Differential drivers of intraspecific and interspecific competition during malaria-helminth co-infection.

Various host and parasite factors interact to determine the outcome of infection. We investigated the effects of two factors on the within-host dynamics of malaria in mice: initial infectious dose and co-infection with a helminth that limits the availability of red blood cells (RBCs). Using a statistical, time-series approach to model the within-host 'epidemiology' of malaria, we found that increasing initial dose reduced the time to peak cell-to-cell parasite propagation, but also reduced its magnitude, while helminth co-infection delayed peak cell-to-cell propagation, except at the highest malaria doses. Using a mechanistic model of within-host infection dynamics, we identified dose-dependence in parameters describing host responses to malaria infection and uncovered a plausible explanation of the observed differences in single vs co-infections. Specifically, in co-infections, our model predicted a higher background death rate of RBCs. However, at the highest dose, when intraspecific competition between malaria parasites would be highest, these effects of co-infection were not observed. Such interactions between initial dose and co-infection, although difficult to predict a priori, are key to understanding variation in the severity of disease experienced by hosts and could inform studies of malaria transmission dynamics in nature, where co-infection and low doses are the norm.

Myiasis (fly disease) and insectal disease generally are causing mental illness.

Mental illness by which psychosis is meant here is known to be caused mainly by imbalances of certain neurotransmitters in the brain. But, what is causing these imbalances? There has been a recent flurry of interest focusing on the possibility of parasitical disease. The appropriateness of this is based on the fact that organisms of the animal kingdom produce the same neurotransmitters. In fact stinging insects release them in their venoms. The proposal here is that insect larval parasites acting on the human brain and body may release such neurotransmitters and cause imbalances and altered mental states and is supported by the occurrence of previously unexplained physical symptoms such as; diarrhoea, constipation, spasms, anaemia, bloating, insomnia, headache, migraine, weight loss, low blood pressure, low grade fever, amnesia and signs of allergy which may accompany mental illness. Some of these symptoms have been previously attributed to the medications prescribed to alleviate the psychotic symptoms but, many are also parasitical signs. It is proposed that the minute larvae may make sudden movements and may be highly motile and may move from pressure, hence evading the phlebotomist's needle. There is also the testimony of those with delusional parasitosis and related addictions, I propose the regularity with which humans are bitten, stung and have their foods infected with insects at all stages as a demonstration of how insectal disease may have the potential for common infection and disease; mental and physical.

Diminished metabolic responses to centrally-administered apelin-13 in diet-induced obese rats fed a high-fat diet.

The central administration of apelin, a recently identified adipokine, has been shown to affect food and water intake. The present study investigated whether body weight could affect an animal's response to apelin. The effects of centrally-administered apelin-13 on food and water intake, activity and metabolic rate were investigated in adult male diet-induced obese (DIO) rats fed either a high fat (32%) or control diet. Rats were administered i.c.v. apelin-13, 15-30 min prior to lights out, and food and water intake, activity and metabolic rate were assessed. Intracerebroventricular administration of apelin-13 decreased food and water intake and respiratory exchange ratio in DIO rats on the control diet, but had no effect in DIO rats on the high-fat diet. In an effort to identify potential central mechanisms explaining the observed physiological responses, the mRNA level of the apelin receptor, APJ, was examined in the hypothalamus. A high-fat diet induced an up-regulation of the expression of the receptor. Apelin induced a down-regulation of the receptor, but only in the DIO animals on the high-fat diet. In conclusion, we have demonstrated a diminished central nervous system response to apelin that is coincident with obesity.

Biodiversity of terrestrial protozoa appears homogeneous across local and global spatial scales.

