Haemolytic substances produced by ichthyotoxic algae often are unknown in molecular structure or specific mechanism of toxicity. Detection and quantification of such substances are dependent on bioassays, using markers that are sensitive for haemolytic impairment and generation of a recordable response. The erythrocyte lysis assay (ELA) represents an advantageous bioassay in this respect, because the lytic response can be measured photometrically by the amount of released haemoglobin. The aim of the present study was to establish an improved assay based on the ELA principle, for sensitive determination of haemolytic substances of microalgae and for high sample throughput. For this purpose we adapted the ELA to a 96-well microtitre plate format, which significantly reduced the sample volumes and allowed rapid processing of samples. Further improvement was achieved by measuring absorption of lysed erythrocytes at 414 nm, which significantly increased the sensitivity of the ELA compared to the measurements at 540 nm that are usually applied in this type of assay. Using carp (Cyprinus carpio) erythrocytes it was possible to detect haemolysis induced by 4 microg ml(-1) of saponin and as little as two haemolytic Alexandrium tamarense cells. It is suggested that this improved ELA in microtitre plates be used as a low-cost monitoring tool for detection and analysis of potential harmful algae. Furthermore, this ELA can be useful as a sensitive screening system for substances of pharmacological interest, e.g. selectively acting cytolytic antibiotics.

In addition to ecological factors, evolutionary processes can determine the invasion success of a species. In particular, genetic admixture has the potential to induce rapid evolutionary change, which can result from natural or human-assisted secondary contact between differentiated populations. We studied the recent range expansion of zander in Germany focusing on the interplay between invasion and genetic admixture. Historically, the rivers Elbe and Danube harboured the most north-western source populations from which a north-westward range expansion occurred. This was initiated by introducing zander outside its native range into rivers and lakes, and was fostered by migration through artificial canals and stocking from various sources. We analysed zander populations of the native and invaded ranges using nuclear and mitochondrial genetic markers. Three genetic lineages were identified, which were traced to ancestral ranges. Increased genetic diversity and admixture in the invaded region highlighted asymmetric gene flow towards this area. We suppose that the adaptive potential of the invading populations was promoted by genetic admixture, whereas competitive exclusion in the native areas provided a buffer against introgression by novel genotypes. These explanations would be in line with evidence that hybridization can drive evolutionary change under conditions when new niches can be exploited.

Very high cell densities and optimal vascularization characterize among others organs and tissues in vivo. In order to study organ-specific functions in vitro or to make use of them in medical devices/treatments in the future, this natural architecture should be rebuilt. An important aspect in this context is the appropriate ratio of medium to cell volume being so far not optimally reestablished in most of the currently available in vitro systems. To improve such culture conditions, we constructed a microstructure to culture hepatocytes and (without any addition of extracellular matrix material) characterized liver tissue in the form of evenly sized aggregates. The liver-specific differentiation status of such aggregates was monitored by their ability to perform CYP450 dependent xenobiotic metabolism along with the measurement of albumin secretion. Freshly isolated adult rat hepatocytes show an initial loss of total CYP450 content and of associated activities (mixed function oxidases). However, in the aggregate system, this level did not decrease further but remained stable or even increased throughout the culture period of 10-13 days. The CYP450 dependent metabolism of the hepatocytes is able to respond to classic inducing agents. The described culture efficiently supports liver-specific functions of adult rat hepatocytes and seems to be suited not only for use in an extracorporeal liver device but also for the formation of evenly sized small aggregates to be of use in transplantation of differentiated liver tissue. Moreover, after design variations, the microstructure can be applied for functional analysis of metabolically active hepatocytes as well as for toxicological and pharmacological validation.

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The coupling of toxicity expression with cell-cycle phases was studied in the toxic marine prymnesiophyte Chrysochromulina polylepis Manton & Parke, Clone B1511. Cell synchronisation of cultures in exponential or early stationary growth phases under nutrient-replete conditions was achieved by manipulation of the photoperiod. Chlorophyll a (chl a) and cell number increased in a stepwise manner, but were asynchronous, with chl a increasing during the light period and cell number increasing during the dark period. In the course of the light period, nearly all cells clustered in the G1 (Gap 1) phase, which lasted for about 20 h. DNA synthesis (S phase) occurred mainly in the dark during a discrete period (about 4 h) and G2 (Gap 2) and mitosis (M) were always completed before the end of the dark period. Toxicity expression, measured by the erythrocyte lysis assay (ELA), exhibited a dramatic drop in LC 50 values (increase in toxicity) during the light period, although this effect was less pronounced after the first 2 generations of cell division when the cultures had entered the stationary phase. Similarly, haemolytic activity per unit cell volume decreased by a factor of 3 to 4 during the dark period over the first 48 h, but became irregular towards the end of the experiment. In this study, the light-dependent effect on toxicity and relationship to discrete phases of the cell cycle are demonstrated for the first time in a prymnesiophyte. KEY WORDS: Cell cycle · Ichthyotoxins ·Phytoflagellates ·Prymnesiophytes ·Chrysochromulina polylepis Resale or republication not permitted without written consent of the publisherAquat Microb Ecol 39: [85][86][87][88][89][90][91][92][93][94][95] 2005 Paasche 1998 [review], Johnsen et al. 1999). The definitive causes of these blooms remain unknown, but in addition to meteorological, hydrographical and chemical conditions that promote growth and toxin production in Chrysochromulina spp., adverse effects on planktonic grazers may also have played a role in bloom development (Nielsen et al. 1990, John et al. 2002.The chemical characterisation of Chrysochromulina polylepis toxin(s) is still lacking. The mode of action of these toxins is apparently non-selective, causing interference mainly with membrane functions, and thus organisms ranging from protozoans to fish are known to be affected (Skjoldal & Dundas 1991, Gjø-saeter et al. 2000. Yasumoto et al. (1990) described the toxins as glucolipids and/or fatty acids, but John et al. (2002) showed that a toxic and an apparently non-toxic clone of C. polylepis exhibited the same lipid and fatty acid composition.Curiously, prior to the major ichthytoxic event in Scandinavia, Chrysochromulina polylepis had been considered to be non-toxic to fish (Manton & Parke 1962) and only slightly toxic to a bryozoan, Electra pilosa (Jebram 1980). Toxicity of C. polylepis was demonstrated to be highly variable within and among strains of this species (review by Edvardsen & Paasche 1998). Little is known of the factors triggering toxicity, but un...

Special microenvironmental conditions are required to induce and/or maintain specific qualities of differentiated cells. An important parameter is the three-dimensional tissue architecture that cannot be reproduced in conventional monolayer systems. Advanced tissue culture systems will meet many of these demands, but may reach their limits, especially when gradients of specific substances over distinct tissue layers must be established for long-term culture. These limitations may be overcome by incorporating microstructures into tissue-like culture systems. The microstructured cell support presented consists of a flat array of 625 cubic microcontainers with porous bottoms, in which cells can be supplied with specific media from both sides of the tissue layer. Permanent cell lines and primary rat hepatocytes have been used to test the culture system. In order to define reproducible conditions for tissue formation and for cell adherence to the structure, several ECM (extracellular matrix) components were tested for coating of microstructured substrata. The described tissue culture system offers great flexibility in adapting the cell support to specific needs.

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