Strengths and weaknesses of microarray approaches to detect Pseudo-nitzschia species in the field

The planktonic diatom genus Pseudo-nitzschia contains several genetically closely related species. Some of these can produce domoic acid, a potent neurotoxin. Thus, monitoring programs are needed to screen for the presence of these toxic species. Unfortunately, many are impossible to distinguish using light microscopy. Therefore, we assessed the applicability of microarray technology for detection of toxic and non-toxic Pseudo-nitzschia species in the Gulf of Naples (Mediterranean Sea). Here, 11 species have been detected, of which at least 5 are potentially toxic. A total of 49 genus- and species-specific DNA probes were designed in silico against the nuclear LSU and SSU rRNA of 19 species, and spotted on the microarray. The microarray was tested against total RNA of monoclonal cultures of eight species. Only three of the probes designed to be species-specific were indeed so within the limits of our experimental design. To assess the effectiveness of the microarray in detecting Pseudo-nitzschia species in environmental samples, we hybridized total RNA extracted from 11 seasonal plankton samples against microarray slides and compared the observed pattern with plankton counts in light microscopy and with expected hybridization patterns obtained with monoclonal cultures of the observed species. Presence of species in field samples generally resulted in signal patterns on the microarray as observed with RNA extracted from cultures of these species, but many a-specific signals appeared as well. Possible reasons for the numerous cross reactions are discussed. Calibration curves for Pseudo-nitzschia multistriata showed linear relationship between signal strength and cell number.     

Molecular probes for the detection and identification of ichthyotoxic marine microalgae of the genus Pseudochattonella (Dictyochophyceae, Ochrophyta)

Phytoflagellates of the genus Pseudochattonella (Dictyochophyceae, Ochrophyta) form blooms in marine coastal waters in northern Europe, Japan, and New Zealand that at times cause fish kills with severe losses for the aquaculture industry. The aim of this study was to develop molecular probes for the detection and identification of Pseudochattonella at the genus and species level. A variety of probes were developed and applied to either dot blot hybridization, (q)PCR, or microarray format. In the dot blot hybridization assay, five different oligonucleotide probes targeting the small subunit (SSU) rDNA were tested against DNA from 18 microalgal strains and shown to be specific to the genus Pseudochattonella. A genus-specific PCR assay was developed by identifying an appropriate primer pair in the SSU—internal transcribed spacer 1 (ITS1) rDNA region. Its specificity was tested by screening against both target and non-target strains, and the assay was used to confirm the presence or absence of Pseudochattonella species in environmental samples. In order to distinguish between the two species of the genus, two PCR primer pairs each biased towards one of the species were designed in the large subunit (LSU) rDNA D1 domain and used for quantitative real-time PCR. Five selected probes (three SSU and two LSU rDNA) were adapted for the use on microarrays and included on a prototype multi-species microarray for the detection of harmful algae (http://www.midtal.com). Finally, microarrays and qPCR were used for the monthly monitoring of a sampling site in outer Oslofjorden during a 1-year period. Members of Pseudochattonella are difficult to identify by light microscopy in Lugol’s preserved samples, and the two species Pseudochattonella verruculosa and Pseudochattonella farcimen can be morphologically distinguished only by transmission electron microscopy. The molecular probes designed in this study will be a valuable asset to microscopical detection methods in the monitoring of harmful algae and for biogeographical and ecological studies of this genus.     

Note: steps taken to optimise probe specificity and signal intensity prior to field validation of the MIDTAL (Microarray for the Detection of Toxic Algae)

A microarray for the detection of toxic algal species was developed in the European Union 7th Framework project MIDTAL. We initially tested all available fluorescence in situ hybridisation probes for toxic algae, which are normally designed to a length of 18 nt, and found that in most cases the signal was rather weak or all probes designed from the second half of the molecule were inaccessible in a microarray format because of secondary structure of the ribosomal RNA molecule We modified the length of the probes, the fragmentation of the rRNA, the stringency of the washing buffers and the length of the spacer molecules linking the probes to the glass surface of the microarray. Because of the secondary structure of the rRNA molecule, regions of the molecule can be difficult to access by the probes. Each of these modifications has improved probe accessibility and probe specificity to reduce false positives.     

