map infected surface waters.

Roof collection and storage of rainwater for drinking

Collecting rainwater from house roofs can provide both a reliable supply and one that complies with World Health Organisation drinking water guidelines. Mass balance allowance allows the storage volume to be assessed. Pathogen die-off in accordance with first order kinetics shows that two or more tanks operating in series will remove substantially more pathogens than a single tank of the same capacity. Drop inlet pipe, bottom overflow and a floating arm draw-off can provide the quiescent conditions for Cryptosporidium oocysts to settle and achieve drinking water protozoa compliance without microfiltration or ozone disinfection.

Cell volume control inParamecium: factors that activate the control mechanisms

A fresh water protozoan Paramecium multimicronucleatum adapted to a given solution was found to swell until the osmotic pressure difference between the cytosol and the solution balanced the cytosolic pressure. The cytosolic pressure was generated as the cell swelled osmotically. When either one or both of these pressures was somehow modified, cell volume would change until a new balance between these pressures was established. A hypothetical osmolyte transport mechanism(s) was presumably activated when the cytosolic pressure exceeded the threshold value of approximately 1.5 x 10(5) Pa as the cell swelled after its subjection to a decreased osmolarity. The cytosolic osmolarity thereby decreased and the volume of the swollen cell resumed its initial value. This corresponds to regulatory volume decrease (RVD). By contrast, another hypothetical osmolyte transport mechanism(s) was activated when the cell shrank after its subjection to an increased osmolarity. The cytosolic osmolarity thereby increased and volume of the shrunken cell resumed its initial value. This corresponds to regulatory volume increase (RVI). The osmolyte transport mechanism responsible for RVD might be activated again when the external osmolarity decreases further, and the cytosolic osmolarity thereby decreases to the next lower level. Similarly, another osmolyte transport mechanism responsible for RVI might be activated again when the external osmolarity increases further, and the cytosolic osmolarity thereby increases to the next higher level. Stepwise changes in the cytosolic osmolarity caused by a gradual change in the adaptation osmolarity found in P. multimicronucleatum is attributable to these osmolyte transport mechanisms. An abrupt change in the amount of fluid discharged from the contractile vacuole seen immediately after changing the external osmolarity reduces an abrupt change in cell volume and thereby protects the cell from the disruption of the plasma membrane by excessive stretch or dehydration during shrinkage.

Occurrence of Cryptosporidium oocysts and Giardia cysts in raw water from the Atibaia River, Campinas, Brazil.

Cryptosporidium parvum and Giardia duodenalis are waterborne parasites that have caused several outbreaks of gastrointestinal disease associated with drinking water. Due to the lack of studies about the occurrence of these protozoa in water in the Southeast of Brazil, an investigation was conducted to verify the presence of cysts and oocysts in superficial raw water of the Atibaia River. The water samples were submitted to membrane filtration (3.0 microm) and elution was processed by (1) scraping and rinsing of membrane (RM method) and (2) acetone-dissolution (ADM method). Microbiologic and chemical parameters were analyzed. Aliquots of the pellets were examined by immunofluorescence (Merifluor, Meridian Diagnostics, Cincinnati, Ohio). All water samples were positive for Cryptosporidium and Giardia, in spite of the high turbidity. Higher recovery rates occurred in samples treated by the RM method than by the ADM technique. The goal for future work is the assessment of viability of cysts and oocysts to determine the public health significance of this finding.

Cellular membranes that undergo cyclic changes in tension: Direct measurement of force generation by an in vitro contractile vacuole of Paramecium multimicronucleatum

The contractile vacuole of the fresh water protozoan Paramecium is a membrane-bound vesicle that expels excess cytosolic water, acquired osmotically, through its periodic exocytotic activity. The in vitro contractile vacuole, isolated in a small amount of cytosol from the Paramecium cell and confined under mineral oil, showed periodic rounding and slackening at regular intervals for an extended time. The contractile vacuole rounded against the cytosol-mineral oil boundary tension. The tension at the surface of the contractile vacuole is, therefore, assumed to increase during the rounding phase. We first estimated the tension relative to the boundary tension from the degree of compression of the contractile vacuole by the boundary. We then determined the absolute value for the tension at the surface of the contractile vacuole from the degree of bending of an elastic carbon fiber microcantilever (8 microm thick; 2 mm long), whose free end was placed at the surface of an in vitro contractile vacuole. The tension was found to increase to its maximum value of approximately 5 mN m(-)(1) when the contractile vacuole rounded. This value was more than 35 times higher than that for the slackened contractile vacuole. Electron micrographs of conventional thin sections of chemically fixed in vitro contractile vacuoles as well as those of in vivo contractile vacuoles obtained from rapid frozen and cryosubstituted cells revealed the lack of any ultrastructural evidence for the presence of a fibrous network system surrounding the contractile vacuole. Thus we conclude that the mechanism(s) by which tension is developed at the surface of the contractile vacuole membrane resides in the contractile vacuole membrane itself. We propose a hypothesis that periodic changes in the spontaneous curvature of the contractile vacuole's lipid bilayer membrane is involved in the periodic development of higher contractile vacuole membrane tension. The isolated CV promises to be an excellent model system for understanding the molecular mechanisms of the dynamics of biological membrane.

Biological and molecular characterization of cellular differentiation in Tetrahymena vorax: a potential biocontrol protozoan.

Tetrahymena vorax (T. vorax) is an indigenous fresh water protozoan with the natural biological potential to maintain a specific aquatic microbial flora by ingesting and eliminating specific microorganism. To investigate the molecular mechanisms controlling Tetrahymena vorax (T. vorax) cellular differentiation from a small-mouth vegetative cell to a voracious large-mouth carnivore capable of ingesting prey ciliates and bacteria from aquatic environments, we use DNA subtraction and gene discovery techniques to identify and isolate T. vorax differentiation-specific genes. The physiological necessity for one newly discovered gene, SUBII-TG, was determined in vivo using an antisense oligonucleotide directed against the 5' SUBII-TG DNA sequence. The barriers to delivering antisense oligonucleotides to the cytoplasm of T. vorax were circumvented by employing a new but simple procedure of processing the oligonucleotide with the differentiation stimulus, stomatin. In these studies, the antisense oligonucleotide down-regulated SUBII-TG mRNA expression, and blocked differentiation and ingestion of prey ciliates. The ability to down-regulate SUBII-TG expression with the antisense oligonucleotide suggests that the molecular mechanisms controlling the natural biological activities of T. vorax can be manipulated to further study its cellular differentiation and potential as a biocontrol microorganism.