Electrically induced Ca2+ transport across the membrane of Paramecium caudatum measured by means of flow-through technique

Transmembrane calcium fluxes related to excitation were studied in Paramecium caudatum. Radioactive (⁴⁵Ca²⁺) or inactive solution was flowed through a dense suspension of unlabelled or labelled cells, and radioactivity was monitored in the solution. The organisms were electrically stimulated by means of extracellular electrodes. As a result of stimulation Ca²⁺ uptake and release was measured. The uptake response dropped with increasing number of successive stimulation periods and increased with growing stimulus amplitude and duration. Maximum uptake was obtained at 20 V/cm and at least 60 s duration and for temperatures between 10 and 15°C. A Ca²⁺ influx of 700 pmol/1000 cells upon 1 min stimulation was measured at 15°C. This corresponds to an increment of the intraciliary [Ca²⁺] of about $\text{5 · 10}{}^{-4}\text{M}$. Ca²⁺ release was dependent on the stimulus amplitude in a similar manner as was Ca²⁺ uptake. Photographic recordings of the swimming behaviour of the organisms were used to interpret the flux data. At temperatures up to 15°C the cells swam backward perpendicular to the applied electric field of 10 to 20 V/cm. At 25°C they showed forward spiralling movement. For the first time evidence was brought for stimulated Ca²⁺ influx in Paramecium at physiological temperatures. It is concluded from the results that a strong active Ca²⁺ extrusion from the intraciliary space counteracts the influx. The Ca²⁺ pump rate must be at least $\text{8 · 10}{}^{12}$ calcium ions per s per cm² ciliary surface.     

第四双小核草履虫减数分裂产物的退化特征分析

通过吖啶橙和Hoechst 33342两种活体荧光染料双染的方法对第四双小核草履虫(Paramecium tetraurelia)接合生殖过程中小核减数分裂产物进行观察,结果发现位于口旁锥外的小核分裂产物呈蓝绿色或黄绿色,表明它们以凋亡的方式发生退化.     

A kinome of 2600 in the ciliate Paramecium tetraurelia

Protein kinases play a crucial role in the regulation of cellular processes. Most eukaryotes reserve about 2.5% of their genes for protein kinases. We analysed the genome of the single-celled ciliate Paramecium tetraurelia and identified 2606 kinases, about 6.6% of its genes, representing the largest kinome to date. A gene tree combined with human kinases revealed a massive expansion of the calcium calmodulin regulated subfamily, underlining the importance of calcium in the physiology of P. tetraurelia. The kinases are embedded in only 40 domain architectures, contrasting 134 in human. This might indicate different mechanisms to achieve target specificity.     

The effect of temperature change on gene expression in Paramecium primaurelia

When paramecia grown at 24°C are transferred rapidly to 32°C, DNA and protein synthesis continue uninterrupted but at higher rates. Electron microscopic observations indicate that more of the macronuclear chromatin is transcribed at the elevated temperature. This interpretation is supported by hybridization experiments which show that the percentage of the macronuclear genome transcribed into poly(A)⁺ RNA is 24°C and 35% at 32°C. Kinetic analysis of cDNA-poly(A)⁺ RNA hybridizations reveals three abundance classes of poly(A)⁺ RNA and indicates that the number of genes expressing low abundance sequences is about 9000 at 24°C and 13000 at 32°C. The intermediately abundant and highly abundant classes are represented by 100–200 and 1–3 different kinds of RNA sequence, respectively. Cross hybridization shows that changes occur throughout the distribution of abundance classes of poly(A)⁺ RNA with increase in temperature.     

Axenization and optimization of in vitro growth of clonal cultures of Tetratrichomonas gallinarum and Trichomonas gallinae

A rapid and simple procedure was established to obtain clonal axenic cultures of Tetratrichomonas gallinarum and Trichomonas gallinae and to optimize their in vitro growth conditions. Medium 199 was used for axenization of two genetically different clones of T. gallinarum and T. gallinae. Six different media were used to optimize the growth behaviour of axenically grown parasites: Medium 199, TYM, TYI-S-33, Hollander fluid (HF), Trichomonas vaginalis (TV) and modified TV media. The highest cell yields for both axenic clones of T. gallinarum were obtained in modified TV medium without antibiotics. The maximum numbers of trophozoites of T. gallinae were obtained in an optimized HF medium. This study demonstrated that axenic cultures for T. gallinarum and T. gallinae could be obtained avoiding the migration technique through a V-tube. Following axenization and optimization, both clones of T. gallinarum and T. gallinae could be propagated both aerobically and anaerobically.     