Parallel detection of harmful algae using reverse transcription polymerase chain reaction labeling coupled with membrane-based DNA array

Harmful algal blooms (HABs) are a global problem, which can cause economic loss to aquaculture industry's and pose a potential threat to human health. More attention must be made on the development of effective detection methods for the causative microalgae. The traditional microscopic examination has many disadvantages, such as low efficiency, inaccuracy, and requires specialized skill in identification and especially is incompetent for parallel analysis of several morphologically similar microalgae to species level at one time. This study aimed at exploring the feasibility of using membrane-based DNA array for parallel detection of several microalgae by selecting five microaglae, including Heterosigma akashiwo, Chaetoceros debilis, Skeletonema costatum, Prorocentrum donghaiense, and Nitzschia closterium as test species. Five species-specific (taxonomic) probes were designed from variable regions of the large subunit ribosomal DNA (LSU rDNA) by visualizing the alignment of LSU rDNA of related species. The specificity of the probes was confirmed by dot blot hybridization. The membrane-based DNA array was prepared by spotting the tailed taxonomic probes onto positively charged nylon membrane. Digoxigenin (Dig) labeling of target molecules was performed by multiple PCR/RT-PCR using RNA/DNA mixture of five microalgae as template. The Dig-labeled amplification products were hybridized with the membrane-based DNA array to produce visible hybridization signal indicating the presence of target algae. Detection sensitivity comparison showed that RT-PCR labeling (RPL) coupled with hybridization was tenfold more sensitive than DNA-PCR-labeling-coupled with hybridization. Finally, the effectiveness of RPL coupled with membrane-based DNA array was validated by testing with simulated and natural water samples, respectively. All of these results indicated that RPL coupled with membrane-based DNA array is specific, simple, and sensitive for parallel detection of microalgae which shows promise for monitoring natural samples in the future.     

Evaluation of the MIDTAL microarray chip for monitoring toxic microalgae in the Orkney Islands, U.K.

Harmful or nuisance algal blooms can cause economic damage to fisheries and tourism. Additionally, toxins produced by harmful algae and ingested via contaminated shellfish can be potentially fatal to humans. The seas around the Orkney Islands, UK currently hold a number of toxic algal species which cause shellfishery closures in most years. Extensive and costly monitoring programs are carried out to detect harmful microalgae before they reach action levels. However, the ability to distinguish between toxic and non-toxic strains of some algae is not possible using these methods. The microarrays for the detection of toxic algae (MIDTAL) microarray contains rRNA probes for toxic algal species/strains which have been adapted and optimized for microarray use. In order to investigate the use of the chip for monitoring in the Orkney Islands, samples were collected between 2009 and 2011 from Brings Deep, Scapa Flow, Orkney Islands, UK; RNA was extracted and hybridized with generation 2 and 3.1 of the chip. The data were then compared to cell counts performed under light microscopy and in the case of Alexandrium tamarense to qPCR data targeting the saxitoxin gene and the LSU-rRNA gene. A good agreement between cell numbers and microarray signal was found for A. tamarense, Pseudo-nitzschia sp., Dinophysis sp. (r?<?0.5, for all) in addition to this there the chip successfully detected a large bloom of Karenia mikimotoi (r?<?0.70) in August and September 2011. Overall, there was good improvement in probe signal between generation 2 and generation 3.1 of the chip with much less variability and more consistent results and better correlation between the probes. The chip performed well for A. tamarense group I signal to cell numbers in calibrations (r?>?0.9). However, in field samples, this correlation was slightly lower suggesting interactions between all species in the sample may affect signal. Overall, the chip showed it could identify the presence of target species in field samples although some work is needed to improve the quantitative nature of the chip before it would be suitable for monitoring in the Orkney Islands.     