Free-living microbes are by far the most abundant group of organisms in the biosphere, yet estimates of global species richness remain nebulous, and there is no consensus regarding the likely geographical distribution of species. Both uncertainties are addressed by the suggestion that the vast abundance of microbes may drive their ubiquitous random dispersal; for this would also make it likely that global species richness is relatively low. Here we test the idea of ubiquitous dispersal of testate amoebae and ciliates living in soil. We analysed their abundance and species richness in 150 soil samples collected from the one-hectare grassland site at Sourhope in Scotland, and in comparable published data from 1500 soil samples collected worldwide. Following taxonomic revision and removal of synonyms, there remained a total of 186 taxa (91 testate and 95 ciliate) recorded from both Sourhope and other places in the world. A fundamental pattern of random spatial distribution of species was revealed in species that are relatively rare. This probably arises from random dispersal, for when localised population growth occurs, the distributions become aggregated, as in virtually all metazoan species. We find no evidence for geographically-restricted protozoan morphospecies at spatial scales of 4 m2, 10,000 m2, or worldwide. Species that are locally rare or abundant are similarly rare or abundant on a global scale. Approximately one third of the global diversity of soil protozoa was found at the one-hectare grassland site in Scotland, but this is a minimum figure, for recorded species richness is proportional to sampling effort, as shown here.

Estimating the growth potential of the soil protozoan community.

We have developed a method for determining the potential abundance of free-living protozoa in soil. The method permits enumeration of four major functional groups (flagellates, naked amoebae, testate amoebae, and ciliates) and it overcomes some limitations and problems of the usual 'direct' and 'most probable number' methods. Potential abundance is determined using light microscopy, at specific time intervals, after quantitative re-wetting of air-dried soil with rain water. No exogenous carbon substrates or mineral nutrients are employed, so the protozoan community that develops is a function of the resources and inhibitors present in the original field sample. The method was applied to 100 soil samples (25 plots x 4 seasons) from an upland grassland (Sourhope, Southern Scotland) in the UK. Median abundances for all four functional groups lie close to those derived from the literature on protozoa living in diverse soil types. Flagellates are the most abundant group in soil, followed by the naked amoebae, then the testate amoebae and ciliates. This order is inversely related to typical organism size in each group. Moreover, preliminary evidence indicates that each functional group contains roughly the same number of species. All of these observations would be consistent with soil having fractal structure across the size-scale perceived by protozoa. The method described will be useful for comparing the effects on the soil protozoan community of different soil treatments (e.g. liming and biocides).

Apparent global ubiquity of species in the protist genus Paraphysomonas.

Evidence is presented for the ubiquity of protist species. Using the example of protists that leave traces (siliceous scales) of their recent population growth, we show that most - perhaps all species in the genus Paraphysomonas, are ubiquitous. Of the species recorded in surveys carried out worldwide, we have identified 78% of their number in 0.1 cm2 of sediment collected from a freshwater pond (total area 10(8) cm2) in England. Moreover, the pond appears to act like a microcosm of aquatic environments in general, for species that are globally rare or abundant, are likewise rare or abundant in the pond. We assume that the rate of neutral migration to the pond is greatest for the globally abundant species. As these species are probably capable of growth in a broad range of conditions, they will more frequently encounter the environment they require for population growth. Thus globally abundant species are also locally abundant in the pond - a pattern that will be amplified by periodic cyst production. Ubiquitous dispersal is probably driven by very high absolute abundance of individuals, and the water column of the pond was estimated to support >10(14) Paraphysomonas individuals. Ubiquity will dampen rates of speciation, and the evidence presented here indicates that global species richness of Paraphysomonas is indeed modest - perhaps close to what is already known.

The phylogenetic position and ultrastructure of the uncultured bacterium Achromatium oxaliferum.

Achromatium oxaliferum is a large, morphologically conspicuous, sediment-dwelling bacterium. Nothing is known concerning its phylogeny and it has eluded all attempts at laboratory cultivation. The limited physiological description of A. oxaliferum has been based on morphological features of the bacterium such as the presence of intracellular sulphur inclusions. A. oxaliferum cells were purified from a wetland region close to Rydal Water (Cumbria, UK). Scanning and transmission electron microscopy revealed that a number of morphologically distinct A. oxaliferum cell-types, based on cell surface features and the size and abundance of calcite and sulphur inclusions within the cells, were present in a single sample of purified cells. PCR was used to amplify almost full-length 16S rRNA gene sequences from DNA extracted from A. oxaliferum cells directly purified from sediments. The PCR products were cloned and partial sequences (approx. 400 bp) were determined for seven of the clones. Three different sequence clusters were recovered from the clone libraries. A near full-length (1489 bp) 16S rRNA gene sequence was determined for a representative clone of the most dominant sequence-type (52% of the sequences). Comparative sequence analysis showed A. oxaliferum to form a deep branching lineage within the gamma-subdivision of the Proteobacteria. A. oxaliferum was related most closely to the Chromatium assemblage that includes sulphur-oxidizing symbiotic bacteria, purple sulphur bacteria, and sulphur- and iron-oxidizing thiobacilli. Phylogenetic inferences made using distance, parsimony and maximum likelihood methods all placed A. oxaliferum with this group of bacteria. Bootstrap support for a relationship with any particular lineage within the assemblage was weak. The seven clone sequences recovered from the A. oxaliferum cells however formed a monophyletic group well supported by bootstrap analysis (85-100% support depending on the analysis done). It was concluded that A. oxaliferum was related to organisms of the Chromatium assemblage but constituted a novel lineage within this group of bacteria. A. oxaliferum cells were confirmed as the source of the 16S rRNA sequence obtained, by the use of a fluorescently-labelled 16S rRNA-targeted oligonucleotide specific for the A. oxaliferum rRNA sequence.

