Molecular and biochemical markers for monitoring dynamic shifts of cellulolytic protozoa in Reticulitermes flavipes

Lower termites rely on cellulolytic protozoa to aid in the digestion of their wood-based diet. However, despite the major contribution of protozoa to the lower termite digestive system, few techniques have been developed to monitor shifts in protozoan populations. This study investigated whether quantitative real-time PCR (qRT-PCR) and/or cellulase enzyme assays can be used to monitor changes of cellulolytic protozoan populations in the lower termite, Reticulitermes flavipes (Kollar). Previously developed cellulase primer sets were used to test for changes in cellulase gene expression, while three different cellulase enzyme assays were used to assess changes in cellulase enzyme activity. The results from this study indicate that qRT-PCR is a reliable method to monitor shifts in cellulolytic protozoan populations. Specifically, qRT-PCR can serve as a useful monitoring technique during high-throughput screening of novel termite control agents such as cellulase inhibitors, and help to answer questions relating to whether or not such control agents impact cellulolytic protozoan populations.     

The Role of Protozoa in the Activated-Sludge Process

Ciliates are the dominant protozoa in activated sludge, andthose which either attach themselves to or crawl over the surfaceof the sludge are the most common. Experimental work has demonstratedthat ciliated protozoa are essential for the production of agood quality effluent. Without ciliates, highly turbid, lowquality effluents are obtained. Experimental evidence furthersuggests that the predatory activities of the ciliates uponthe dispersed growths of bacteria are responsible for clarificationand for the reduction of coliform numbers during the activated-sludgeprocess. Mathematical models have been devised which take intoaccount both the bacterial and the protozoan populations. Thepopulations have been simulated on a computer and the resultsobtained in this way have helped to explain many of the observationsthat have been made upon the microbial populations found infull-scale activated-sludge plants.     

Colony-dependent sex differences in protozoan communities of the lower termite <Emphasis Type="Italic">Reticulitermes speratus</Emphasis> (Isoptera: Rhinotermitidae)

In many animals, sex differences in hormones, behavior, and immunity lead to differences in their gut microbial communities. One of the best-known examples of mutualistic symbiosis is that between lower termites and their intestinal protozoa. Although differences in the protozoan communities of different castes have been studied in lower termites, nothing is known about the sex differences in protozoan communities in neuter castes. Here, we show that termite workers have different protozoan communities according to sex depending on the colony. We investigated the communities of symbiotic protozoa living in lower termites, Reticulitermes speratus, and how they are affected by sex and caste. Workers had the largest numbers of protozoa, followed by soldiers, whereas reproductives (primary kings and secondary queens) had no protozoa. Workers showed colony-dependent sex differences in the total abundance of protozoa, whereas soldiers showed no such sex differences. There were significant sex effect and/or interaction effect between colony and sex in abundances of five species of protozoa in workers. Workers also showed significant sex differences and/or colony-dependent sex differences in proportion of six species of protozoa. These may result in sex differences in the host–symbiont interaction due to physiological or behavioral sex differences in workers that have not been recognized previously. This study has an important implication: although workers are not engaged in reproduction, their potential sex difference may affect various aspects of social interactions.     

4-Methylumbelliferyl-beta-N-Acetylglucosaminide Hydrolysis by a High-Affinity Enzyme, a Putative Marker of Protozoan Bacterivory

Hydrolysis of an artificial fluorogenic substrate, 4-methylumbelliferyl-beta-N-acetylglucosaminide, has been studied in a monoculture predator-prey system with either a flagellate (Bodo saltans) or a ciliate (Cyclidium sp.) fed upon pure bacterial culture (Aeromonas hydrophila or Alcaligenes xylosoxidans). Aeromonas hydrophila produced a low-affinity beta-N-acetylglucosaminidase-like enzyme (K(m), >100 mumol liter) but Alcaligenes xylosoxidans did not. Inoculation of both bacterial strains with bacterivorous protozoa induced the occurrence of another, high-affinity, beta-N-acetylglucosaminidase-like enzyme (K(m), <0.5 mumol liter). The latter enzyme showed significant, close correlations with total grazing rates of both B. saltans (r = 0.96) and Cyclidium sp. (r = 0.89) estimated by using uptake of fluorescently labelled bacteria. Further significant correlations between several protozoan parameters and kinetic parameters of this enzyme suggest its likely protozoan origin. If both types of enzyme occurred together, they could be satisfactorily distinguished by using kinetic data analysis. Hence, measurements of beta-N-acetylglucosaminidase-like activities might be promising to use to improve estimations of protozoan bacterivory.     