Seasonal dynamics of harmful algae in outer Oslofjorden monitored by microarray, qPCR, and microscopy

Monitoring of marine microalgae is important to predict and manage harmful algal blooms. Microarray Detection of Toxic ALgae (MIDTAL) is an FP7-funded EU project aiming to establish a multi-species microarray as a tool to aid monitoring agencies. We tested the suitability of different prototype versions of the MIDTAL microarray for the monthly monitoring of a sampling station in outer Oslofjorden during a 1-year period. Microarray data from two different versions of the MIDTAL chip were compared to results from cell counts (several species) and quantitative real-time PCR (qPCR; only Pseudochattonella spp.). While results from generation 2.5 microarrays exhibited a high number of false positive signals, generation 3.3 microarray data generally correlated with microscopy and qPCR data, with three important limitations: (1) Pseudo-nitzschia cells were not reliably detected, possibly because cells were not sufficiently retained during filtration or lysed during the extraction, and because of low sensitivity of the probes; (2) in the case of samples with high concentrations of non-target species, the sensitivity of the arrays was decreased; (3) one occurrence of Alexandrium pseudogonyaulax was not detected due to a 1-bp mismatch with the genus probe represented on the microarray. In spite of these shortcomings our data demonstrate the overall progress made and the potential of the MIDTAL array. The case of Pseudochattonella — where two morphologically similar species impossible to separate by light microscopy were distinguished — in particular, underlines the added value of molecular methods such as microarrays in routine phytoplankton monitoring.     

Molecular probes and microarrays for the detection of toxic algae in the genera Dinophysis and Phalacroma (Dinophyta)

Dinophysis and Phalacroma species containing diarrheic shellfish toxins and pectenotoxins occur in coastal temperate waters all year round and prevent the harvesting of mussels during several months each year in regions in Europe, Chile, Japan, and New Zealand. Toxicity varies among morphologically similar species, and a precise identification is needed for early warning systems. Molecular techniques using ribosomal DNA sequences offer a means to identify and detect precisely the potentially toxic species. We designed molecular probes targeting the 18S rDNA at the family and genus levels for Dinophysis and Phalacroma and at the species level for Dinophysis acuminata, Dinophysis acuta, and Dinophysis norvegica, the most commonly occurring, potentially toxic species of these genera in Western European waters. Dot blot hybridizations with polymerase chain reaction (PCR)-amplified rDNA from 17 microalgae were used to demonstrate probe specificity. The probes were modified along with other published fluorescence in situ hybridization and PCR probes and tested for a microarray platform within the MIDTAL project (http://www.midtal.com). The microarray was applied to field samples from Norway and Spain and compared to microscopic cell counts. These probes may be useful for early warning systems and monitoring and can also be used in population dynamic studies to distinguish species and life cycle stages, such as cysts, and their distribution in time and space.     

Microarray testing for the presence of toxic algae monitoring programme in Galicia (NW Spain)

Rapid and reliable detection of harmful algae in coastal areas and shellfish farms is an important requirement of monitoring programmes. Monitoring of toxic algae by means of traditional methods, i.e., light microscopy, can be time consuming when many samples have to be routinely analysed. Reliable species identification requires expensive equipment and trained personnel to carry out the analyses. However, all techniques for the monitoring of harmful algae usually require transportation of samples to specialised laboratories. In many monitoring laboratories, results are usually obtained within five working days after receiving the sample and therefore preventative measures are not always possible. Molecular technologies are rapidly improving the detection of phytoplankton and their toxins and the speed at which the results can be obtained. Assays are based on the discrimination of the genetic differences of the different species and species-specific probes can be designed. Such probes have been adapted to a microarray or phylochip format and assessed in several EU monitoring sites. Microarray results are presented for 1 year of field samples validated with cell counts from concentrated samples taken during toxic events from the weekly sampling of the Galician Monitoring Programme done by INTECMAR. The Galician monitoring laboratory does their own counting and their results are posted on their web site within 24 h. There was good correlation between cells present and microarray signals. In the few cases of false negatives, these can be attributed to poor RNA extraction of the target species, viz. Prorocentrum or Dinophysis. Where potential false positives were encountered, the smaller volume taken for cell counts as compared to the upto 300 times more volume taken for RNA extraction for the microarray is likely the cause for these differences, making the microarray more sensitive. The microarray was able to provide better species resolution in Alexandrium and Pseudo-nitzschia. In all cases, the toxins recovered by the toxin array were matched by target species in the array or in the cell counts.     

