Microbial diversity in lake sediments detected by PCR-DGGE

In this study, PCR-denaturing gradient gel electrophoresis (DGGE) was applied to analyze the microbial communities in lake sediments from Lake Xuanwu, Lake Mochou in Nanjing and Lake Taihu in Wuxi. Sediment samples from seven locations in three lakes were collected and their genomic DNAs were extracted. The DNA yields of the sediments of Lake Xuanwu and Lake Mochou were high (10 μg/g), while that of sediments in Lake Taihu was relatively low. After DNA purification, the 16S rDNA genes (V3 to V5 region) were amplified and the amplified DNA fragments were separated by parallel DGGE. The DGGE profiles showed that there were five common bands in all the lake sediment samples indicating that there were similarities among the populations of microorganisms in all the lake sediments. The DGGE profiles of Lake Xuanwu and Lake Mochou were similar and about 20 types of microorganisms were identified in the sediment samples of both lakes. These results suggest that the sediment samples of these two city lakes (Xuanwu, Mochou) have similar microbial communities. However, the DGGE profiles of sediment samples in Lake Taihu were significantly different from these two lakes. Furthermore, the DGGE profiles of sediment samples in different locations in Lake Taihu were also different, suggesting that the microbial communities in Lake Taihu are more diversified than those in Lake Xuanwu and Lake Mochou. The differences in microbial diversity may be caused by the different environmental conditions, such as redox potential, pH, and the concentrations of organic matters. Seven major bands of 16S rDNA genes fragments from the DGGE profiles of sediment samples were further re-amplified and sequenced. The results of sequencing analysis indicate that five sequences shared 99%–100% homology with known sequences (Bacillus and Brevibacillus, uncultured bacteria), while the other two sequences shared 93%–96% homology with known sequences (Acinetobacter, and Bacillus). The study shows that the PCR-DGGE technique combined with sequence analysis is a feasible and efficient method for the determination of microbial communities in sediment samples. __________ Translated from Acta Ecologica Sinica, 2006, 26(11): 3610–3616 [译自: 生态学报]     

Modulation of the Calmodulin-induced Inhibition of Sarcoplasmic Reticulum Calcium Release Channel (Ryanodine Receptor) by Sulfhydryl Oxidation in Single Channel Current Recordings and [3H]Ryanodine Binding

The modulation of the calmodulin-induced inhibition of the calcium release channel (ryanodine receptor) by two sulfhydryl oxidizing compounds, 4-(chloromercuri)phenyl–sulfonic acid (4-CMPS) and 4,4′-dithiodipyridine (4,4′-DTDP) was determined by single channel current recordings with the purified and reconstituted calcium release channel from rabbit skeletal muscle sarcoplasmic reticulum (HSR) and [³H]ryanodine binding to HSR vesicles. 0.1 μm CaM reduced the open probability (P _o ) of the calcium release channel at maximally activating calcium concentrations (50–100 μm) from 0.502 ± 0.02 to 0.137 ± 0.022 (n= 28), with no effect on unitary conductance. 4-CMPS (10–40 μm) and 4,4′-DTDP (0.1–0.3 mm) induced a concentration dependent increase in P _o (> 0.9) and caused the appearance of longer open states. CaM shifted the activation of the calcium release channel by 4-CMPS or 4,4′-DTDP to higher concentrations in single channel recordings and [³H]ryanodine binding. 40 μm 4-CMPS induced a near maximal (P _o > 0.9) and 0.3 mm 4,4′-DTDP a submaximal (P _o = 0.74) channel opening in the presence of CaM, which was reversed by the specific sulfhydryl reducing agent DTT. Neither 4-CMPS nor 4,4′-DTDP affected Ca-[¹²⁵I]calmodulin binding to HSR. 1 mm MgCl₂ reduced P _o from 0.53 to 0.075 and 20–40 μm 4-CMPS induced a near maximal channel activation (P _o > 0.9). These results demonstrate that the inhibitory effect of CaM or magnesium in a physiological concentration is diminished or abolished at high concentrations of 4-CMPS or 4,4′-DTDP through oxidation of activating sulfhydryls on cysteine residues of the calcium release channel. Received: 22 July 1999/Revised: 15 November 1999     