Some rumen ciliates have endosymbiotic methanogens.

Most of the small ciliate protozoa, including Dasytricha ruminantium and Entodinium spp. living in the rumen of sheep, were found to have intracellular bacteria. These bacteria were not present in digestive vacuoles. They showed characteristic coenzyme F420 autofluorescence and they were detected with a rhodamine-labelled Archaea-specific oligonucleotide probe. The measured volume percent of autofluorescing bacteria (1%) was close to the total volume of intracellular bacteria estimated from TEM stereology. Thus it is likely that all of the bacteria living in the cytoplasm of these ciliates were endosymbiotic methanogens, using H2 evolved by the host ciliate to form methane. Intracellular methanogens appear to be much more numerous than those attached to the external cell surface of ciliates.

Cyclidium porcatum n. sp.: a Free-living anaerobic scuticociliate containing a stable complex of hydrogenosomes, eubacteria and archaeobacteria.

A new ciliate species (Cyclidium porcatum) is the first freshwater anaerobic scuticociliate to be cultured and described. It contains a unique tripartite structure consisting of hydrogenosomes (confirmed by cytochemical staining for hydrogenase), interspersed with methanogens (confirmed by auto fluorescence and in situ hybridisation with an archaeobacterial 16S rRNA-specific probe) and unidentified eubacteria (confirmed with a eubacterial 16S rRNA-specific probe). This complex structure is stable and persistent, indicating that it is an anaerobic symbiotic consortium incorporating three functional partners.

Non-culturable Aeromonas salmonicida in lake water.

The survival of Aeromonas salmonicida subsp. salmonicida was investigated in lake water. During a 21-day study A. salmonicida became non-culturable in sterile lake water held at 10 degrees C. The incubation of replicate samples between 5 degrees C and 25 degrees C produced similar results. The recovery of colony-forming units of A. salmonicida from different lake water systems indicated that they survived longer in water that was naturally enriched (eutrophic) or enriched with tryptone soya broth. Flow cytometry, fluorescence light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) indicated that non-culturable cells were present. These cells could not be revived in dilutions of tryptone soya broth, whole dead fish or dissected fish tissue. Although viability could not be proven, it was shown that the morphological integrity found in viable cells was also maintained in non-culturable cells.

Anaerobic ciliates from a sulphide-rich solution lake in Spain.

We have examined and quantified the anaerobic ciliates living in the hypolimnion of a 14 m deep sulphide-rich (up to 0.73 mM) solution lake in Spain. At least seven ciliate species were found, numbering up to 50 ml-1 in total and reaching maximum abundance close to the sediment. Caenomorpha medusula, Lacrymaria elegans, L. sapropelica and Lagynus sp. were the most abundant species. Their vertical distributions were not related to the sulphide profile. Most ciliates were dependent on the sedimentation of cryptomonads, photosynthetic bacteria (especially Chromatium and Oscillatoria) and other bacteria from their sites of production in closely-juxtaposed mid-water plates. All anaerobic ciliates contained at least one type of symbiotic bacterium which showed methanogen autofluorescence. C. medusula, Lagynus sp. and Lacrymaria sapropelica also contained a large, non-fluorescing rod-shaped bacterium. In C. medusula, the methanogens and the non-fluorescing rods were both attached to the hydrogenosomes. In this ciliate alone, a third bacterial type was attached to the external ventral surface of the ciliate. Digestion of sulphide-oxidising bacteria by ciliates which harbour methanogenic bacteria provides a short bridge between the anaerobic sulphur and carbon cycles. Theoretical considerations of the rate of ciliate consumption of microbial carbon in the anoxic hypolimnion indicate that it is significant and that it may amount to 4 × 10(-5) g cm(-2)d(-1).