Capillary electrophoresis finger print technique (CE-SSCP): an alternative tool for the monitoring activities of HAB species in Baja California Sur Costal

In Mexican waters, there is no a formal and well-established monitoring program of harmful algal blooms (HAB) events. Until now, most of the work has been focused on the characterization of organisms present in certain communities. Therefore, the development of new techniques for the rapid detection of HAB species is necessary. Capillary electrophoresis finger print technique (CE-SSCP) is a fingerprinting technique based on the identification of different conformers dependent of its base composition. This technique, coupled with capillary electrophoresis, has been used to compare and identify different conformers. The aim of this study was to determine if CE-SSCP analysis of ribosomal RNA (rRNA) gene fragments could be used for a rapid identification of toxic and harmful HAB species to improve monitoring activities along the coasts of Baja California Sur, Mexico.Three different highly variable regions of the 18S and 28S rRNA genes were chosen and their suitability for the discrimination of different dinoflagellate species was assessed by CE-SSCP.The CE-SSCP results obtained for the LSU D7 fragment has demonstrated that this technique with this gene region could be useful for the identification of the ten dinoflagellates species of different genera.We have shown that this method can be used to discriminate species and the next step will be to apply it to natural samples to achieve our goal of molecular monitoring for toxic algae in Mexican waters. This strategy will offer an option to improve an early warning system of HAB events for coastal BCS, allowing the possible implementation of mitigation strategies. A monitoring program of HAB species using molecular methods will permit the analysis of several samples in a short period of time, without the pressure of counting with a taxonomic expert in phytoplankton taxonomy.     

GPR-Analyzer: a simple tool for quantitative analysis of hierarchical multispecies microarrays

Monitoring of marine microalgae is important to predict and manage harmful algae blooms. It currently relies mainly on light-microscopic identification and enumeration of algal cells, yet several molecular tools are currently being developed to complement traditional methods. MIcroarray Detection of Toxic ALgae (MIDTAL) is an FP7-funded EU project aiming to establish a hierarchical multispecies microarray as one of these tools. Prototype arrays are currently being tested with field samples, yet the analysis of the large quantities of data generated by these arrays presents a challenge as suitable analysis tools or protocols are scarce. This paper proposes a two-part protocol for the analysis of the MIDTAL and other hierarchical multispecies arrays: Signal-to-noise ratios can be used to determine the presence or absence of signals and to identify potential false-positives considering parallel and hierarchical probes. In addition, normalized total signal intensities are recommended for comparisons between microarrays and in order to relate signals for specific probes to cell concentrations using external calibration curves. Hybridization- and probe-specific detection limits can be calculated to help evaluate negative results. The suggested analyses were implemented in “GPR-Analyzer”, a platform-independent and graphical user interface-based application, enabling non-specialist users to quickly and quantitatively analyze hierarchical multispecies microarrays. It is available online at http://folk.uio.no/edvardse/gpranalyzer.     

Introduction to project MIDTAL: its methods and samples from Arcachon Bay, France

Microalgae worldwide regularly cause harmful effects, considered from the human perspective, in that they cause health problems and economic damage to fisheries and tourism. Cyanobacteria cause similar problems in freshwaters. These episodes encompass a broad range of phenomena collectively referred to as “harmful algal blooms” (HABs). For adequate management of these phenomena, monitoring of microalgae is required. However, effective monitoring is time-consuming because cell morphology as determined by light microscopy may be insufficient to give definitive species and toxin attribution. In the European Union FP7 project MIDTAL (Microarrays for the Detection of Toxic Algae), we achieved rapid species identification using rRNA genes as the target. These regions can be targeted for probe design to recognise species or even strains. We also included antibody reactions to specific toxins produced by these microalgae because, even when cell numbers are low, toxins can be present and can accumulate in the shellfish. Microarrays are the state-of-the-art technology in molecular biology for the processing of bulk samples for detection of target RNA/DNA sequences. After 36 months, we have completed RNA-cell number–signal intensity calibration curves for 18 HAB species and the analysis of monthly field samples from five locations from year 1. Results from one location, Arcachon Bay (France), are reported here and compared favourably with cell counts in most cases. In general, the microarray was more sensitive than the cell counts, and this is likely a reflection in the difference in water volume analysed with the volume filtered for the microarray an order of magnitude greater.     