Aquaporins from pathogenic protozoan parasites: structure, function and potential for chemotherapy

Infectious diseases, caused by protozoa, such as malaria, sleeping sickness, Chagas' disease or leishmaniasis, are a global threat. The increase in the number of affected individuals and the rapid spread of drug-resistant strains call for specific novel strategies to combat human pathogenic parasites. In the search for novel drug targets, transport proteins for nutrients and metabolites of the parasite-host interface are getting into focus. The present review summarizes and discusses the currently available results on protozoan aquaporins. Various genes coding for aquaporin water and solute channels have been identified in the protozoan genomes and they are probable elements of the parasite's cell membrane. Phylogenetic analysis reveals that individual aquaporin genes are of bacterial or plant origin. So far, six protozoan aquaporins have been cloned and functionally characterized. Typically, these are bifunctional channels and pass water at intermediate to high rates as well as uncharged solutes. In the present review, amino acid compositions of the individual pore entries are compared and permeability properties are attributed to specific protein features. Furthermore, possible physiological roles in osmotic protection and metabolism are discussed. Finally, the potential of protozoan aquaporins for use as a target or entry pathway for chemotherapeutic compounds is reviewed.     

Dynamics of grazing protozoa follow that of microalgae in natural biofilm communities

This study investigates the dynamics of protozoan community in biofilms formed on inert artificial surfaces suspended in various freshwater environments. The results also test the hypothesis that the dynamics of protozoan and microalgal communities in biofilms are interdependent because the latter form one of the major food items of benthic protozoa. Cleaned glass slides were suspended in surface waters at four sampling locations to collect biofilm communities. The glass slides after retrieval were observed under a microscope for diatom and protozoan density and their generic composition. Members of protozoa belonging to phylum Sarcomastigophora dominated the protozoan community followed by phylum Ciliophora in all sampling locations. The variation of protozoan feeding groups showed an initial abundance of autotrophs/holophytes which gave way to heterotrophs, predators, and bacterivores towards the end of the study. The density and generic composition of protozoa varied significantly with the age of biofilm and sampling location. The density variation of protozoa followed that of diatoms in all four sampling locations and this has resulted in a significant positive correlation between diatom and protozoan densities. This suggests the dependency and/or food web connectedness of these two communities in natural biofilms.     

Protozoan growth rates on secondary-metabolite-producing Pseudomonas spp. correlate with high-level protozoan taxonomy

Different features can protect bacteria against protozoan grazing, for example large size, rapid movement, and production of secondary metabolites. Most papers dealing with these matters focus on bacteria. Here, we describe protozoan features that affect their ability to grow on secondary-metabolite-producing bacteria, and examine whether different bacterial secondary metabolites affect protozoa similarly. We investigated the growth of nine different soil protozoa on six different Pseudomonas strains, including the four secondary-metabolite-producing Pseudomonas fluorescens DR54 and CHA0, Pseudomonas chlororaphis MA342 and Pseudomonas sp. DSS73, as well as the two nonproducers P. fluorescens DSM50090(T) and P. chlororaphis ATCC43928. Secondary metabolite producers affected protozoan growth differently. In particular, bacteria with extracellular secondary metabolites seemed more inhibiting than bacteria with membrane-bound metabolites. Interestingly, protozoan response seemed to correlate with high-level protozoan taxonomy, and amoeboid taxa tolerated a broader range of Pseudomonas strains than did the non-amoeboid taxa. This stresses the importance of studying both protozoan and bacterial characteristics in order to understand bacterial defence mechanisms and potentially improve survival of bacteria introduced into the environment, for example for biocontrol purposes.     

