Uncovering potential mangrove microbial bioindicators to assess urban and agricultural pressures on Martinique island in the eastern Caribbean Sea.

Martinique's mangroves, which cover 1.85 ha of the island (<0.1 % of the total area), are considerably vulnerable to local urban, agricultural, and industrial pollutants. Unlike for temperate ecosystems, there are limited indicators that can be used to assess the anthropogenic pressures on mangroves. This study investigated four stations on Martinique Island, with each being subject to varying anthropogenic pressures. An analysis of mangrove sediment cores approximately 18 cm in depth revealed two primary types of pressures on Martinique mangroves: (i) an enrichment in organic matter in the two stations within the highly urbanized bay of Fort-de-France and (ii) agricultural pressure observed in the four studied mangrove stations. This pressure was characterized by contamination, exceeding the regulatory thresholds, with dieldrin, total DDT, and metals (As, Cu and Ni) found in phytosanitary products. The mangroves of Martinique are subjected to varying degrees of anthropogenic pressure, but all are subjected to contamination by organochlorine pesticides. Mangroves within the bay of Fort-de-France experience notably higher pressures compared to those in the island's northern and southern regions. In these contexts, the microbial communities exhibited distinct responses. The microbial biomass and the abundance of bacteria and archaea were higher in the two less-impacted stations, while in the mangrove of Fort-de-France, various phyla typically associated with polluted environments were more prevalent. These differences in the microbiota composition led to the identification of 65 taxa, including Acanthopleuribacteraceae, Spirochaetaceae, and Pirellulaceae, that could potentially serve as indicators of an anthropogenic influence on the mangrove sediments of Martinique Island.

Deciphering environmental forcings in the distribution of meiofauna and nematodes in mangroves of the Atlantic-Caribbean-East Pacific and Indo-West Pacific regions.

Mangroves develop under environmental conditions and anthropogenic pressures whose impact on benthic meiofauna remains poorly understood. It is unclear how meiofauna communities are structured according to local sedimentary conditions. This study was designed to characterize the community structure of meiofauna and nematodes (dominant taxa) and the associated environmental forcings in intertidal mangrove sediments from Mayotte (Indo-West-Pacific), Martinique and Guadeloupe (Caribbean). Sediment cores were sampled at the end of the dry season at low tide on adult mangrove stands with similar immersion time. In each sediment layer, we analyzed redox potential, pH, porewater salinity, grain size, organic matter, metals, organic contaminants, prokaryotes and meiofauna. Our results show that sediments far from cities and agricultural fields trapped site-specific contaminants due to local water transport processes. Some metals, PAHs or pesticides exceeded toxicity thresholds in most of the studied stations, thus being harmful to benthic fauna. The sedimentary environment acts as a filter selecting specific meiofauna communities at station scale only in the Caribbean. In Mayotte, horizontal homogeneity contrasts with vertical heterogeneity of the sedimentary environment and the meiofauna. Nematode genera showed particular distribution patterns horizontally and vertically, suggesting the presence of sediment patches suitable for a restricted pool of genera on each island. Results in the Caribbean are consistent with nested diversity patterns due to environmental filtering. Conversely, horizontal homogeneity at Mayotte would reflect greater dispersal between stations or more spatially homogeneous anthropogenic pressures. The nematode genera present at depth may not be the most specialized, but the most versatile, capable of thriving in different conditions. Terschellingia and Daptonema showed contrasted responses to environmental forcing, likely due to their versatility, while Desmodora showed uniform responses between study areas, except when toxicity thresholds were exceeded. Our results emphasize that a given genus of nematode may respond differently to sedimentary conditions depending on sites.

Mangrove microbiota along the urban-to-rural gradient of the Cayenne estuary (French Guiana, South America): Drivers and potential bioindicators.