Scaling of Pelagic Metabolism to Size,Trophy and Forest Cover in Small Danish Lakes

ABSTRACT We tested whether pelagic light and nutrient availability, metabolism, organic pools and CO₂-supersaturation were related to lake size and surrounding forest cover in late summer–autumn measurements among 64 small (0.02–20 ha), shallow seepage lakes located in nutrient-rich, calcareous moraine soils in North Zealand, Denmark. We found a strong implicit scaling to lake size as light availability increased significantly with lake size while nutrient availability, phytoplankton biomass and dissolved organic matter declined. Forest lakes had significantly stronger net heterotrophic traits than open lakes as higher values were observed for light attenuation above and in the water, dissolved organic matter, pelagic community respiration (R) relative to maximum gross primary production (R/GPP) and CO₂-supersaturation. Total-phosphorus was the main predictor of phytoplankton biomass (Chl) despite a much weaker relationship than observed in previous studies of larger lakes. Maximum gross primary production increased with algal biomass and decreased with dissolved organic matter, whereas community respiration increased with dissolved organic matter and particularly with gross primary production. These results suggest that exogenous organic matter supplements primary production as an energy source to heterotrophs in these small lakes, and particularly so in forest lakes experiencing substantial shading from the forest and dissolved humic material. This suggestion is supported by 20–30-fold CO₂ supersaturation in the surface water of the smallest forest lakes and more than sixfold supersaturation in 75% of all measurements making these lakes among the most supersaturated temperate lakes examined so far.     

Electroencephalographic characteristics of cognitive-specific alerting attention in verbal learning: III. Localized characteristics of EEG spatial synchronization

Electroencephalograms (EEGs) were recorded in 19 standard derivations in 88 healthy subjects (students) in the state of rest with eyes open and during memorization (learning) of verbal bilingual semantic pairs (the Latin and Russian languages) and retrieval of information from memory (control). The estimates of EEG coherence in these states were compared for the following frequency bands: θ (4–7 Hz), α₁ (7–10 Hz), α₂ (10–13 Hz), β₁ (13–18 Hz), β₂ (18–30 Hz), and γ (30–40 Hz). Compared to the state of rest, the decrease in coherence in the pairs of derivations from the frontal and central cortical areas in all EEG frequency bands was the most pronounced for memorization, and the increase in coherence in the interhemispheric derivation pairs of the parietal-occipital region in most of the frequency bands was the most pronounced for retrieval. In addition, in the pairs formed by derivations from the parietal-occipital region with derivations from the frontal and central regions, retrieval is also characterized by an increase in coherence in the β₂ and γ bands along with its decrease in the low-frequency ranges. The dynamics of EEG coherence, when comparing the states of memorization and retrieval, is more statistically significant in the interhemispheric and cross-hemispheric pairs of derivations than in the intrahemispheric pairs. The revealed topographic specificity of the dynamics of EEG coherence owing to the change of state is considered in terms of the notion on cognitive-specific forms of sustained goal-directed mental attention.     

Comparative analysis of bacterioplankton assemblages from maritime Antarctic freshwater lakes with contrasting trophic status