Persistence of free-living protozoan communities across rearing cycles in commercial poultry houses

The introduction and survival of zoonotic bacterial pathogens in poultry farming have been linked to bacterial association with free-living protozoa. To date, however, no information is available on the persistence of protozoan communities in these environments across consecutive rearing cycles and how it is affected by farm- and habitat-specific characteristics and management strategies. We therefore investigated the spatial and temporal dynamics of free-living protozoa in three habitats (pipeline, water, and miscellaneous samples) in three commercial poultry houses across three rearing cycles by using the molecular fingerprinting technique denaturing gradient gel electrophoresis (DGGE). Our study provides strong evidence for the long-term (ca. 6-month) persistence of protozoa in broiler houses across consecutive rearing cycles. Various free-living protozoa (flagellates, ciliates, and amoebae), including known vectors of bacterial pathogens, were observed during the down periods in between rearing cycles. In addition, multivariate analysis and variation partitioning showed that the protozoan community structure in the broiler houses showed almost no change across rearing cycles and remained highly habitat and farm specific. Unlike in natural environments, protozoan communities inside broiler houses are therefore not seasonal. Our results imply that currently used biosecurity measures (cleaning and disinfection) applied during the down periods are not effective against many protozoans and therefore cannot prevent potential cross-contamination of bacterial pathogens via free-living protozoa between rearing cycles.     

Signaling of immune system cells by glycosylphosphatidylinositol (GPI) anchor and related structures derived from parasitic protozoa

Glycosylphosphatidylinositol (GPI) anchor and glycoinositolphospholipid (GIPL) are abundant molecules present in the membrane of parasitic protozoa that are common etiologic agents of medical and veterinary diseases. Recent studies have documented the immunostimulatory/regulatory activity of protozoan-derived GPI-anchors and related structures. Among the bioactivity displayed by the protozoan-derived GPI-anchor is the ability to elicit the synthesis of pro-inflammatory cytokines as well as nitric oxide by host macrophages. In contrast, at high concentrations GIPL and lipophosphoglycan (LPG) derived from protozoan parasites suppress several functions of the host immune system. Additionally, the protozoan-derived GPI-anchor and GIPL have been shown to serve as targets for both specific B and NK-T lymphocyte responses. This information extends our knowledge about parasite molecules that stimulate/regulate the host immune system during protozoan infection. The identification of receptor(s) and signaling pathways triggered by these GPI-related glycolipids may provide new insights for the development of therapies that inhibit detrimental immune responses or potentiate beneficial immune responses observed during infection with protozoan parasites.     

Plasmid encoded antibiotics inhibit protozoan predation of Escherichia coli K12

Bacterial plasmids and phages encode the synthesis of toxic molecules that inhibit protozoan predation. One such toxic molecule is violacein, a purple pigmented, anti-tumour antibiotic produced by the Gram-negative soil bacterium Chromobacterium violaceum. In the current experiments a range of Escherichia coli K12 strains were genetically engineered to produce violacein and a number of its coloured, biosynthetic intermediates. A bactivorous predatory protozoan isolate, Colpoda sp.A4, was isolated from soil and tested for its ability to ‘graze’ on various violacein producing strains of E. coli K12. A grazing assay was developed based on protozoan “plaque” formation. Using this assay, E. coli K12 strains producing violacein were highly resistant to protozoan predation. However E. coli K12 strains producing violacein intermediates, showed low or no resistance to predation. In separate experiments, when either erythromycin or pentachlorophenol were added to the plaque assay medium, protozoan predation of E. coli K12 was markedly reduced. The inhibitory effects of these two molecules were removed if E. coli K12 strains were genetically engineered to inactivate the toxic molecules. In the case of erythromycin, the E. coli K12 assay strain was engineered to produce an erythromycin inactivating esterase, PlpA. For pentachlorophenol, the E. coli K12 assay strain was engineered to produce a PCP inactivating enzyme pentachlorophenol-4-monooxygenase (PcpB). This study indicates that in environments containing large numbers of protozoa, bacteria which use efflux pumps to remove toxins unchanged from the cell may have an evolutionary advantage over bacteria which enzymatically inactivate toxins.     