The microbial communities inhabiting the Atlantic-East Pacific (AEP) mangroves have been poorly studied, and mostly comprise chronically polluted mangroves. In this study, we characterized changes in the structure and diversity of microbial communities of mangroves along the urban-to-rural gradient of the Cayenne estuary (French Guiana, South America) that experience low human impact. The microbial communities were assigned into 50 phyla. Proteobacteria, Chloroflexi, Acidobacteria, Bacteroidetes, and Planctomycetes were the most abundant taxa. The environmental determinants found to significantly correlated to the microbial communities at these mangroves were granulometry, dieldrin concentration, pH, and total carbon (TC) content. Furthermore, a precise analysis of the sediment highlights the existence of three types of anthropogenic pressure among the stations: (i) organic matter (OM) enrichment due to the proximity to the city and its wastewater treatment plant, (ii) dieldrin contamination, and (iii) naphthalene contamination. These forms of weak anthropogenic pressure seemed to impact the bacterial population size and microbial assemblages. A decrease in Bathyarchaeota, "Candidatus Nitrosopumilus", and Nitrospira genera was observed in mangroves subjected to OM enrichment. Mangroves polluted with organic contaminants were enriched in Desulfobacteraceae, Desulfarculaceae, and Acanthopleuribacteraceae (with dieldrin or polychlorobiphenyl contamination), and Chitinophagaceae and Geobacteraceae (with naphthalene contamination). These findings provide insights into the main environmental factors shaping microbial communities of mangroves in the AEP that experience low human impact and allow for the identification of several potential microbial bioindicators of weak anthropogenic pressure.

Metatranscriptomes of oil-contaminated marine coastal sediment affected by oil addition and/or by the bioturbating activity of the marine polychaete Hediste diversicolor: Who are the microbial players?

In coastal marine sediment, oxygen fluctuations induced by bioturbating activities are widespread and exert a great influence, not only on the structure and diversity of the microbenthic communities, but also on their activities. Thus, the activity of benthic organisms can have a significant influence on the degradation of hydrocarbons (HC) and can favor the development of hydrocarbonoclastic microorganisms in contaminated marine sediments. Here, we have generated metatranscriptomic data from coastal marine sediments affected by oil addition and/or by the reworking activity of the marine polychaete Hediste diversicolor to gain insights into the active microbial groups involved in the response to oil addition under the oxygen-fluctuating conditions. The preliminary results suggest that the macrofauna promote the diversity of active aerobic hydrocarbonoclastic bacteria in marine sediments, even if its influence cannot be strongly observed at the microbial community expression profiles level.

Oil removal and effects of spilled oil on active microbial communities in close to salt-saturation brines.

Abiotic and biotic processes associated with the degradation of a light petroleum in brines close to the salt-saturation (~31 %) and the effect of labile organic matter (LOM) supply (casaminoacids/citrate; 0.2 and 0.1 % w/v, respectively) were followed during an incubation of 30 days. After 4-week incubation at 40 °C under light/dark cycles, a 24 % of abiotic degradation was observed in untreated brines. The stimulation of native brines community with LOM addition allowed an additional 12.8 % oil attenuation due to biodegradation processes. Successional changes in the active microbial community structure due to the oil contamination (16S rRNA DGGE approach) showed the selection of one phylotype affiliated to Salinibacter and the disappearance of Haloquadratum walsbyi in untreated brines. In LOM-amended microcosms, phylotypes related to Salinibacter, Haloarcula, Haloterrigena and Halorhabdus were selected. An effect of hydrocarbon contamination was only observed in the bacterial community with the inhibition of two dominant proteobacterial phylotypes. This study further confirms that short-term and moderate oil biodegradation is possible in LOM-stimulated brines. Biodegradation should be much more reduced under in situ conditions. Self-cleaning capacities of close to saturation hypersaline lakes appears, therefore very limited compared to non-extreme haline environments.

Ecological significance of Synergistetes in the biological treatment of tuna cooking wastewater by an anaerobic sequencing batch reactor.

Lab-scale 2L-anaerobic sequencing batch reactor was operated under mesothermic conditions. The degradation of protein-rich organic matter was determined by chemical oxygen demand, biogas production, and protein-removal activity over the operation. The structure of the microbial community was determined by qPCR and next-generation sequencing on 16S rRNA genes. At the steady state, a very efficient removal of protein (92%) was observed. Our results demonstrate a decrease of archaeal and bacterial abundance over time. Members of the phylum Synergistetes, with a peculiar emphasis for those pertaining to families Dethiosulfovibrionaceae and Aminiphilaceae, are of major ecological significance regarding the treatment of this industrial wastewater. The prominent role to be played by members of the phylum Synergistetes regarding protein and/or amino acid degradation is discussed.