The bacterioplankton assemblages of eight maritime Antarctic lakes with a wide range of trophic status and geographic span (six lakes from Hope Bay, Antarctic Peninsula and two from Potter Peninsula, King George Island) were described using denaturing gradient gel electrophoresis and band sequencing during two consecutive austral summers (2003–2004). Analyses of the gels identified a total of 230 bands spread across 57 different positions. Among those bands, 14 were shared between lakes from Hope Bay and Potter Peninsula, 17 were observed only in particular lakes, and 17 were registered both years in the same lake. We successfully reamplified and sequenced 43 bands located in 36 different positions belonging to Bacteroidetes, Actinobacteria, Betaproteobacteria and Cyanobacteria. The closest matches for 63% of the sequenced bands were from Antarctic or from other cold environment clones and sequences already in the databases, suggesting the widespread dominance of microbial communities adapted to cold habitats. The results of the multivariate analyses (Cluster Analysis and CCA) indicated that the nutrient status of the lake influences the bacterioplankton assemblages. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of a biosurfactant, mannosylerythritol lipid produced from Candida sp. SY16

One yeast strain, SY16, was selected as a potential producer of a biosurfactant, and identified as a Candida species. A biosurfactant produced from Candida sp. SY16 was purified and confirmed to be a glycolipid. This glycolipid-type biosurfactant lowered the surface tension of water to 29 dyne/cm at critical micelle concentration of 10 mg/l (1.5 × 10⁻⁵ M), and the minimum interfacial tension was 0.1 dyne/cm against kerosene. Thin-layer and high-pressure liquid chromatography studies demonstrated that the glycolipid contained mannosylerythritol as a hydrophilic moiety. The hydrophilic sugar moiety of the biosurfactant was determined to be β-d-mannopyranosyl-(1 → 4)-O-meso-erythritol by nuclear magnetic resonance (NMR) and fast atom bombardment mass–spectroscopy analyses. The hydrophobic moiety, fatty acids, of the biosurfactant was determined to be hexanoic, dodecanoic, tetradecanoic, and tetradecenoic acid by gas chromatography–mass spectroscopy. The structure of the native biosurfactant was determined to be 6-O-acetyl-2,3- di-O-alkanoyl-β-d-mannopyranosyl-(1 → 4)-O-meso-erythritol by NMR analyses. We newly determined that an acetyl group was linked to the C-6 position of the d-mannose unit in the hydrophilic sugar moiety. Received: 18 December 1999 / Received last revision: 2 June 1999 / Accepted: 4 June 1999     

Genetic Diversity of Eukaryotic Plankton Assemblages in Eastern Tibetan Lakes Differing by their Salinity and Altitude

Eukaryotic plankton assemblages in 11 high-mountain lakes located at altitudes of 2,817 to 5,134 m and over a total area of ca. one million square kilometers on the Eastern Tibet Plateau, spanning a salinity gradient from 0.2 (freshwater) to 187.1 g l⁻¹ (hypersaline), were investigated by cultivation independent methods. Two 18S rRNA gene-based fingerprint approaches, i.e., the terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis (DGGE) with subsequent band sequencing were applied. Samples of the same lake type (e.g., freshwater) generally shared more of the same bands or T-RFs than samples of different types (e.g., freshwater versus saline). However, a certain number of bands or T-RFs among the samples within each lake were distinct, indicating the potential presence of significant genetic diversity within each lake. PCA indicated that the most significant environmental gradient among the investigated lakes was salinity. The observed molecular profiles could be further explained (17–24%) by ion percentage of chloride, carbonate and bicarbonate, and sulfate, which were also covaried with change of altitude and latitude. Sequence analysis of selected major DGGE bands revealed many sequences (largely protist) that are not related to any known cultures but to uncultured eukaryotic picoplankton and unidentified eukaryotes. One fourth of the retrieved sequences showed ≤97% similarity to the closest sequences in the GenBank. Sequences related to well-known heterotrophic nanoflagellates were not retrieved from the DGGE gels. Several groups of eukaryotic plankton, which were found worldwide and detected in low land lakes, were also detected in habitats located above 4,400 m, suggesting a cosmopolitan distribution of these phylotypes. Collectively, our study suggests that there was a high beta-diversity of eukaryotic plankton assemblages in the investigated Tibetan lakes shaped by multiple geographic and environmental factors.     