Purine and pyrimidine transport in pathogenic protozoa: from biology to therapy

Purine salvage is an essential function for all obligate parasitic protozoa studied to date and most are also capable of efficient uptake of preformed pyrimidines. Much progress has been made in the identification and characterisation of protozoan purine and pyrimidine transporters. While the genes encoding protozoan or metazoan pyrimidine transporters have yet to be identified, numerous purine transporters have now been cloned. All protozoan purine transporter-encoding genes characterised to date have been of the Equilibrative Nucleoside Transporter family conserved in a great variety of eukaryote organisms. However, these protozoan transporters have been shown to be sufficiently different from mammalian transporters to mediate selective uptake of therapeutic agents. Recent studies are increasingly addressing the structure and substrate recognition mechanisms of these vital transport proteins.     

Sociobiology of biodegradation and the role of predatory protozoa in biodegrading communities

Predatory protozoa are known to enhance biodegradation by bacteria in a variety of systems including rumen. This is apparently counterintuitive since many protozoa do not themselves produce extracellular degradative enzymes and prey upon bacterial degraders. We propose a mechanism of protozoal enhancement of bacterial biodegradation based on the sociobiology of biodegradation. Since extracellular enzyme production by degraders involves a cost to the bacterial cell, cheaters that do not make the enzyme will have a selective advantage. In the presence of cheaters, degraders that physically attach to water-insoluble substrate will have a selective advantage over free-floating degraders. On the other hand, cheaters will benefit by being free floaters since they consume the solubilized products of extracellular enzymes. Predatory ciliated protozoa are more likely to consume free-floating cheaters. Thus, due to protozoan predation a control is exerted on the cheater population. We illustrate the dynamics of such a system with the help of a computer simulation model. Available data on rumen and other biodegradation systems involving protozoa are compatible with the assumptions and predictions of the model.     

Response of Fresh-Water Protozoan Artificial Communities to Metals*

An artificial fresh-water protozoan community was subjected to different concentrations of Zn and Cu in a test system consisting of Plexiglas troughs through which pond water flowed continuously. Although the % survival of colonizing species exposed to Cu or Zn fluctuated greatly at each concentration, the range of toxicity for each compound allowed comparison of protozoa with other organisms with respect to resistance to heavy metal toxicity. Individual protozoan species also were exposed for 3 hr to Zn, Cu, Cr, phenol, Pb, Mn, Co, HNO₃, acetic acid, Al, Sn, and HCl to derive time to death curves. Protozoa tested appeared to be more resistant than Daphnia to phenol, K₂C₂O₇, and Cu; however, some species were more sensitive than Daphnia to Zn, nitric acid, and HCl. This suggests that the sensitivity of protozoa to toxicants may be either more or less than that of macroinvertebrates and that information does not suffice to predict sensitivity. Moreover, the relative sensitivity of protozoa to various toxicants will not always be the same, i.e. species X may be twice as tolerant to a toxicant as species Y but its relative sensitivity may be quite different for another toxicant.     

哈尔滨人工湖泊中原生动物群落变化规律

2003年3月~2004年2月调查了哈尔滨人工湖泊原生动物群落多样性的变化规律,应用PFU法研究了原生动物群落季节变化演替的特征。共观察到各类原生动物120种,其中鞭毛虫53种,占原生动物总数的44%,肉足虫9种占原生动物总数的8%,纤毛虫58种占原生动物总数的48%,分析了原生动物在该湖的组成特点。原生动物在此人工湖泊中1个周年内的表现规律为植物性鞭毛虫多,动物性鞭毛虫少;纤毛虫多,肉足虫少。原生动物群落的结构参数(种类组成、个体丰度和多样性指数)因水质的变化而发生变化。在此湖中原生动物的优势种为喇叭虫属(S tentor)、眼虫属(E ug lena)、游仆虫属(E up lote)、隐滴虫属(C ryp tom onas),常年的居留种为眼虫属(E ug lena)、弹跳虫属(H a lteria)、侠盗虫属(S trobilid ium)、隐滴虫属(C ryp tom onas)。全年3个采样站的生物多样性指数在1.912~7.473之间。原生动物在1个周年中平均个体丰度出现的规律依次递减的顺序是秋季、夏季、春季、冬季。采样站1的平均个体丰度在四个季节中均高于采样站2、3。     

