For several hundred years freshwater crayfish (Crustacea—Decapoda—Astacidea) have played an important ecological, cultural and culinary role in Scandinavia. However, many native populations of noble crayfish Astacus astacus have faced major declines during the last century, largely resulting from human assisted expansion of non-indigenous signal crayfish Pacifastacus leniusculus that carry and transmit the crayfish plague pathogen. In Denmark, also the non-indigenous narrow-clawed crayfish Astacus leptodactylus has expanded due to anthropogenic activities. Knowledge about crayfish distribution and early detection of non-indigenous and invasive species are crucial elements in successful conservation of indigenous crayfish. The use of environmental DNA (eDNA) extracted from water samples is a promising new tool for early and non-invasive detection of species in aquatic environments. In the present study, we have developed and tested quantitative PCR (qPCR) assays for species-specific detection and quantification of the three above mentioned crayfish species on the basis of mitochondrial cytochrome oxidase 1 (mtDNA-CO1), including separate assays for two clades of A. leptodactylus. The limit of detection (LOD) was experimentally established as 5 copies/PCR with two different approaches, and the limit of quantification (LOQ) were determined to 5 and 10 copies/PCR, respectively, depending on chosen approach. The assays detected crayfish in natural freshwater ecosystems with known populations of all three species, and show promising potentials for future monitoring of A. astacus, P. leniusculus and A. leptodactylus. However, the assays need further validation with data 1) comparing traditional and eDNA based estimates of abundance, and 2) representing a broader geographical range for the involved crayfish species.

A new microarray method, the isotope array approach, for identifying microorganisms which consume a 14 C-labeled substrate within complex microbial communities was developed. Experiments were performed with a small microarray consisting of oligonucleotide probes targeting the 16S rRNA of ammonia-oxidizing bacteria (AOB). Total RNA was extracted from a pure culture of Nitrosomonas eutropha grown in the presence of [ 14 C]bicarbonate. After fluorescence labeling of the RNA and microarray hybridization, scanning of all probe spots for fluorescence and radioactivity revealed that specific signals were obtained and that the incorporation of 14 C into rRNA could be detected unambiguously. Subsequently, we were able to demonstrate the suitability of the isotope array approach for monitoring community composition and CO 2 fixation activity of AOB in two nitrifying activated-sludge samples which were incubated with [ 14 C]bicarbonate for up to 26 h. AOB community structure in the activated-sludge samples, as predicted by the microarray hybridization pattern, was confirmed by quantitative fluorescence in situ hybridization (FISH) and comparative amoA sequence analyses. CO 2 fixation activities of the AOB populations within the complex activated-sludge communities were detectable on the microarray by 14 C incorporation and were confirmed independently by combining FISH and microautoradiography. AOB rRNA from activated sludge incubated with radioactive bicarbonate in the presence of allylthiourea as an inhibitor of AOB activity showed no incorporation of 14 C and thus was not detectable on the radioactivity scans of the microarray. These results suggest that the isotope array can be used in a PCR-independent manner to exploit the high parallelism and discriminatory power of microarrays for the direct identification of microorganisms which consume a specific substrate in the environment.Deciphering the structure and function of complex microbial communities is a central theme in microbial ecology. However, traditional cultivation-dependent methods are inadequate to fulfill this task because most members of microbial communities in natural and engineered systems cannot be cultured (2, 69). In contrast, the cultivation-independent 16S rRNA approach allows one to obtain more complete inventories of such microorganisms present in a particular system. If this approach is combined with quantitative gene probe techniques, such as dot blot (66) or fluorescence in situ hybridization (FISH) (14), the compositions of microbial communities can be determined accurately (27,37,56,57,61,62). However, these techniques are very time-consuming; therefore, the number of samples for which quantitative data can be obtained is relatively limited. This limitation can be (at least partially) overcome by the inclusion of DNA microarray technology for the detection of bacterial 16S rRNA or its genes (3,7,23,29,32,48,64,77). Although precise quantitative data are difficult to obtain with this format, it allows one to apply many rRNA-targeted oligonucl...

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14 CO 2 assimilation. This is a metabolic feature that has not been described previously for filamentous bacteria. Such information could not have been derived by using the traditional MAR procedure, whereas the new HetCO 2 -MAR approach differentiates better between substrate uptake and substrate metabolism that result in growth. The HetCO 2 -MAR results were supported by stable isotope analysis of 13 C-labeled phospholipid fatty acids from activated sludge incubated under aerobic and anaerobic conditions in the presence of 13 CO 2 . In conclusion, the novel HetCO 2 -MAR approach expands the possibility for studies of the ecophysiology of uncultivated microorganisms.

