Evidence for the Presence of "Metabolic Sterols" in Pneumocystis: Identification and Initial Characterization of Pneumocystis carinii Sterols

Mixed life cycle stages of rat-derived Pneumocystis carinii were isolated from host lungs and their sterols were compared with those present in lungs from normal and immunosuppressed uninfected rats. Gas-liquid chromatography consistently detected, resolved, and quantified 9, 10, and 20 sterol components in the total nonsaponifiable neutral lipid fraction of lungs from normal rats, lungs from immunosuppressed uninfected rats, and P. carinii preparations, respectively. In all samples, cholesterol was the most abundant sterol present, comprising 97%, 93%, and 78% of total sterols in lungs from normal rats, lungs from immunosuppressed uninfected rats, and P. carinii , respectively. Tentative identifications of several rat lung and P. carinii minor sterols were made based on gas-liquid chromatogram retention times and fragmentation patterns from mass spectral analyses. Campesterol (ergost-5-en-3-ol), cholest-5-en-3-one, and β -sitosterol (stigmast-5-en-3-ol) were among the minor components present in both types of lung controls, and were also components of P. carinii sterols. In contrast to lung controls, the sterols of P. carinii were enriched in C₂₈ and C₂₉ sterols with one or two double bonds, and a hydroxyl group at C-3 (ergost-5-en-3-ol, ergost-7-en-3-ol, ergosta-dien-3-ol, stigmast-5-en-3-ol, stigmast-7-en-3-ol and stigmasta-dien-3-ol). Steryl esters of P. carinii , probably stored in cytoplasmic lipid droplets, were dominated by those present in the host lung. In separate studies. 3-hydroxy-3-methylglutaryl coenzyme A activity, a key enzyme in the regulation of sterol biosynthesis, was detected in purified P. carinii preparations and incorporation of radiolabeled squalene and mevalonate was observed. Together, these results suggest that the parasite readily takes up and incorporates host sterols, and that the organism synthesizes some of its own "metabolic sterols"     

Comparative Study of Microsporidian Spores by Flow Cytometric Analysis

ABSTRACT. Spore suspensions of microsporidian parasites of fish (Microsporidium ovoideum, Glugea stephani, Glugea atherinae and Spraguea lophii ) have been analyzed by flow cytometry. Spore nuclei were dyed either by propidium iodide or bis-benzimide (Hoechst 33342). By observation of forward light scatter and fluorescence the four species could be distinguished and the mono- and diplokaryotic populations of S. lophii identified. Staining of DNA by bis-benzimide was better and easier than propidium iodide. Forward light scatter and fluorescence values were characteristic of each species and remained unchanged throughout the year, so flow cytometry can be used for distinction of spores of some microsporidian parasites once their flow cytometric parameters are known. However, special care has to be taken in tool calibration and material preparation for analysis because of the high precision of the technique.     

Improved Coated Vesicle Isolation Allows Better Characterization of Clathrin Polypeptides

Postmicrosomal pellets from plant sources are grossly contaminatedwith nbosomes. Previous purifications of clathrin coated vesicles(CCV) from such subcellular fractions have therefore often involvedan RNase treatment. Performed at 30°C, this step inherentlycarries with it the dangers of proteolysis. We document herea method for CCV isolation which avoids this. Through the inclusionof suitable antiproteases in the homogenizing and subsequentisolation media, we have also been able to improve the qualityof CCV recovered from plant tissues. As a result we have tentativelybeen able to identify clathrin light chains from zucchini hypocotyland pea cotyledon CCV. Similar to light chains from bovine brainthese polypeptides are heat stable, can be solubilized fromneutralized TCA precipitates, bind calcium and clathrin heavychains. However, in contrast to brain CCV the two light chainsof plant CCV are some 10 kDa heavier. Key words: Antiproteases, Ca²⁺-binding, clathrin coated vesicles, clathrin heavy chains, clathrin light chain(s), heat stability, pea cotyledons, RNase, zucchini hypocotyls     

The evolutionary significance of bimodal emergence in the butterfly, Maniola jurtina (Lepidoptera: Satyrinae) (L.).

Most temperate butterflies exhibit a tightly synchronized unimodal adult emergence to facilitate mate location. Exceptions are presumably subject to unusual selection pressure. This study examines the pattern of emergence in Maniola jurtina , which was found to exhibit both unimodal and bimodal emergence patterns at different sites in south-east England. The bimodal pattern was found on chalk grassland; elsewhere the emergence was unimodal. Adults from each emergence peak rarely meet, suggesting that there may be some degree of reproductive isolation. Morphological measurements and electrophoretic analysis of allozyme frequencies are carried out to quantify differentiation between emergence peaks. Captive stock was reared to examine differences in the immature stages. Butterflies from each emergence differ significantly in most morphological variables measured, those from the second peak tending to be smaller. The immature stages differ in morphology and longevity of the egg stage. Allozyme frequencies did not differ between peaks, suggesting that they are not reproductively isolated. Explanations for the maintenance of differences between emergence peaks despite gene flow are discussed. I propose that division of offspring between two emergence times may have evolved to avoid the risk inherent in placing all offspring in one peak which may be rendered inviable by temporal fluctuations in habitat quality.     