Impact of protozoan grazing on nitrification and the ammonia- and nitrite-oxidizing bacterial communities in activated sludge

In activated sludge, protozoa feed on free-swimming bacteria and suspended particles, inducing flocculation and increasing the turnover rate of nutrients. In this study, the effect of protozoan grazing on nitrification rates under various conditions in municipal activated sludge batch reactors was examined, as was the spatial distribution of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) within the activated sludge. The reactors were monitored for ammonia, nitrite, nitrate, and total nitrogen concentrations, and bacterial numbers in the presence and absence of cycloheximide (a protozoan inhibitor), allylthiourea (an inhibitor of ammonia oxidation), and EDTA (a deflocculating agent). The accumulations of nitrate, nitrite, and ammonia were lower in batches without than with protozoa grazing. Inhibition of ammonia oxidation also decreased the amount of nitrite and nitrate accumulation. Inhibiting protozoan grazing along with ammonia oxidation further decreased the amounts of nitrite and nitrate accumulated. Induction of deflocculation led to high nitrate accumulation, indicating high levels of nitrification; this effect was lessened in the absence of protozoan grazing. Using fluorescent in situ hybridization and confocal laser scanning microscopy, AOB and NOB were found clustered within the floc, and inhibiting the protozoa, inhibiting ammonia oxidation, or inducing flocculation did not appear to lower the number of AOB and NOB present or affect their position within the floc. These results suggest that the AOB and NOB are present but less active in the absence of protozoa.     

Protozoan Response to the Addition of Bacterial Predators and Other Bacteria to Soil

Representatives of several categories of bacteria were added to soil to determine which of them might elicit responses from the soil protozoa. The various categories were nonobligate bacterial predators of bacteria, prey bacteria for these predators, indigenous bacteria that are normally present in high numbers in soil, and non-native bacteria that often find their way in large numbers into soil. The soil was incubated and the responses of the indigenous protozoa were determined by most-probable-number estimations of total numbers of protozoa. Although each soil was incubated with only one species of added bacteria, the protozoan response for the soil was evaluated by using most-probable-number estimations of several species of bacteria. The protozoa did not respond to incubation of the soil with either Cupriavidus necator, a potent bacterial predator, or one of its prey species, Micrococcus luteus. C. necator also had no effect on the protozoa. Therefore, in this case, bacterial and protozoan predators did not interact, except for possible competition for bacterial prey cells. The soil protozoa did not respond to the addition of Arthrobacter globiformis or Bacillus thuringiensis. Therefore, the autochthonous state of Arthrobacter species in soil and the survival of B. thuringiensis were possibly enhanced by the resistance of these species to protozoa. The addition of Bacillus mycoides and Escherichia coli cells caused specific responses by soil protozoa. The protozoa that responded to E. coli did not respond to B. mycoides or any other bacteria, and vice versa. Therefore, addition to soil of a nonsoil bacterium, such as E. coli, did not cause a general increase in numbers of protozoa or in protozoan control of the activities of other bacteria in the soil.     

An automated technique for most-probable-number (MPN) analysis of densities of phagotrophic protists with lux AB labelled bacteria as growth medium.

An automated modification of the most-probable-number (MPN) technique has been developed for enumeration of phagotrophic protozoa. The method is based on detection of prey depletion in micro titre plates rather than on presence of protozoa. A transconjugant Pseudomonas fluorescens DR54 labelled with a luxAB gene cassette was constructed, and used as growth medium for the protozoa in the micro titre plates. The transconjugant produced high amounts of luciferase which was stable and allowed detection for at least 8 weeks. Dilution series of protozoan cultures and soil suspensions were inoculated into micro titre plates amended with a suspension of the transconjugant. After 45 days measurement of light emission allowed detection of individual wells in the titre plates, where protozoan grazing had removed the inoculated bacteria.     