Strong inhibitory effects of the anionic surfactant linear alkylbenzene sulfonate (LAS) on four strains of autotrophic ammonia-oxidizing bacteria (AOB) are reported. Two Nitrosospira strains were considerably more sensitive to LAS than two Nitrosomonas strains were. Interestingly, the two Nitrosospira strains showed a weak capacity to remove LAS from the medium. This could not be attributed to adsorption or any other known physical or chemical process, suggesting that biodegradation of LAS took place. In each strain, the metabolic activity (50% effective concentration [EC 50 ], 6 to 38 mg liter ؊1) was affected much less by LAS than the growth rate and viability (EC 50 , 3 to 14 mg liter ؊1 ) were. However, at LAS levels that inhibited growth, metabolic activity took place only for 1 to 5 days, after which metabolic activity also ceased. The potential for adaptation to LAS exposure was investigated with Nitrosomonas europaea grown at a sublethal LAS level (10 mg liter ؊1); compared to control cells, preexposed cells showed severely affected cell functions (cessation of growth, loss of viability, and reduced NH 4 ؉ oxidation activity), demonstrating that long-term incubation at sublethal LAS levels was also detrimental. Our data strongly suggest that AOB are more sensitive to LAS than most heterotrophic bacteria are, and we hypothesize that thermodynamic constraints make AOB more susceptible to surfactant-induced stress than heterotrophic bacteria are. We further suggest that AOB may comprise a sensitive indicator group which can be used to determine the impact of LAS on microbial communities.Autotrophic ammonia-oxidizing bacteria (AOB) have been considered ideal microbial indicators of perturbations caused by pollutants in natural environments (20,54,58). At least three reasons for this can be formulated: (i) AOB perform a vital bottleneck role in N cycling in many natural environments because of their unique ability to oxidize NH 4 ϩ to NO 2 Ϫ (1, 45, 51); (ii) AOB are generally sensitive to pollutants and often require a long time for recovery (20, 25, 33, 54); and (iii) there is a simple and cheap method for measuring NH 4 ϩ oxidation activities in environmental samples (25). In addition, the AOB constitute a very promising model group for studies of microbial diversity and activity, since the methods used for studying these bacteria have been drastically improved recently (9). Given the impressive development of methods, it is likely that AOB will continue to be considered attractive indicators of environmental perturbations in the years to come.Inhibitory effects of various compounds on AOB in various environments have been extensively reported. However, due to the difficulties involved in cultivation of AOB, there is still a general lack of data concerning the effects of toxic chemicals on the general physiology of these bacteria. Previous studies have dealt mostly with specific nitrification inhibitors targeting the NH 4 ϩ monooxygenase enzyme (5, 42), and very little is known about other targets of tox...

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Sewage sludge is frequently applied as fertilizers to cultivated land. However, municipal sewage sludge often contains organic contaminants including nonylphenol (NP), an intermediate from nonionic surfactant degradation. Knowledge about NP degradation in sludge-amended soil is an important prerequisite for adequate risk assessments. In this study, mineralization of 14C-labeled NP in homogenized and nonhomogenized sludge-soil mixtures was investigated. NP was degraded within 38 days in aerobic homogenized mixtures. In nonhomogeneous mixtures containing sludge aggregates, the degradation of NP was retarded and was generally not completed within 3 months (119-126 days). No detectable amounts of NP were transported from the sludge aggregates to the surrounding soil (detection limit: <0.04 mg of NP/kg dw of soil). Oxygen penetration into sludge aggregates was monitored for 50 days with an oxygen microelectrode. An extrapolation of the oxygen data suggested that more than 1 year was required to obtain fully aerobic conditions in a 2-cm sludge aggregate. Since NP is considered persistent in the absence of oxygen, residual amounts of NP may be present in the anaerobic center of aggregates for prolonged periods. The results demonstrate that sludge aggregate size and thus oxygen availability will be a major controlling factor for NP degradation in soil amended with sewage sludge and that the mobility of NP from sludge aggregates to the surrounding soil is negligible.

The current extinction and climate change crises pressure us to predict population dynamics with ever‐greater accuracy. Although predictions rest on the well‐advanced theory of age‐structured populations, two key issues remain poorly explored. Specifically, how the age‐dependency in demographic rates and the year‐to‐year interactions between survival and fecundity affect stochastic population growth rates. We use inference, simulations and mathematical derivations to explore how environmental perturbations determine population growth rates for populations with different age‐specific demographic rates and when ages are reduced to stages. We find that stage‐ vs. age‐based models can produce markedly divergent stochastic population growth rates. The differences are most pronounced when there are survival‐fecundity‐trade‐offs, which reduce the variance in the population growth rate. Finally, the expected value and variance of the stochastic growth rates of populations with different age‐specific demographic rates can diverge to the extent that, while some populations may thrive, others will inevitably go extinct.