Structure of the Microbial Communities in Coniferous Forest Soils in Relation to Site Fertility and Stand Development Stage

Abstract The structure, biomass, and activity of the microbial community in the humus layer of boreal coniferous forest stands of different fertility were studied. The Scots pine dominated CT (Calluna vulgaris type) represented the lowest fertility, while VT (Vaccinium vitis-idaéa type), MT (Vaccinium myrtillus type), and OMT (Oxalis acetocella–Vaccinium myrtillus type) following this order, were more fertile types. The microbial community was studied more closely by sampling a succession gradient (from a treeless area to a 180-years-old Norway spruce stand) at the MT type site. The phospholipid fatty acid (PLFA) analysis revealed a gradual shift in the structure of the microbial community along the fertility gradient even though the total microbial biomass and respiration rate remained unchanged. The relative abundance of fungi decreased and that of bacteria increased with increasing fertility. The structure of the bacterial community also changed along the fertility gradient. Irrespective of a decrease in fungal biomass and change in bacterial community structure after clear-cutting, the PLFA analysis did not show strong differences in the microbial communities in the stands of different age growing on the MT type site. The spatial variation in the structure of the microbial community was studied at a MT type site. Semivariograms indicated that the bacterial biomass, the ratio between the fungal and bacterial biomasses, and the relative amount of PLFA 16:1ω5 were spatially autocorrelated within distances around 3 to 4 m. The total microbial and fungal biomasses were autocorrelated only up to 1 m. The spatial distribution of the humus microbial community was correlated mainly with the location of the trees, and consequently, with the forest floor vegetation. Received: 9 November 1998; Accepted: 26 April 1999     

Temporal and Spatial Distribution of the nifH Gene of N2 Fixing Bacteria in Forests and Clearcuts in Western Oregon

Abstract Decomposition of plant litter is a primary mechanism of nutrient recycling and redistribution in most terrestrial ecosystems. Previously we demonstrated by a nested PCR protocol that 20 distinctive nifH (the gene encoding nitrogenase reductase) HaeIII restriction fragment length polymorphism (RFLP) patterns were derived from bulk DNA associated with samples of plant litter and soil collected at one Douglas Fir (DF) forest [33]. Five of the nifH DNA patterns (II–VI) were dominant types in DF litter with characteristic fragments of 237–303 bp length, whereas samples from soil contained primarily seven other patterns 131–188 bp length (IX–XV). Here we report that the 237–303 bp fragments characteristic for forest litter could generally not be detected in plant litter or soil samples collected in clearcuts that adjoin the forest sites. The same fragments (237–303 bp) were also found in the litter at this DF forest site over 16 months and were consistently found in litter at 12 other DF forest or recent (<2 yrs) clearcut sites. However, trace to none of these fragments were detected in 6 clearcut (5–10 yrs) or different forest types (oak, alder) collected over a 200 km east–west direction in western Oregon, USA. Data suggest that the logging practice in DF forests that creates a clearcut removes a unique gene pool of nitrogen-fixing microorganisms. These organisms could potentially contribute more to nitrogen fixation in forest litter than litter from natural or invasive plants that grow in clearcuts [26]. Received: 1 June 1999; Accepted: 7 October 1999; Online Publication: 23 February 2000     

