The Use of Protamine-Coated Slides for Immobilizing Protozoa*

Fresh-water, marine or parasitic protozoa can be immobilized for microscopic observation on protaminecoated slides. They appear not to be harmed by this procedure. The type of protamine derivative and its concentration are important, best results being obtained with 0.015–0.05% (w/v) of protamine sulfate.     

Cytologische Studien an Trichocysten

Zusammenfassung Die Nesselkapseltrichocysten der Ciliaten Loxophyllum und Prorodon wurden hinsichtlich ihrer Feinstruktur sowie des Ausschleuderungsablaufes untersucht. Loxophyllum besitzt zwei morphologisch unterscheidbare Sorten von Toxicysten, wohingegen Prorodon nur eine Art dieser Organelle aufweist (Krüger, 1936).Wie an Hand gehemmter Stadien gezeigt werden kann, verläuft die Ausschleuderung bis auf geringfügige Variationen bei den drei Sorten in der gleichen Art: Schläuche, die im ruhenden Zustand bereits in den endgültigen Dimensionen in der Toxicystenkapsel eng ineinandergeschachtelt vorliegen, werden handschuhfingerförmig umgestülpt und verlassen hierdurch die Kapsel und damit den Zellkörper. Dieser Prozeß ist mit der Ausscheidung einer teils fädigen, teils amorphen Substanz gekoppelt.Der Umstülpungsvorgang der Innenstruktur der Nesselkapseltrichocysten wird mit den völlig andersartigen, während der Ausschleuderung von Mucocysten und Spindeltrichocysten ablaufenden morphologischen Veränderungen verglichen.Es zeigte sich, daß aufgrund ihrer differierenden Feinstrukturen und Funktionsweisen in den Nesselkapseltrichocysten einerseits und in den Mucocysten sowie Spindeltrichocysten andererseits zwei grundsätzlich voneinander verschiedene Organellentypen gesehen werden müssen.

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Cytological studies on trichocystsVIII. Fine structure and function of toxicysts in Loxophyllum meleagris and Prorodon teres

Summary The toxicysts of the ciliates Loxophyllum meleagris and Prorodon teres were examined with regard to fine structure and expulsion mechanism. Loxophyllum possesses two morphologically distinct types of toxicysts, whereas in Prorodon only one type is present.As can be shown by discharge inhibition experiments, the expulsion mechanisms, except for small variations, are identical in all three types: Tubes, which in their resting state lie closely packed one within the other possess already at this state their final dimensions; they are inverted, thereby leaving the capsule and thus the cell body. This process is correlated with the excretion of a substance partly filamentous, partly amorphous.The inversion of the tubes during the expulsion is compared to the fundamentally different morphological alterations during discharge of mucocysts and spindle trichocysts.The differences and similarities in fine structure and function between toxicysts on the one hand and mucocysts and spindle trichocysts on the other, indicate that two rather than three fundamentally different organelle types must be distinguished.

Herrn Prof. Dr. Friedrich Krüger (Hamburg-Blankenese) zu seinem 71. Geburtstag am 18. 8. 1973 gewidmet.     

Phylogenomics of non-model ciliates based on transcriptomic analyses

Ciliates are one of the oldest living eukaryotic unicellular organisms, widely distributed in the waters around the world. As a typical marine oligotrich ciliate, Strombidium sulcatum plays an important role in marine food webs and energy flow. Here we report the first deep sequencing and analyses of RNA-Seq data from Strombidium sulcatum. We generated 42,640 unigenes with an N50 of 1,451 bp after de novo assembly and removing rRNA, mitochondrial and bacteria contaminants. We employed SPOCS to detect orthologs from S. sulcatum and 17 other ciliates, and then carried out the phylogenomic reconstruction using 127 single copy orthologs. In phylogenomic analyses, concatenated trees have similar topological structures with concordance tree on the class level. Together with phylogenetic networks analysis, it aroused more doubts about the placement of Protocruzia, Mesodinium and Myrionecta. While epiplasmic proteins are known to be related to morphological characteristics, we found the potential relationship between gene expression of epiplasmic proteins and morphological characteristics. This work supports the use of high throughput approaches for phylogenomic analysis as well as correlation analysis between expression level of target genes and morphological characteristics.     