Use of dispersant in mudflat oil-contaminated sediment: behavior and effects of dispersed oil on micro- and macrobenthos.

The present study aimed to examine whether the use of dispersant would be suitable for favoring the hydrocarbon degradation in coastal marine sediments without impacting negatively micro- and macrobenthic organisms. Mudflat sediments, maintained during 286 days in mesocosms designed to simulate natural conditions, were contaminated or not with Ural blend crude oil (REBCO) and treated or not with third-generation dispersant (Finasol OSR52). While the dispersant did not lead to an increase of hydrocarbon biodegradation, its use enables an attenuation of more than 55 % of the sediment concentration of total petroleum hydrocarbons (TPH). Canonical correspondence analysis (CCA) correlating T-RFLP patterns with the hydrocarbon content and bacterial abundance indicated weak differences between the different treatments except for the mesocosm treated with oil and dispersant for which a higher bacterial biomass was observed. The use of the dispersant did not significantly decrease the macrobenthic species richness or macroorganisms' densities in uncontaminated or contaminated conditions. However, even if the structure of the macrobenthic communities was not affected, when used in combination with oil, biological sediment reworking coefficient was negatively impacted. Although the use of the dispersant may be worth considering in order to accelerate the attenuation of hydrocarbon-contaminated mudflat sediments, long-term effects on functional aspects of the benthic system such as bioturbation and bacterial activity should be carefully studied before.

Dynamics of bacterial assemblages and removal of polycyclic aromatic hydrocarbons in oil-contaminated coastal marine sediments subjected to contrasted oxygen regimes.

To study the impact of oxygen regimes on the removal of polycylic aromatic hydrocarbons (PAHs) in oil-spill-affected coastal marine sediments, we used a thin-layer incubation method to ensure that the incubated sediment was fully oxic, anoxic, or was influenced by oxic-anoxic switches without sediment stirring. Hydrocarbon content and microbial assemblages were followed during 60 days to determine PAH degradation kinetics and microbial community dynamics according to the oxygenation regimes. The highest PAH removal, with 69 % reduction, was obtained at the end of the experiment under oxic conditions, whereas weaker removals were obtained under oscillating and anoxic conditions (18 and 12 %, respectively). Bacterial community structure during the experiment was determined using a dual 16S rRNA genes/16S rRNA transcripts approach, allowing the characterization of metabolically active bacteria responsible for the functioning of the bacterial community in the contaminated sediment. The shift of the metabolically active bacterial communities showed that the selection of first responders belonged to Pseudomonas spp. and Labrenzia sp. and included an unidentified Deltaproteobacteria-irrespective of the oxygen regime-followed by the selection of late responders adapted to the oxygen regime. A novel unaffiliated phylotype (B38) was highly active during the last stage of the experiment, at which time, the low-molecular-weight (LMW) PAH biodegradation rates were significant for permanent oxic- and oxygen-oscillating conditions, suggesting that this novel phylotype plays an active role during the restoration phase of the studied ecosystem.

Effect of physical sediments reworking on hydrocarbon degradation and bacterial community structure in marine coastal sediments.

The present study aimed to examine whether the physical reworking of sediments by harrowing would be suitable for favouring the hydrocarbon degradation in coastal marine sediments. Mudflat sediments were maintained in mesocosms under conditions as closer as possible to those prevailing in natural environments with tidal cycles. Sediments were contaminated with Ural blend crude oil, and in half of them, harrowing treatment was applied in order to mimic physical reworking of surface sediments. Hydrocarbon distribution within the sediment and its removal was followed during 286 days. The harrowing treatment allowed hydrocarbon compounds to penetrate the first 6 cm of the sediments, and biodegradation indexes (such as n-C18/phytane) indicated that biodegradation started 90 days before that observed in untreated control mesocosms. However, the harrowing treatment had a severe impact on benthic organisms reducing drastically the macrofaunal abundance and diversity. In the harrowing-treated mesocosms, the bacterial abundance, determined by 16S rRNA gene Q-PCR, was slightly increased; and terminal restriction fragment length polymorphism (T-RFLP) analyses of 16S rRNA genes showed distinct and specific bacterial community structure. Co-occurrence network and canonical correspondence analyses (CCA) based on T-RFLP data indicated the main correlations between bacterial operational taxonomic units (OTUs) as well as the associations between OTUs and hydrocarbon compound contents further supported by clustered correlation (ClusCor) analysis. The analyses highlighted the OTUs constituting the network structural bases involved in hydrocarbon degradation. Negative correlations indicated the possible shifts in bacterial communities that occurred during the ecological succession.