Age and composition of carbonate shoreface sediments, Kailua Bay, Oahu, Hawaii

The origin, age, and dynamics of carbonate sediments in Kailua Bay on Oahu, Hawaii, are described. The shoreface (from shoreline to 4 km offshore) consists of a broad (5 km²) fringing coral reef ecosystem bisected by a sinuous, shore-normal, sand-filled paleostream channel 200–300 m wide. The median grain diameter of surface sands is finest on the beach face (<0.3 mm) and increases offshore along the channel axis. Kailua sands are >90% biogenic carbonate, dominated by skeletal fragments of coralline algae (e.g. Porolithon, up to 50%) followed by the calcareous green alga Halimeda (up to 32%), coral fragments (1–24%), mollusc fragments (6–21%), and benthic foraminifera (1–10%). Sand composition and age across the shoreface are correlated to carbonate production. Corals and coralline algae, principal builders of the reef framework, are younger and more abundant in sands along the channel axis and in offshore reefal areas, while Halimeda, molluscs, and foraminifera are younger and more dominant in nearshore waters shoreward of the main region of framework building. Shoreface sediments are relatively old. Of 20 calibrated radiocarbon dates on skeletal constituents of sand, only three are younger than 500 years b.p.; six are 500–1000 years b.p.; six are 1000–2000 years b.p.; and five are 2000–5000 years b.p. Dated fine sands are older than medium to coarse sands and hence may constitute a reservoir of fossil carbonate that is distributed over the entire shoreface. Dominance of fossiliferous sand indicates long storage times for carbonate grains, which tend to decrease in size with age, such that the entire period of relative sea-level inundation (∼5000 years) is represented in the sediment. Despite an apparently healthy modern coral ecosystem, the surficial sand pool of Kailua Bay is dominated by sand reflecting an antecedent system, possibly one that existed under a +1–2 m sea-level high stand during the mid- to late Holocene. Accepted: 20 December 1999     

Somatic embryogenesis, plant regeneration and cyanogenesis in Manihot glaziovii Muell. Arg. (ceara rubber)

 The report describes a system for somatic embryogenesis and direct plant regeneration from the embryos of Manihot glaziovii. Somatic embryos were obtained by culturing young leaf lobes (3–6 mm long) adjacent to the apex in Murashige and Skoog medium containing 18 μm 2,4-dichlorophenoxy acetic acid for 20 days and then transferring them to a maturation medium with 0.5 μm 6-benzylaminopurine. Secondary embryogenesis was induced from cotyledonary segments of somatic embryos by using the same protocol as that for primary embryogenesis. For regeneration, somatic embryos were cultured in medium supplemented with 10⁻⁴ m kinetin and 53.4% of them developed into plantlets. Linamarin and linamarase were not detected in calli or in somatic embryos. Linamarin content was found to be highest in leaves of regenerated plantlets, followed by stem and root tissues. Levels of linamarase activity were almost the same in leaves and stem tissues and very low in roots. Received: 19 April 1999 / Revision received: 11 August 1999 / Accepted: 17 August 1999     

Transition zones in small lakes: the importance of dilution and biological uptake on lake-wide heterogeneity

Community structure and diversity patterns of pelagic copepods were investigated for the coastal areas and in the marine lakes of the Palau islands in the West Pacific. We conducted field surveys during 2004–2007 and collected zooplankton samples from eight coastal areas and 16 marine lakes. The marine lakes in the islands of Palau are limnologically classified into two types, meromictic and holomictic lakes. Species diversity indices (Margalef’s species richness d′, Shannon–Wiener index H′, and Simpson’s dominance) were measured at each sampling site. The copepod community structure was analyzed using multivariate analyses, hierarchical cluster analysis, and non-metric multidimensional scaling (MDS) from the PRIMER package. A total of 36 copepod taxa were identified to the genus or species level from Calanoida, Cyclopoida, Harpacticoida, and Poecilostomatoida. Multivariate analysis based on the Bray–Curtis similarity index revealed that copepod assemblages could be classified into three different groups according to their habitat: (I) meromictic lakes, (II) holomictic lakes with an exceptionally shallow and flat basin (type A holomictic lakes), (III) holomictic lakes with a deep basin (type B holomictic lakes), and all coastal areas. The meromictic lakes were characterized by markedly decreased species diversity (d′ = 0.15, H′ = 0.41) in which only two brackish-water species, Bestiolina similis and Oithona dissimilis, were dominant. Type A holomictic lakes were characterized by relatively low levels of species diversity (d′ = 1.25, H′ = 1.35). In contrast, type B holomictic lakes were characterized by relatively high levels of species diversity, which was comparable to that of coastal areas. This result indicated that zooplankton assemblages in the type B holomictic lakes were closely related to a coastal community. The present study showed that the species diversity of pelagic copepods varies according to the level of isolation and the local environmental conditions for each marine lake.     