Allometric relationships of field populations of two clonal species with contrasting life histories, Cladium jamaicense and Typha domingensis

Allometric analysis was used to examine morphological relationships in field populations of two clonal plants, Cladium jamaicense and Typha domingensis, in a Florida Everglades wetland. We found that allometric relationships of individuals sampled from field populations could be adequately derived and applied to analyzing both leaf and ramet growth responses to site differences along a nutrient gradient. Overall, the allometric relationships showed a significant departure from isometry which indicates that the relationships were size-dependent. Leaf-level morphological relationships were significantly different between species and between sites along the nutrient gradient. These differences, however, were not expressed on the ramet-level. Neither species expressed a plastic allocation response to site differences along the nutrient gradient. Biomass allocation between above- and below-ground for both species indicated significant size-dependent relationships with decreasing relative allocation below-ground with increasing size. Models for predicting total plant biomass (above- and below-ground) for both C. jamaicense and T. domingensis were developed based on two non-destructive measurements that are easily obtainable in the field. The models followed the equation log (biomass) = α + β₁ × log (height) + β₂ × log (basal area), where α was species specific while β₁ and β₂ were similar for both species but significantly different according to site along the nutrient gradient. Analysis of this model showed that plant height had a relatively greater influence on biomass than basal area at all sites. This difference was greatest at the un-enriched area where plants tend to be short and thick and the least at the moderately enriched site where the relative influence of both parameters was similar.     

Life-history variation in agricultural and wild populations of Erucastrum nasturtiifolium (Brassicaceae)

In the present study we relate the variability in life-history traits (such as flowering time and lifespan) of the herbaceous biennial–perennial Erucastrum nasturtiifolium (Brassicaceae) to habitat type. We studied plant populations from arable fields and from eroded mountain habitats, such as badlands and rocky slopes. Collection sites ranged from low basin to sub-alpine regions in the NE Iberian Peninsula. Plants were grown under common garden conditions to evaluate genetic variation among and within populations. Plants were also subjected to a resource gradient to detect genetic variation in phenotypic plasticity. The populations exhibited differentiation across a number of life-history traits (mainly flowering time and lifespan) and morphological traits related to growth (basal stem diameter, plant height and number of branches). This suggests that life-history differences among populations are genetically based. Moreover, our results show that variation in flowering time and lifespan are affected by habitat type independent of other abiotic factors such as altitude or continentality. Thus, populations from arable fields started flowering in their first year and displayed annual cycles, whereas those from wild habitats generally flowered in their second year and showed biennial or even perennial cycles. Intra-population differences in flowering time were observed in only one population, and were related to nutrient availability. We suggest that early-flowering and shorter lifespan populations of E. nasturtiifolium may have been selected from late-flowering and longer lifespan populations as part of a selective process ensuring survival and future offspring amidst unpredictable and frequently disturbed environments such as exist in many agricultural habitats.     

Biochemical studies of the excitable membrane of Paramecium tetraurelia. II. Phospholipids of ciliary and other membranes