Changes in susceptibility of beech (Fagus sylvatica) seedlings towards Phytophthora citricola under the influence of elevated atmospheric CO2 and nitrogen fertilization

The growth-differentiation balance hypothesis (GDBH) predicts changes in susceptibility of plants against herbivores with changing resource availability. In the presented study we tested the validity of the GDBH for trees infected with a root pathogen. For this purpose Fagus sylvatica seedlings grown under different atmospheric CO₂- and soil nitrogen regimes were infected with the root pathogen Phytophthora citricola. High nitrogen supply increased total biomass of beech regardless of the CO₂-treatment, whereas elevated CO₂ enhanced biomass only in the high nitrogen treatment. The responses of beech under the different growing regimes to the Phytophthora root infection were not in line with the predictions of the GDBH. Enhanced susceptibility of beech against P. citricola was found in seedlings grown under elevated CO₂ and low nitrogen supply. Fifteen months after inoculation these plants were characterized by enhanced water use efficiency, by altered root-shoot ratios, and by enhanced specific root tip densities.     

荧光原位杂交技术在环境微生物生态学解析中的应用研究

近年来 ,诸多国家在环境微生物领域先后开展了分子生物学研究方法的建立和生物学评价工作。一些不依靠纯培养的微生物群落的分析方法已得到广泛应用和发展。荧光原位杂交 (FISH)技术 ,具有细胞在测定过程中不被破坏、形状不改变、特异性强、能够真实反映在自然环境下微生物的情况及分布等特点 ,在环境微生物群落探测分析中已逐渐被广泛应用。该技术利用带有荧光标记的特异性寡核苷酸探针 ,与细胞内相应的靶核糖体结合 ,能将微生物探测、鉴定到属和种的水平。运用于硝化细菌、除磷细菌和丝状微生物等废水处理中常见的特征性微生物种群和群落生态学研究中 ,颇为高效。该技术的应用避免了传统培养方法进行鉴定和计数的局限性 ,在环境微生物生态学解析中具有较高应用价值。     

Response of Brazilian native trees to acute ozone dose

Ozone (O₃) is a toxic secondary pollutant able to cause an intense oxidative stress that induces visual symptoms on sensitive plant species. Controlled fumigation experiment was conducted with the aim to verify the O₃ sensibility of three tropical species: Piptadenia gonoachanta (Mart.) Macbr. (Fabaceae), Astronium graveolens Jacq. (Anacardiaceae), and Croton floribundus Spreng. (Euphorbiaceae). The microscopical features involved in the oxidative stress were recognized based on specific histochemical analysis. The three species showed visual symptoms, characterized as necrosis and stippling between the veins, mostly visible on the adaxial leaf surface. All the studied species presented hypersensitive-like response (HR-like), and peroxide hydrogen accumulation (H₂O₂) followed by cell death and proanthocyanidin oxidation in P. gonoachanta and A. graveolens. In P. gonoachanta, a decrease in chlorophyll autofluorescence occurred on symptomatic tissues, and in A. graveolens and C. floribundus, a polyphenol compound accumulation occurred. The responses of Brazilian native species were similar to those described for sensitive species from temperate climate, and microscopical markers may be useful for the detection of ozone symptoms in future studies in the field.     