Phylogenetic positions of several amitochondriate protozoa

Several groups of parasitic protozoa, as represented by Giardia, Trichomonas, Entamoeba and Microsporida, were once widely considered to be the most primitive extant eukaryotic group―Archezoa. The main evidence for this is their ‘lacking mitochondria’ and possessing some other primitive features between prokaryotes and eukaryotes, and being basal to all eukaryotes with mitochondria in phylogenies inferred from many molecules. Some authors even proposed that these organisms diverged before the endosymbiotic origin of mitochondria within eukaryotes. This view was once considered to be very significant to the study of origin and evolution of eukaryotic cells (eukaryotes). However, in recent years this has been challenged by accumulating evidence from new studies. Here the sequences of DNA topoisomerase II in G. lamblia, T. vaginalis and E. histolytica were identified first by PCR and sequencing, then combining with the sequence data of the microsporidia Encephalitozoon cunicul and other eukaryotic groups of different evolutionary positions from GenBank, phylogenetic trees were constructed by various methods to investigate the evolutionary positions of these amitochondriate protozoa. Our results showed that since the characteristics of DNA topoisomerase II make it avoid the defect of ‘long-branch attraction’ appearing in the previous phylogenetic analyses, our trees can not only reflect effectively the relationship of different major eukaryotic groups, which is widely accepted, but also reveal phylogenetic positions for these amitochondriate protozoa, which is different from the previous phylogenetic trees. They are not the earliest-branching eukaryotes, but diverged after some mitochondriate organisms such as kinetoplastids and mycetozoan; they are not a united group but occupy different phylogenetic positions. Combining with the recent cytological findings of mitochondria-like organelles in them, we think that though some of them (e.g. diplo-monads, as represented by Giardia) may occupy a very low evolutionary position, generally these organisms are not as extremely primitive as was thought before; they should be poly-phyletic groups diverging after the endosymbiotic origin of mitochondrion to adapt themselves to anaerobic parasitic life.     

Changes in microbial community structure, methanogenesis and rumen fermentation in response to saponin-rich fractions from different plant materials

Aims:  Investigation of the effects of saponin-rich fractions on rumen fermentation, methane production and the microbial community.
Methods and Results:  Saponins were extracted from Carduus , Sesbania and Knautia leaves and fenugreek seeds. Two levels of saponin-rich fractions with a substrate were incubated using the Hohenheim gas method. Methane was measured using an infrared-based methane analyser and microbial communities using quantitative PCR. On addition of saponin-rich fractions, methane and short-chain fatty acid production was not affected. The protozoal counts decreased by 10–39%. Sesbania saponins decreased methanogen population by 78%. Decrease in ruminal fungal population (20–60%) and increase in Fibrobacter succinogenes (21–45%) and Ruminococcus flavefaciens (23–40%) were observed.
Conclusions:  The saponins evaluated possessed anti-protozoal activity; however, this activity did not lead to methane reduction. Fenugreek saponins seemed to have potential for increasing rumen efficiency. The saponins altered the microbial community towards proliferation of fibre-degrading bacteria and inhibition of fungal population.
Significance and Impact of the Study:  The uni-directional relationship between protozoal numbers and methanogenesis, as affected by saponins, is not obligatory. All saponins might not hold promise for decreasing methane production from ruminants.     

Comparison of fatty acid composition of bacterial and protozoal fractions in rumen fluid of sheep fed diet supplemented with sunflower, rapeseed and linseed oils

The objective of this study was to investigate effects of oil supplements on the composition of fatty acids (FA), especially of trans11-C18:1 (vaccenic acid, TVA) and cis9, trans11-C18:2 conjugated linoleic acid (c9,t11-CLA), in bacterial (BF) and protozoal (PF) fractions of rumen fluid of sheep that was fractionated centrifugation. Four sheep were fed a diet consisting of meadow hay (960 g dry matter (DM)/day) and of barley grain (240 g DM/day), with sunflower oil (SO), rapeseed oil (RO) or linseed oil (LO) as supplements (60 g/day) in a Latin square design. The oils were used as they are rich in linoleic acid (SO, 533 g/kg of FA), oleic acid (RO, 605 g/kg of FA) and α-linolenic acid (LO, 504 g/kg of FA). Compared to the control (i.e., without oils), oil supplements influenced the concentration of unsaturated (UFA) and saturated fatty acids (SFA). In both BF and PF, the main fatty acids were palmitic and stearic, but PF contained a higher proportion of TVA and c9,t11-CLA than BF. In PF, TVA concentrations, ranked by oil supplement, were SO > RO > LO > Control (174, 150, 118, 74 g/kg of FA, respectively) and the c9,t11-CLA concentrations were RO > SO > LO > Control (59, 51, 27 and 15 g/kg of FA, respectively). Concentrations of c9,t11-CLA in PF were two to three times higher than in BF with all the oil supplements versus the control. Oil treatments impacted the c9,t11-CLA concentration in the fractions, especially SO and RO. The protozoal fraction contained a higher proportion of TVA and c9,t11-CLA than did the bacterial fraction, and dietary addition of SO, RO and LO resulted in a higher incorporation of TVA into both bacterial and protozoal microbial fractions, which probably positively affected TVA flow from the rumen.     