Optical study of spatiotemporal inhibition evoked by two-tone sequences in the guinea pig auditory cortex

Spatiotemporal response patterns in the anterior and dorsocaudal fields of the guinea pig auditory cortex after two-tone sequences were studied in anesthetized animals (Nembutal 30 mg kg⁻¹) using an optical recording method (voltage-sensitive dye RH795, 12 × 12 photodiode array). Each first (masker) and second (probe) tone was 30 ms long with a 10-ms rise-fall time. Masker-probe pair combinations of the same or different frequencies with probe delays of 30–150 ms were presented to the ear contralateral to the recording side. With same-frequency pairs, responses to the probe were inhibited completely after probe delays of less than 50 ms and the inhibition lasted for more than 150 ms, and the inhibition magnitudes in different isofrequency bands of the anterior field were essentially the same. With different-frequency (octave-separated) pairs, responses to the probe were not inhibited completely even after probe delays as short as 30 ms, and the inhibition lasted only for 110–130 ms. Inhibition magnitudes were different from location to location. Accepted: 4 August 1997     

AFLP analysis of genetic diversity and relationship among some Chinese domestic ducks and wild ducks

The amplified fragment length polymorphic (AFLP) technique was used to analyze the genome DNA polymorphism among 8 breeds of domestic ducks and 2 species of wild ducks. Nine of the 17 selected primers pairs gave reproducible polymorphic DNA amplification bands. The amplified bands ranged from 44 to 83 per primer pair. Of the 513 AFLP markers obtained, 498 were polymorphic. The proportion of polymorphic loci was 97.1%. The genetic distance (D) and similarity coefficients (GS) were calculated based on the polymorphic data. Between domestic ducks D ranged from 0.331 to 0.589, while between domestic ducks and the wild ducks, it ranged from 0.298 to 0.520 (vs. Anas Platyrhynchos) and from 0.316 to 0.522 (vs. A. Poecilorhyncha), respectively. The variance analysis showed no significant difference between the two groups of data, which indicated that both mallard and spot-billed ducks made contributions to domestic duck evolution. A dendrogram was constructed according to the D value. __________ Translated from Journal of Xiamen University (Natural Science), 2005, 44(5): 729–733 [译自: 厦门大学学报 (自然科学版), 2005, 44(5): 729–733]     

Localization and Identification of Populations of Phosphatase-Active Bacterial Cells Associated with Activated Sludge Flocs

Abstract The majority of phosphatase (PO₄ase) activity detected in fresh aerobic activated sludge from a municipal wastewater treatment plant was associated with suspended floc material. PO₄ase activity appeared to be localized in discrete bacteria-containing areas of the floc matrix based on the distribution of nucleic acid–stained cells and precipitated fluorescent crystals produced as a result of reaction of the enzyme(s) with the artificial substrate ELF™-PO₄. Of the total floc-associated bacterial cells that stained positive with the nucleic acid–binding fluorochrome acridine orange (AO), 8.8 ± 1.2% displayed PO₄ase activity based on the proximity of AO-stained cells to precipitated ELF crystals. Using a 16S rRNA oligonucleotide probe specific for the cytophaga–flavobacteria group, it was determined that 17–20% of the floc-associated bacteria that probed positive also displayed PO₄ase activity. Furthermore, 35–45% of the ELF fluorescence was associated with bacterial cells that probed positive for the cytophaga–flavobacteria group. The results suggest that the cytophaga–flavobacteria, as a group, is important in mediating the liberation of inorganic orthophosphate (P_i) from phosphomonoesters of detrital organic phosphate (organic-P) in the aerobic activated sludge process of wastewater treatment. Received: 17 March 1999; Accepted: 9 June 1999     

Bacterial 'cosmopolitanism' and importance of local environmental factors for community composition in remote high-altitude lakes