Oil spill effects on macrofaunal communities and bioturbation of pristine marine sediments (Caleta Valdés, Patagonia, Argentina): experimental evidence of low resistance capacities of benthic systems without history of pollution.

The Patagonian coast is characterized by the existence of pristine ecosystems which may be particularly sensitive to oil contamination. In this study, a simulated oil spill at acute and chronic input levels was carried out to assess the effects of contamination on the macrobenthic community structure and the bioturbation activity of sediments sampled in Caleta Valdés creek. Superficial sediments were either noncontaminated or contaminated by Escalante crude oil and incubated in the laboratory for 30 days. Oil contamination induced adverse effects on macrobenthic community at both concentrations with, for the highest concentration, a marked decrease of approximately 40 and 55 % of density and specific richness, respectively. Besides the disappearance of sensitive species, some other species like Oligochaeta sp. 1, Paranebalia sp., and Ostracoda sp. 2 species have a higher resistance to oil contamination. Sediment reworking activity was also affected by oil addition. At the highest level of contamination, nearly no activity was observed due to the high mortality of macroorganisms. The results strongly suggest that an oil spill in this protected marine area with no previous history of contamination would have a deep impact on the non-adapted macrobenthic community.

Substrates specialization in lipid compounds and hydrocarbons of Marinobacter genus.

The impact of petroleum contamination and of burrowing macrofauna on abundances of Marinobacter and denitrifiers was tested in marine sediment mesocoms after 3 months incubation. Quantification of this genus by qPCR with a new primer set showed that the main factor favoring Marinobacter abundance was hydrocarbon amendment followed by macrofauna presence. In parallel, proportion of nosZ-harboring bacteria increased in the presence of marcrofauna. Quantitative finding were explained by physiological data from a set of 34 strains and by genomic analysis of 16 genomes spanning 15 different Marinobacter-validated species (Marinobacter hydrocarbonoclasticus, Marinobacter daeopensis, Marinobacter santoriniensis, Marinobacter pelagius, Marinobacter flavimaris, Marinobacter adhaerens, Marinobacter xestospongiae, Marinobacter algicola, Marinobacter vinifirmus, Marinobacter maritimus, Marinobacter psychrophilus, Marinobacter lipoliticus, Marinobacter manganoxydans, Marinobacter excellens, Marinobacter nanhaiticus) and 4 potential novel ones. Among the 105 organic electron donors tested in physiological analysis, Marinobacter pattern appeared narrow for almost all kinds of organic compounds except lipid ones. Strains of this set could oxidize a very large spectrum of lipids belonging to glycerolipids, branched, fatty acyls, and aromatic hydrocarbon classes. Physiological data were comforted by genomic analysis, and genes of alkane 1-monooxygenase, haloalkane dehalogenase, and flavin-binding monooxygenase were detected in most genomes. Denitrification was assessed for several strains belonging to M. hydrocarbonoclasticus, M. vinifirmus, Marinobacter maritinus, and M. pelagius species indicating the possibility to use nitrate as alternative electron acceptor. Higher occurrence of Marinobacter in the presence of petroleum appeared to be the result of a broader physiological trait allowing this genus to use lipids including hydrocarbon as principal electron donors.

Impact of oil on bacterial community structure in bioturbated sediments.