The phospholipids of cilia and deciliated bodies of Paramecium tetraurelia were isolated and characterized. 1-alkyl-2-acyl-sn-glycero-3-(2′-aminoethyl) phosphonate (GAEPL), phosphatidylethanolamine, and 1-alkyl-2-acyl-sn-glycero-3-phosphorylcholine (GPC) were the major lipids of Paramecium, and the minor lipids included phosphatidylinositol, cardiolipin, ceramide-(2-aminoethyl) phosphonate (CAEP), ceramide phosphorylethanolamine (COPE) and four sphingolipids whose identity was not established. The deciliated bodies contained 4% cardiolipin, 15% GAEPL, 41% phosphatidylethanolamine, 30% GPC and 3% each of CAEP and phosphatidylinositol; the cilia contained no cardiolipin, 24% GAEPL, 37% phosphatidylethanolamine, 15% GPC, 15% CAEP, 3% phosphatidylinositol, 2% COPE and small amounts (approx. 1%) of the four uncharacterized sphingolipids. No alteration in phospholipid composition was found among cells harvested in the various stages of growth. The phospholipids of six Paramecium mutants of three distinct phenotypes (pawn, paranoiac and fast) were also examined. Only one significant difference was found on comparison of the whole cell, deciliated body and cilia fraction of the mutants with the analogous fractions from wild type cells: the fast mutant, fA 97, had two extra, minor phospholipids (approx. 2%) in the deciliated body fraction that were tentatively identified as 1,2-diacyl-sn-glycero-3-(2′-aminoethyl) phosphonate (AEPL) and 1-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamine (GPE).     

Biochemical studies of the excitable membrane of Paramecium tetraurelia. V. effects of proteases on the ciliary membrane

The swimming behavior of Paramecium is regulated by an excitable membrane that covers the body and cilia of the protozoan. In order to obtain information on the topology and function of ciliary membrane proteins, Paramecia were treated with trypsin, chymotrypsin or pronase and the effects of these proteases were analyzed using electron microscopy, gel electrophoresis of ciliary fractions and behavioral tests. At the concentrations used, trypsin and chymotrypsin had little or no effect on the cells while pronase removed the cell surface coat, visible as fuzzy material covering the cell membrane. The same pronase treatment caused the specific removal of a high molecular weight protein (250 000), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This protein, the ‘immobilization antigen’, constitutes the major protein of the ciliary membrane. Although the immobilization antigen was removed (or markedly decreased), no marked and reproducible difference was observed in the swimming behavior of the treated cells. We also determined the effects of proteases on isolated ciliary fractions to explore the sidedness of ciliary membrane proteins. A set of proteins relatively resistant to protease digestion was identified; they may be intrinsic membrane proteins.     

Outgrowth of nerve fibres from Drosophila central nervous system cultured in vitro

Explants of the central nervous system of Drosophila have been shown to produce nerve fibres in vitro. The effects of various culture conditions on fibre outgrowth have been examined. Nervous tissue could form nerve fibres in vitro when the explants were obtained from mid-embryonic or early- and mid-pupal stages, but not when they were obtained from larvae or late-pupae. The effect of the temperature-sensitive mutation shibire^ts has been investigated by placing mutant explants into culture at permissive (17°C) or restrictive (28°C) temperatures. No differences in the extent of fibre outgrowth between wild-type and shibire^ts were observed, regardless of the temperature of cultivation.     

Corynebacterium heidelbergense sp. nov., isolated from the preen glands of Egyptian geese (Alopochen aegyptiacus)

Two strains (pedersoli^T and girotti) of a new species of bacteria were isolated from the preen glands of wild Egyptian geese (Alopochen aegyptiacus) from the river Neckar in southern Germany in two subsequent years. The strains were lipophilic, fastidious, Gram-positive rods and belonged to the genus Corynebacterium. Phylogenetically, the isolates were most closely related to Corynebacterium falsenii DSM 44353^T which has been found to be associated with birds before. 16S rRNA gene sequence similarity to all known Corynebacterium spp. was significantly <97%. Corresponding values of rpoB showed low levels of similarity <87% and ANIb was <73%. G + C content of the genomic DNA was 65.0 mol% for the type strain of the goose isolates, as opposed to 63.2 mol% in Corynebacterium falsenii. MALDI-TOF MS analysis of the whole-cell proteins revealed patterns clearly different from the related species, as did biochemical tests, and polar lipid profiles. We therefore conclude that the avian isolates constitute strains of a new species, for which the name Corynebacterium heidelbergense sp. nov. is proposed. The type strain is pedersoli^T (=DSM 104638^T = LMG 30044^T).     