1. In October 2004, plankton samples were collected from six permanent lakes located between 4960 and 5440 m a.s.l. in the Mount Everest region (Nepal) to assess how spatial and local environmental factors affect natural bacterial community composition. Fingerprinting analysis of the bacterial 16S rRNA gene fragment was done by denaturing gradient gel electrophoresis (DGGE).
2. The number of DGGE bands (range: 12–23) was not correlated with lake area or remoteness, but there was a strong negative correlation with the ratio of catchment to lake area ( r  = −0.826, P  <   0.05), suggesting that hydraulic retention time affects the establishment of the bacterial community in these seepage lakes.
3. Most dominant sequences belonged to Betaproteobacteria except in two lakes where members of Bacteroidetes made the largest relative contribution. Up to 81% of the phylotypes had high similarity (>98 to 100%) in partial 16S rRNA gene sequence to those reported from other alpine lakes and glaciers around the world, suggesting the presence of 'cosmopolitan' bacteria.
4. An analysis based on dissimilarity matrices and the Mantel test revealed the existence of dissimilarities in bacterial community composition related to geographical distance over a small spatial scale (<6 km), but determined by local environmental constraints.
5. Our results suggest that several bacterial phylotypes are ubiquitous in the freshwater aquatic realm, but taxon sorting by local environmental constraints is important.     

Agrobacterium-mediated genetic transformation of a phalaenopsis orchid

 Genetically transformed plants of a phalaenopsis orchid [Doritaenopsis Coral Fantasy×Phalaenopsis (Baby Hat×Ann Jessica)] were regenerated after cocultivation of cell clumps with Agrobacterium tumefaciens strains LBA4404 (pTOK233) and EHA101 (pIG121Hm) that harbored genes for β-glucuronidase (GUS) and hygromycin resistance. The efficiency of transformation was markedly increased by 10 h cocultivation of cell clumps with A. tumefaciens that had been induced with 200 μm acetosyringone, and by inclusion of 500 μm acetosyringone in the cocultivation medium. Hygromycin-resistant cell clusters (0.5–3 mm in diameter) were selected from the infected cell clumps after 4–6 weeks of culture on agar (8 g/l)-solidified new Dogashima medium (NDM) containing 20 g/l sucrose, 0.1 mg/l naphthaleneacetic acid, 1.0 mg/l benzyladenine (BA), 50 mg/l hygromycin and 300 mg/l cefotaxime. The cell clusters proliferated 4 weeks after transfer onto the same medium. To induce callus greening, the carbon source was changed from sucrose to maltose. The green calli obtained produced protocorm-like bodies (PLBs) after 4 weeks of culture on phytohormone-free NDM medium. Regeneration of transgenic plantlets was enhanced by incubating PLBs on NDM medium supplemented with 0.1 mg/l abscisic acid, followed by partial desiccation for 10–30 min. Successful transformation was confirmed by histochemical GUS assay, PCR analysis and Southern hybridization of transformants. With this transformation system, more than 100 hygromycin-resistant phalaenopsis plantlets were produced about 7 months following infection of the cell aggregates. Received: 10 November 1998 / Revision received: 4 June 1999 / Accepted: 22 June 1999     

Seasonal abundance and depth distribution of Blennius fluviatilis and introduced Lepomis gibbosus, in Lake Banyoles (Catalonia, Spain)