Six years of in situ CO2 enrichment evoke changes in soil structure and soil biota of nutrient-poor grassland

Nutrient‐poor grassland on a silty clay loam overlying calcareous debris was exposed to elevated CO₂ for six growing seasons. The CO₂ exchange and productivity were persistently increased throughout the experiment, suggesting increases in soil C inputs. At the same time, elevated CO₂ lead to increased soil moisture due to reduced evapotransporation. Measurements related to soil microflora did not indicate increased soil C fluxes under elevated CO₂. Microbial biomass, soil basal respiration, and the metabolic quotient for CO₂ (qCO₂) were not altered significantly. PLFA analysis indicated no significant shift in the ratio of fungi to bacteria. 0.5 m KCl extractable organic C and N, indicators of changed DOC and DON concentrations, also remained unaltered. Microbial grazer populations (protozoa, bacterivorous and fungivorous nematodes, acari and collembola) and root feeding nematodes were not affected by elevated CO₂. However, total nematode numbers averaged slightly lower under elevated CO₂ (?16%, ns) and nematode mass was significantly reduced (?43%, P = 0.06). This reduction reflected a reduction in large‐diameter nematodes classified as omnivorous and predacious. Elevated CO₂ resulted in a shift towards smaller aggregate sizes at both micro‐ and macro‐aggregate scales; this was caused by higher soil moisture under elevated CO₂. Reduced aggregate sizes result in reduced pore neck diameters. Locomotion of large‐diameter nematodes depends on the presence of large enough pores; the reduction in aggregate sizes under elevated CO₂ may therefore account for the decrease in large nematodes. These animals are relatively high up the soil food web; this decline could therefore trigger top‐down effects on the soil food web. The CO₂ enrichment also affected the nitrogen cycle. The N stocks in living plants and surface litter increased at elevated CO₂, but N in soil organic matter and microbes remained unaltered. Nitrogen mineralization increased markedly, but microbial N did not differ between CO₂ treatments, indicating that net N immobilization rates were unaltered. In summary, this study did not provide evidence that soils and soil microbial communities are affected by increased soil C inputs under elevated CO₂. On the contrary, available data (¹³C tracer data, minirhizotron observations, root ingrowth cores) suggests that soil C inputs did not increase substantially. However, we provide first evidence that elevated CO₂ can reduce soil aggregation at the scale from µ m to mm scale, and that this can affect soil microfaunal populations.     

Natural Chagas disease in four baboons

Background  Chagas disease is common in Central and South America and the southern United States. The causative agent is Trypanosoma cruzi (order Kinetoplastida, family Trypanosomatidae), a kinetoplastid protozoan parasite of humans and other vertebrates. It is a serious public health issue and the leading cause of heart disease and cardiovascular death in Central and South America. In 1984, a colony baboon was discovered to be infected with T. cruzi .
Methods  As the initial diagnosis was made by microscopic observation of the amastigote forms of T. cruzi in myocardial fibers, T. cruzi amastigotes have been identified in three additional baboons.
Results  The primary findings were similar in all four baboons and were congestive heart failure with edema of dependent areas, hydrothorax, hydropericardium, and multifocal to diffuse lymphoplasmacytic myocarditis.
Conclusions  A baboon animal model of Chagas disease could contribute significantly to the development of therapies for the disease in humans.     

Vertical Micro‐Zonation of Testate Amoebae and Ciliates in Peatland Waters in Relation to Potential Food Resources and Grazing Pressure

The aim of this study was to examine the community structure and vertical micro‐distribution of testate amoebae and ciliates in a raised bog in eastern Poland, as well as to assess the influence of potential food resources (Chl‐a, bacteria, heterotrophic flagellates) and predators (rotifers and copepods) on protozoa communities. Samples were taken from surface, bottom and interstitial waters. At each type of micro‐habitat and each sampling date water was sampled using a plexiglass corer or mini‐piezometers. Additionally, in order to evaluate grazing pressure, field enclosures were used in which metazoan abundance and composition was manipulated by size‐fractionation. Over experiments, metazoan populations shifted from dominance of rotifers to copepods. In the first experiment, with rotifers dominating, metazoa had only a modest predatory impact on the protozoa. In contrast, the second experiment, with copepods prevailing, demonstrated a clear top‐down control of the protozoa communities by metazoan. The density and biomass of protozoa significantly differed between the studied stations, with the lowest numbers in the interstitial water and the highest in the surface water. Surface sampling were dominated by mixotrophic taxa, whereas the deepest sampling level was characterized by increase in the proportion of bacterivore species. These differences between micro‐habitats may be due to differences in environmental conditions (food resources and grazing pressure). Ordination analysis revealed that bacteria can strongly regulate the abundance and taxonomic composition of protozoa in the bottom and interstitial waters. Metazoan predators could be the main regulators of protozoa communities in surface water. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)