Kocuria uropygioeca sp. nov. and Kocuria uropygialis sp. nov., isolated from the preen glands of Great Spotted Woodpeckers (Dendrocopos major)

Two new species of Gram-positive cocci were isolated from the uropygial glands of wild woodpeckers (Dendrocopos major) originating from different locations in Germany. A polyphasic approach confirmed the affiliation of the isolates to the genus Kocuria. Phylogenetic analysis based on the 16S rRNA gene showed high degree of similarity to Kocuria koreensis DSM 23367^T (99.0% for both isolates). However, low ANIb values of <80% unequivocally separated the new species from K. koreensis. This finding was further corroborated by DNA fingerprinting and analysis of polar lipid profiles. Furthermore, growth characteristics, biochemical tests, MALDI-TOF MS analysis, and G + C contents clearly differentiated the isolates from their known relatives. Besides, the woodpecker isolates significantly differed from each other in their whole-cell protein profiles, DNA fingerprints, and ANIb values. In conclusion, the isolated microorganisms constitute members of two new species, for which the names Kocuria uropygioeca sp. nov. and Kocuria uropygialis sp. nov. are proposed. The type strains are 36^T (DSM 101740^T = LMG 29265^T) and 257^T (=DSM 101741^T = LMG 29266^T) for K. uropygialis sp. nov. and K. uropygioeca sp. nov., respectively.     

Bradyrhizobium valentinum sp. nov., isolated from effective nodules of Lupinus mariae-josephae, a lupine endemic of basic-lime soils in Eastern Spain

Bacterial strains isolated from nitrogen-fixing nodules of Lupinus mariae-josephae have been characterized following genetic, phenotypic and symbiotic approaches. Analysis of 16S rRNA genes placed them in a group together with Bradyrhizobium elkanii USDA 76^T, B. pachyrhizi PAC48^T, B. jicamae PAC68^T, ‘B. retamae’ Ro19^T and B. lablabi CCBAU 23086^T with over 99.0% identity. Phylogenetic analysis of concatenated housekeeping genes, recA, atpD and glnII, suggested that L. mariae-josephae strains represent a new Bradyrhizobium species, closely related to B. lablabi CCBAU 23086^T, B. jicamae PAC68^T and ‘B. retamae’ Ro19^T with 92.1, 91.9 and 90.8% identity, respectively. These results are consistent with overall genomic identities calculated as Average Nucleotide Identity (ANIm) using draft genomic sequences obtained for relevant strains. While L. mariae-josephae strains LmjM3^T/LmjM6 exhibited a 99.2% ANIm value, they were significantly distant (<93% ANIm) from type strains of their closest species (‘B. retamae’ Ro19^T, B. lablabi CCBAU 23086^T and B. jicamae PAC68^T). Whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (WC-MALDI-TOF-MS) analysis of proteomic patterns of the same strains was consistent with these results. The symbiosis-related genes nodC, nodA and nifH genes from strains nodulating L. mariae-josephae were phylogenetically related to those from ‘B. retamae’ Ro19^T, but divergent from those of strains that nodulate other lupine species. Based on genetic, genomic, proteomic and phenotypic data presented in this study, L. mariae-josephae nodulating strains LmjM3^T, LmjM6 and LmjM2 should be grouped within a new species for which the name Bradyrhizobium valentinum sp. nov. is proposed (type strain LmjM3^T = CECT 8364^T, LMG 2761^T)     

Encapsulation reactions in vitro by haemocytes of Heliothis virescens

A simple in vitro system for studying capsule formation by Heliothis virescens haemocytes was devised. The system produced capsules morphologically and ultrastructurally similar to those formed in vivo. Encapsulation proceeded normally when melanization was inhibited and when divalent cations were absent. Capsule development took place in two physiologically distinct phases. Aggregation of haemocytes around a foreign object (phase 1) was a passive process. Consolidation of haemocytes into a smooth, adherent capsule (phase 2) required metabolic energy. Phase 1 was inhibited irreversibly by propranolol and caffeine. Inhibition of phase 1 by mild trypsinization could be reversed by haemolymph lysate. Preliminary evidence indicates that encapsulation promoting factors in the lysate originate in haemocytes.     