Temporal and spatial occurrence of limnetic larval fish in Lake Banyoles in the spring and summer of 1993 and 1994 indicated that pumpkinseed spawned over 15 weeks (from mid-May to mid-August) and freshwater blenny over 14 weeks (from mid-May until early August). We noted that the spawning period of pumpkinseed commenced earlier in Lake Banyoles than in more northern lakes. Pumpkinseed and freshwater blenny larvae were collected by tow net in both the limnetic (at 4–4.5 m) and littoral zones (at 0–0.5 m), during daylight (no samples were taken during night). Freshwater blenny larvae were more abundant than pumpkinseed larvae. Both of them were more abundant in 1993 than in 1994 (pumpkinseed: 1.61 ind. 100 m−3 vs. 0.70 ind. 100 m−3; freshwater blenny: 3.95 ind. 100 m−3 vs. 1.90 ind. 100 m−3) and in both years they were uniformly distributed in the lake, without differences between the areas considered. The pumpkinseed larvae migrated at 3.5 mm TL into the limnetic zone, and returned to the vegetation of the littoral zone as juveniles. The freshwater blenny larvae were also planktonic (3.5–14 mm TL) but were distributed in both the littoral and limnetic zone before benthic settlement. The larval distribution is discussed on the basis of a trade-off between foraging and predation risk. This revised version was published online in July 2006 with corrections to the Cover Date.     

Physical and chemical limnology of alpine lakes and pools in the Rwenzori Mountains (Uganda–DR Congo)

This study describes the physical and chemical properties of 17 Afroalpine lakes (>2 m deep) and 11 pools (<2 m deep) in the Rwenzori mountains, Uganda-DR Congo, with the aim to establish the baseline conditions against which to evaluate future environmental and biological changes in these unique tropical ecosystems, and to provide the foundation for lake-based paleoenvironmental studies. Most Rwenzori lakes are located above 3,500 m elevation, and dilute (5–52 μS/cm specific conductance at 25°C) open systems with surface in- and outflow. Multivariate ordination and pairwise correlations between environmental variables mainly differentiate between (1) lakes located near or above 4,000 m (3,890–4,487 m), with at least some direct input of glacial meltwater and surrounded by rocky catchments or alpine vegetation; and (2) lakes located mostly below 4,000 m (2,990–4,054 m), remote from glaciers and surrounded by Ericaceous vegetation and/or bogs. The former group are mildly acidic to neutral clear-water lakes (surface pH: 5.80–7.82; Secchi depth: 120–280 cm) with often above-average dissolved ion concentrations (18–52 μS/cm). These lakes are (ultra-) oligotrophic to mesotrophic (TP: 3.1–12.4 μg/l; Chl-a: 0.3–10.9 μg/l) and phosphorus-limited (mass TN/TP: 22.9–81.4). The latter group are mildly to strongly acidic (pH: 4.30–6.69) waters stained by dissolved organic carbon (DOC: 6.8–13.6 mg/l) and more modest transparency (Secchi-disk depth: 60–132 cm). Ratios of particulate carbon, particulate nitrogen and chlorophyll a in these lakes indicate that organic matter in suspension is primarily derived from the lakes’ catchments rather than aquatic primary productivity. Since key features in the Rwenzori lakes’ abiotic environment are strongly tied to temperature and catchment hydrology, these Afroalpine lake ecosystems can be expected to respond sensitively to climate change and glacier melting. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Nutrient–chlorophyll relationships in tropical–subtropical lakes: do temperate models fit?

In tropical lakes relatively little is known about the general relationship between nutrient concentration and phytoplankton biomass. Using data from 192 lakes from tropical and subtropical regions we examine the relationship between total P (TP) and chlorophyll (Chl). The lakes are all located between 30° S to 31° N include systems in Asia, Africa, and North and South America but are dominated by Brazilian (n=79) and subtropical N. American (n=67) systems. The systems vary in morphometry (mean depth and lake area), trophic state as well total N (TN) to␣total P (TP) ratios and light extinction. Despite a nearly 500-fold range in TP concentrations (2–970 μg P l⁻¹), there was a poorer relationship between log TP and log Chl (r ²=0.42) than is generally observed for temperate systems from either narrow or broad geographic regions. N limitation is not a likely explanation for the relatively weak TP–Chl relationship in the tropical–subtropical systems. Systems had high average TN:TP ratios and neither a multiple regression with log TP and log TN nor separating systems with high TN:TP (>17 by weight) improved the predictive power of the log TP–log Chl relationship.