On the abundance and distribution of protozoa and their food in a productive freshwater pond.

We have examined and quantified the protozoa living in a productive freshwater pond during a 2-day period in June 1987. Over 90 species were recognised. The planktonic and benthic communities were dominated by ciliates and heterotrophic flagellates although the large amoeba Pelomyxa palustris was abundant (102 ml(-1)) in anaerobic sediments. Picoplankton averaged 1.4 × 10(7) ml(-1), phototrophic nanoplankton 0.8 × 10(5) ml(-1), heterotrophic nanoplankton 0.9 × 10(5) ml(-1) and planktonic ciliates 1.3 × 10(2) ml(-1). Numbers were about two orders of magnitude higher in the sediment. Protozoan biomass ranged from 3% to 61% of the total plankton biomass. Heterotrophic flagellates were the principal grazers of the picoplankton. Planktonic ciliates fed mainly on phototrophic nanoplankton but they probably also ingested heterotrophic flagellates. Benthic ciliates were predominantly bactivorous. Competition between ciliate species was minimised by both spatial and food niche separation. Ten species of planktonic ciliates appeared to contain algal symbionts: one species (Strombidium viride) contained structures resembling sequestered chloroplasts. These findings concerning the diversity and abundance of protozoa in a freshwater pond are consistent with the consensus opinion expressed in the marine literature that protozoa play a fundamental role in microbial food webs within aquatic ecosystems.

Effect of osmotic stress on the ultrastructure and viability of the yeast Saccharomyces cerevisiae.

Exposure of the yeast Saccharomyces cerevisiae to hypertonic solutions of non-permeating compounds resulted in cell shrinkage, without plasmolysis. The relationship between cell volume and osmolality was non-linear; between 1 and 4 osM there was a plateau in cell volume, with apparently a resistance to further shrinkage; beyond 4 osM cell volume was reduced further. The loss of viability of S. cerevisiae after hypertonic stress was directly related to the reduction in cell volume in the shrunken state. The plasma membrane is often considered to be the primary site of osmotic injury, but on resuspension from a hypertonic stress, which would have resulted in a major loss of viability, all cells were osmotically responsive. The effects of osmotic stress on mitochondrial activity and structure were investigated using the fluorescent probe rhodamine 123. The patterns of rhodamine staining were altered only after extreme stress and are assumed to be a pathological feature rather than a primary cause of injury. Changes in the ultrastructure of the cell envelope were examined by freeze-fracture and scanning electron microscopy. In shrunken cells the wall increased in thickness, the outer surface remained unaltered, whilst the cytoplasmic side buckled with irregular projections into the cytoplasm. On return to isotonic solutions these structural alterations were reversible, suggesting a considerable degree of plasticity of the wall. However, the rate of enzyme digestion of the wall may have been modified, indicating that changes in wall structure persist.

Structural and functional aspects of biological freezing techniques.

The cooling procedures used to prepare samples for ultrastructural examination at low temperatures often differ markedly from those used to recover optimal function of cells on thawing. The implications of these differences are reviewed. Damage and alteration to the structure and function of the cells may be caused by the high concentrations of cryoprotective agents such as glycerol or dimethyl sulphoxide (DMSO) often added to reduce ice crystal artefacts. Under the rapid cooling conditions commonly employed for structural studies, these additives are not cryoprotective; low rates of cooling are necessary for them to be effective. Rapidly cooled cells that contain intracellular ice are only injured during rewarming so their structure may be as yet unaltered by any damaging effects at low temperatures. Most cells able to recover on thawing are grossly shrunken at low temperatures but since they are potentially functional they are of interest structurally. These cryobiological principles are illustrated with freeze-fracture, freeze substitution and functional assays. The cell types chosen were Chlorella sp. and mammalian tissue culture cells.