Primate life histories and dietary adaptations: a comparison of Asian colobines and macaques

Primate life histories are strongly influenced by both body and brain mass and are mediated by food availability and perhaps dietary adaptations. It has been suggested that folivorous primates mature and reproduce more slowly than frugivores due to lower basal metabolic rates as well as to greater degrees of arboreality, which can lower mortality and thus fecundity. However, the opposite has also been proposed: faster life histories in folivores due to a diet of abundant, protein-rich leaves. We compared two primate taxa often found in sympatry: Asian colobines (folivores, 11 species) and Asian macaques (frugivores, 12 species). We first described new data for a little-known colobine (Phayre's leaf monkeys, Trachypithecus phayrei crepusculus) from Phu Khieo Wildlife Sanctuary, Thailand. We then compared gestation periods, ages at first birth, and interbirth intervals in colobines and macaques. We predicted that heavier species would have slower life histories, provisioned populations would have faster life histories, and folivores would have slower life histories than frugivores. We calculated general regression models using log body mass, nutritional regime, and taxon as predictor variables. Body mass and nutritional regime had the predicted effects for all three traits. We found taxonomic differences only for gestation, which was significantly longer in colobines, supporting the idea of slower fetal growth (lower maternal energy) compared to macaques and/or advanced dental or gut development. Ages at first birth and interbirth intervals were similar between taxa, perhaps due to additional factors (e.g., allomothering, dispersal). Our results emphasize the need for additional data from wild populations and for establishing whether growth data for provisioned animals (folivores in particular) are representative of wild ones.     

Definition of a juvenile hormone-sensitive period in Rhodnius prolixus

This paper is an attempt to establish the times of onset and termination of the juvenile hormone-sensitive period for metamorphosis in fifth-instar larvae of Rhodnius. Small regions of the abdominal integument were exposed to discrete pulses of the juvenile hormone analogue ZR-515 (methoprene) by applying small drops of a mixture in paraffin to the dorsum at various times after a bloodmeal and removing these drops after different time intervals. The diffusion coefficient of the analogue in the integument was estimated and used together with estimates of its metabolism to determine the lag times between application of the analogue and its rise to above threshold concentration in the epidermis, and between removal of the analogue source and its fall to below threshold concentration in the epidermis. These lag times were estimated to be 1.5 and 24h, respectively. Knowledge of the lag times makes it possible to establish the limits of the juvenile hormone-sensitive period or metamorphosis from the responses of larvae to variously-timed pulses of the analogue. The juvenile hormone-sensitive period has the following properties. For the population as a whole it lasts from about day 3 to about day 9 after a bloodmeal. Any individual in that population, however, only requires the presence of juvenile hormone during a 2 to 4-day period. The exact duration of an individual's sensitive period within these limits is a stochastic event. Surprisingly, for any individual, a pulse of juvenile hormone is equally effective when experienced early as when experienced late during its juvenile hormone-sensitive period.     

The life cycle of Anguina agrostis: Post-embryonic growth of the second stage larva

The growth and development of the freshly hatched second stage larva (FHL₂) into the infective second stage “dauer” larva (DL₂) has been followed under field conditions and in callus tissue culture. A comparison of this development has been made between specimens originating from widely separated geographic regions. The FHL₂'s from Narrogin (Western Australia) are slightly shorter than those from Murray Bridge (South Australia) and have a mean length of 543 ± 55 μm compared with 580±51 μm. However, the lengths of the DL₂s from these areas are similar, having mean lengths of 841 ± 35 μm and 849 ±26 μm respectively. Furthermore stylet lengths in all these larvae are similar and are approx. 10 μm. Morphological changes associated with the transition from FHL₂ to DL₂ include thickening of the cuticle, a change in shape of the lateral alae associated with stretching and the synthesis of numerous lipid storage granules. Physiological changes include a marked increase in swimming activity and the ability to enter into an anhydrobiotic state. Growth from FHL₂ to DL₂ in Lolium multiflorum callus tissue culture took place within 9 days at 20°C. No moulting was observed and growth did not take place beyond the DL₂ stage under these conditions.