Prey processing in Leurognathus marmoratus and the evolution of form, and function in desmognathine salamanders (Plethodontidae)

Function and biological role of morphological specialization in desmognathine salamanders are analysed in the light of studies of feeding in Leurognatthus marmoratus. Nine morphological features uniquely characterize the Desmognathinae as compared to its sister group, the Plethodontinae, and other salamanders: (1) heavily ossified and strongly articulated skull and mandible; (2) flat, wedgelike head profile; (3) stalked occipital condyles; (4) modified atlas; (5) modified anterior trunk vertebrae; (6) atlanto-mandibular ligaments; (7) enlarged dorsal spinal muscles; (8) enlarged quadrato-pectoralis muscles; and (9) hind limbs relatively larger than forelimbs. Dorsoventral head mobility is increased at the atlanto-occipital joint by the stalked occipital condyles which simultaneously increase the mechanical advantage of the hypertrophied axial muscles that cross the joint. During head depression the atlanto-mandibular ligaments are placed in tension. Force generated by the quadrato-pectoralis muscles is transmitted directly to the mandible, creating a powerful bite with the jaws in full occlusion. Desmognathines use an efficient static pressure system for subduing and/or killing prey items held in the jaws, not a kinetic-inertial mechanism, as previously suggested. Leurognathus exhibits a behaviour ('head-tucking') unique to desmognathines that is consistent with the static-pressure hypothesis. Several desmognathine features (1, 2, 5, 7, 9) are not explicable as adaptations for feeding; these function as locomotory specializations for burrowing, especially for wedging under rocks within and alongside streams. Desmognathines use head-tucking during such wedging and burrowing movements, thus locomotory specializations act in concert with the feeding specializations. We suggest that origin of the atlanto-mandibular ligaments can be considered a 'key innovation' in that it allowed the secondary invasion of stream habitats by adults of ancestral desmognathines.     

Winter Ciliates in a British Columbian Fjord: Six New Species and an Analysis of Ciliate Putative Prey

ABSTRACT. This work provides the first study of North Pacific planktonic ciliates by quantitative protargol staining. Triplicate water bottle samples were collected at a depth of 2 m (above the shallow pycnocline) at six stations in Indian Arm, British Columbia, on February 15, 1990, and February 26, 1991. Thirty-six ciliate species were observed. Six new species are described from protargolstained specimens: Strombidium lynni n. sp., Strombidium taylori n. sp., Strombidium basimorphum n. sp., Slrombidiurn ventropinnum n. sp., Strobilidium undinum n. sp., and Urotricha cyrtonucleata n. sp.
Ciliate abundance varied significantly (ANOVA, α= 0.05) between sampling sites, ranging from 550 to 6,800 cells/liter in 1990 and from 1,800 to 7,900 cells/liter in 1991. Biomass also varied significantly (ANOVA, α= 0.05) ranging from 3.7 × 10⁵ to 3.3 × 10⁶ pg carbon/liter in 1990 and 3.04 × 10⁶− 6.97 × 10⁶ pg carbon/liter in 1991. Putative prey were enumerated in three size fractions (1.5–5 μm, 5–10 μm and 10–25 μm). The source of variation in ciliate abundance and biomass was not identified. Parameters of salinity, temperature, putative prey, chlorophyll a and pycnocline depth did not significantly correlate with ciliate biomass or abundance (α= 0.05).     

Spatial sampling to detect slug abundance in an arable field

Overuse of molluscicides by farmers in arable systems can lead to environmental and product contamination. Here we assess a simple and inexpensive surface trapping method for monitoring populations of slugs (Deroceras reticulatum and Arion intermedius). This method was biased against small slugs, and against A. intermedius, when compared to direct soil sampling. Regression was used to model the relationship between the results of surface trapping and soil sampling methods. Spatial Analysis by Distance IndicEs (SADIE) algorithms were used to describe the spatial relationships between the two sets of samples. Using both traditional statistical methods and spatial statistics, the spatial information collected from surface traps was sufficient to identify patches and gaps in slug numbers and possibly to allow the spot application of slug control, and thus provide land managers who experience slug damage with a way of reducing molluscicides use, whilst maintaining slug control. Further improvements and applications of the model are discussed.