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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