Microbial communities in pesticide-contaminated soils in Kyrgyzstan and bioremediation possibilities

In Kyrgyzstan, many former storehouses and dump sites for obsolete pesticides exist. In 2009/2010, an inventory and assessment of these sites including risks of environmental hazard has been conducted by FAO and the World Bank. Monitoring revealed high concentration of pesticides listed as persistent organic pollutants (POPs). The purpose of this research was to study the microbial structural complexes of the pesticide-contaminated soils in these dumping zones, and to search for and select microorganism’s destructors with cytochrome P450 genes for pesticide degradation. Culture-dependent and culture-independent approaches were used to determine the taxonomic composition of these bacterial communities. The universal primer set for the 16S ribosomal RNA (rRNA) gene and the specific primer set P450R were used to amplify the cytochrome P450 hydroxylase gene. In soils from Suzak A and B and soils from Balykchy dumping sites, the bacteria from the Actinobacteria phylum (Micrococcus genus) were dominant. These bacteria made up 32–47% of the indigenous local microflora; bacteria species from the Pseudomonas genus (Gammaproteobacteria phylum) made up 23% in Suzak, 12% in Balykchy soils. Bacillus species from the Firmicutes phylum were found only in Suzak soils. The 16S rRNA analyses and the specific primer set P450R have revealed bacteria with cytochrome genes which are directly involved in the degradation process of organic carbon compounds. Experiments were carried out to help select active degraders from the bacterial populations isolated and used to degrade Aldrin in laboratory. Active bacterial strains from the Pseudomonas fluorescens and Bacillus polymyxa population were selected which demonstrated high rates of degradation activity on Aldrin.     

Beyond selectivity: Are behavioral avoidance and hormesis likely causes of pyrethroid-induced outbreaks of the southern red mite Oligonychus ilicis?

Secondary pest outbreak is a counterintuitive ecological backlash of pesticide use in agriculture that takes place with the increase in abundance of a non-targeted pest species after pesticide application against a targeted pest species. Although the phenomenon was well recognized, its alternative causes are seldom considered. Outbreaks of the southern red mite Oligonychus ilicis are frequently reported in Brazilian coffee farms after the application of pyrethroid insecticides against the coffee leaf miner Leucoptera coffeella. Selectivity favoring the red mite against its main predatory mites is generally assumed as the outbreak cause, but this theory has never been tested. Here, we assessed the toxicity (and thus the selectivity) of deltamethrin against both mite species: the southern red mite and its phytoseid predator Amblyseius herbicolus. Additionally, behavioral avoidance and deltamethrin-induced hormesis were also tested as potential causes of red mite outbreak using free-choice behavioral walking bioassays with the predatory mite and life-table experiments with both mite species, respectively. Lethal toxicity bioassays indicated that the predatory mite was slightly more susceptible than its prey (1.5×), but in more robust demographic bioassays, the predator was three times more tolerant to deltamethrin than its prey, indicating that predator susceptibility to deltamethrin is not a cause of the reported outbreaks. The predator did not exhibit behavioral avoidance to deltamethrin; however insecticide-induced hormesis in the red mite led to its high population increase under low doses, which was not observed for the predatory mite. Therefore, deltamethrin-induced hormesis is a likely cause of the reported red mite outbreaks.     

Exposure of Brassica juncea (L) to arsenic species in hydroponic medium: comparative analysis in accumulation and biochemical and transcriptional alterations

Arsenic (As) contamination in the environment has attracted considerable attention worldwide. The objective of the present study was to see the comparative effect of As species As(III) and As(V) on accumulation, biochemical responses, and gene expression analysis in Brassica juncea var. Pusa Jaganath (PJn). Hydroponically grown 14-day-old seedlings of B. juncea were treated with different concentrations of As(III) and As(V). Accumulation of total As increased with increasing concentration of both As species and exposure time, mainly in roots. Reduction in seed germination, root–shoot length, chlorophyll, and protein content were observed with increasing concentration and exposure time of both As species, being more in As(III)-treated leaves. PJn variety showed that antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX)) and stress-related parameters (cysteine, proline, and malondialdehyde (MDA)) were stimulated and allows plant to tolerate both As species. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis in leaves showed significant changes in protein profile with more stringent effect with As(III) stress. Semiquantitative RT-PCR analysis showed regulation in expression of phytochelatin synthase (PCS), metallothionine-2 (MT-2), glutathione reductase (GR), and glutathione synthetase (GS) genes under both As(III) and As(V) stresses. Results suggested that accumulation and inhibition on physiological parameters differ according to the As species, while molecular and biochemical parameters showed a combinatorial type of tolerance mechanism against As(III) and As(V) stresses.     