Mobility of protozoa through narrow channels

Microbes in the environment are profoundly affected by chemical and physical heterogeneities occurring on a spatial scale of millimeters to micrometers. Physical refuges are critical for maintaining stable bacterial populations in the presence of high predation pressure by protozoa. The effects of microscale heterogeneity, however, are difficult to replicate and observe using conventional experimental techniques. The objective of this research was to investigate the effect of spatial constraints on the mobility of six species of marine protozoa. Microfluidic devices were created with small channels similar in size to pore spaces in soil or sediment systems. Individuals from each species of protozoa tested were able to rapidly discover and move within these channels. The time required for locating the channel entrance from the source well increased with protozoan size and decreased with channel height. Protozoa of every species were able to pass constrictions with dimensions equal to or smaller than the individual's unconstrained cross-sectional area. Channel geometry was also an important factor affecting protozoan mobility. Linear rates of motion for various species of protozoa varied by channel size. In relatively wide channels, typical rates of motion were 300 to 500 microm s(-1) (or about 1 m per hour). As the channel dimensions decreased, however, motilities slowed more than an order of magnitude to 20 microm s(-1). Protozoa were consistently observed to exhibit several strategies for successfully traversing channel reductions. The empirical results and qualitative observations resulting from this research help define the physical limitations on protozoan grazing, a critical process affecting microbes in the environment.     

Protozoan Grazing Increases Mineralization of Naphthalene in Marine Sediment

Bacterial decomposition of organic matter is frequently enhanced when protozoa are present. Various mechanisms have been proposed to account for this phenomenon, including effects associated with grazing by protozoa (such as increased recycling of limiting nutrients, removal of senescent cells, or reduction of competition among bacteria) and indirect effects of grazers (such as excretion of bacterial growth factors). Few studies have examined the role of protozoa in bacterial degradation of xenobiotic compounds in sediment containing a natural community of microbes. The effect of protozoa on mineralization of naphthalene was investigated in this study. Laboratory experiments were conducted using field-contaminated estuarine sediment, with the indigenous microbial populations. Mineralization of naphthalene was up to four times greater in treatments with actively grazing protozoa than in treatments containing the grazing inhibitor cytochalasin B. Control experiments confirmed that the grazing inhibitor was not toxic to ciliates but did prevent them from grazing. The grazing inhibitor did not affect growth rates of a mixed culture of sediment bacteria or a pure polycyclic-aromatic-hydrocarbon-degrading strain. Once grazing had been inhibited, supplementing treatments with inorganic N and P, glucose, or additional protozoa failed to stimulate naphthalene mineralization. Naphthalene-degrading bacteria were four to nine times less abundant when protozoan grazing was suppressed. We suggest that protozoa enhance naphthalene mineralization by selectively grazing on those sediment bacteria that ordinarily would outcompete naphthalene-degrading bacteria.     

Cell death and human intestinal protozoa: a brief overview

Protozoan programmed cell death or apoptosis is an important factor in the survival of the parasite and its pathogenicity. The most amazing aspect of protozoan cell death is in its molecular architecture. To date, protozoa lack most of the components of the highly complex cell death machinery studied in multicellular organisms. Hence the unique apoptotic machinery in protozoa can be exploited for the development of therapeutic drugs and diagnostic markers. This review focuses on human intestinal protozoa undergoing cell death and inducing or inhibiting host cell apoptosis. The first part of this review focuses on intestinal protozoa that undergo PCD under various stress conditions. The second part focuses on protozoa that induce or inhibit PCD in their host cell. Although these intestinal parasites differ in their mechanism of infection and intracellular localization, they may activate conserved cell death pathways within themselves and in the host cell. Understanding conserved cell death pathways in the intestinal protozoa and their host-parasite PCD relationship may lead to drug targets which can be used for a broad range of parasitic diseases.