Comparison of specific versus literature species sensitivity distributions for herbicides risk assessment

Species sensitivity distributions (SSDs) are an important predictive tool in risk assessment. Usually, literature data are used to build SSDs that are mostly based on planktonic species. But, to get adequate protective thresholds for environmental communities, one could argue that SSD should be built on ecotoxicological data obtained from species found in the ecosystem that should be protected. This is particularly true when benthic algae are of concern. Due to the lack of literature data, building SSD on benthic microalgae is difficult. This paper aims in comparing SSDs, and thus protective thresholds (hazardous concentration that affects 5 % of the species of a community, HC₅), built on ecotoxicological data obtained (1) from literature and (2) with specific bioassays on benthic diatoms from a lake. Thresholds were derived for protection against four herbicides separately and for a mixture of them. Sensitivity data obtained from literature were statistically lower than the specific data for all herbicides: Species tested in the literature were usually more sensitive (mainly chlorophytes), leading to more protective lower HC₅. The HC₅ thresholds (literature or specific) derived for protection against the mixture were also compared to the observed sensitivity of an assemblage of benthic diatom species exposed to an increasing range of herbicide mixture concentrations. We observed that one species within the assemblage (Fragilaria rumpens) was affected at a concentration below both the literature and the specific HC₅ thresholds.     

Long-term Hg pollution-induced structural shifts of bacterial community in the terrestrial isopod (Porcellio scaber) gut

In previous studies we detected lower species richness and lower Hg sensitivity of the bacteria present in egested guts of Porcellio scaber (Crustacea, Isopoda) from chronically Hg polluted than from unpolluted environment. Basis for such results were further investigated by sequencing of 16S rRNA genes of mercury-resistant (Hg^r) isolates and clone libraries. We observed up to 385 times higher numbers of Hg^r bacteria in guts of animals from polluted than from unpolluted environment. The majority of Hg^r strains contained merA genes. Sequencing of 16S rRNA clones from egested guts of animals from Hg-polluted environments showed elevated number of bacteria from Pseudomonas, Listeria and Bacteroidetes relatives groups. In animals from pristine environment number of bacteria from Achromobacter relatives, Alcaligenes, Paracoccus, Ochrobactrum relatives, Rhizobium/Agrobacterium, Bacillus and Microbacterium groups were elevated. Such bacterial community shifts in guts of animals from Hg-polluted environment could significantly contribute to P. scaber Hg tolerance.     

Effects of a sulfonylurea herbicide on the soil bacterial community

Sulfonylurea herbicides are widely used on a wide range of crops to control weeds. Chevalier® OnePass herbicide is a sulfonylurea herbicide intensively used on cereal crops in Algeria. No information is yet available about the biodegradation of this herbicide or about its effect on the bacterial community of the soil. In this study, we collected an untreated soil sample, and another sample was collected 1 month after treatment with the herbicide. Using a high-resolution melting DNA technique, we have shown that treatment with Chevalier® OnePass herbicide only slightly changed the composition of the whole bacterial community. Two hundred fifty-nine macroscopically different clones were isolated from the untreated and treated soil under both aerobic and microaerobic conditions. The strains were identified by sequencing a conserved fragment of the 16S rRNA gene. The phylogenetic trees constructed using the sequencing results confirmed that the bacterial populations were similar in the two soil samples. Species belonging to the Lysinibacillus, Bacillus, Pseudomonas, and Paenibacillus genera were the most abundant species found. Surprisingly, we found that among ten strains isolated from the treated soil, only six were resistant to the herbicide. Furthermore, bacterial overlay experiments showed that only one resistant strain (related to Stenotrophomonas maltophilia) allowed all the sensitive strains tested to grow in the presence of the herbicide. The other resistant strains allowed only certain sensitive strains to grow. On the basis of these results, we propose that there must be several biodegradation pathways for this sulfonylurea herbicide.