Oil spills threaten coastlines where biological processes supply essential ecosystem services. Therefore, it is crucial to understand how oil influences the microbial communities in sediments that play key roles in ecosystem functioning. Ecosystems such as sediments are characterized by intensive bioturbation due to burrowing macrofauna that may modify the microbial metabolisms. It is thus essential to consider the bioturbation when determining the impact of oil on microbial communities. In this study, an experimental laboratory device maintaining pristine collected mudflat sediments in microcosms closer to true environmental conditions--with tidal cycles and natural seawater--was used to simulate an oil spill under bioturbation conditions. Different conditions were applied to the microcosms including an addition of: standardized oil (Blend Arabian Light crude oil, 25.6 mg.g⁻¹ wet sediment), the common burrowing organism Hediste (Nereis) diversicolor and both the oil and H. diversicolor. The addition of H. diversicolor and its associated bioturbation did not affect the removal of petroleum hydrocarbons. After 270 days, 60% of hydrocarbons had been removed in all microcosms irrespective of the H. diversicolor addition. However, 16S-rRNA gene and 16S-cDNA T-RFLP and RT-PCR-amplicon libraries analysis showed an effect of the condition on the bacterial community structure, composition, and dynamics, supported by PerMANOVA analysis. The 16S-cDNA libraries from microcosms where H. diversicolor was added (oiled and un-oiled) showed a marked dominance of sequences related to Gammaproteobacteria. However, in the oiled-library sequences associated to Deltaproteobacteria and Bacteroidetes were also highly represented. The 16S-cDNA libraries from oiled-microcosms (with and without H. diversicolor addition) revealed two distinct microbial communities characterized by different phylotypes associated to known hydrocarbonoclastic bacteria and dominated by Gammaproteobacteria and Deltaproteobacteria. In the oiled-microcosms, the addition of H. diversicolor reduced the phylotype-richness, sequences associated to Actinobacteria, Firmicutes and Plantomycetes were not detected. These observations highlight the influence of the bioturbation on the bacterial community structure without affecting the biodegradation capacities.

Evidence for surfactant production by the haloarchaeon Haloferax sp. MSNC14 in hydrocarbon-containing media.

The potential for surfactant production by the extreme halophilic archaeon Haloferax sp. MSNC14 in the presence of individual hydrocarbon substrates was studied. This strain was selected for its ability to grow on different types of hydrocarbons at high NaCl concentrations. Linear (n-heptadecane or C17) and isoprenoid (pristane) alkanes, a polyaromatic hydrocarbon (phenanthrene) and ammonium acetate (highly water-soluble control compound) were used as growth substrates. The adherence potential was demonstrated by the ability of the cells to adhere to liquid or solid hydrocarbons. The biosurfactant production was indicated by the reduction of the surface tension (ST) and by the emulsification activity (EA) of cell-free supernatants. Growth on acetate was accompanied by a low EA (lower than 0.1) and a high ST (~70 mN/m), whereas an important EA (up to 0.68 ± 0.08) and a reduction of ST (down to 32 ± 2.3 mN/m) were observed during growth on the different hydrocarbons. Both ST and EA varied with the growth phase. The adhesion to hydrocarbons was higher when cells were grown on C17 (by 60-70 %) and pristane (by 30-50 %) than on phenanthrene (~25 %). The results demonstrated that strain MNSC14 was able to increase the bioavailability of insoluble hydrocarbons, thus facilitating their uptake and their biodegradation even at high salt concentration.

Characterization of specificity of bacterial community structure within the burrow environment of the marine polychaete Hediste (Nereis) diversicolor.

Bioturbation is known to stimulate microbial communities, especially in macrofaunal burrows where the abundance and activities of bacteria are increased. Until now, these microbial communities have been poorly characterized and an important ecological question remains: do burrow walls harbor similar or specific communities compared with anoxic and surface sediments? The bacterial community structure of coastal sediments inhabited by the polychaete worm Hediste diversicolor was investigated. Surface, burrow wall and anoxic sediments were collected at the Carteau beach (Gulf of Fos, Mediterranean Sea). Bacterial diversity was determined by analyzing small subunit ribosomal RNA (16S rRNA) sequences from three clone libraries (168, 179 and 129 sequences for the surface, burrow wall and anoxic sediments, respectively). Libraries revealed 306 different operational taxonomic units (OTUs) belonging to at least 15 bacterial phyla. Bioinformatic analyses and comparisons between the three clone libraries showed that the burrow walls harbored a specific bacterial community structure which differed from the surface and anoxic environments. More similarities were nevertheless found with the surface assemblage. Inside the burrow walls, the bacterial community was characterized by high biodiversity, which probably results from the biogeochemical heterogeneity of the burrow system.

Bacterial community changes during bioremediation of aliphatic hydrocarbon-contaminated soil.

The microbial community response during the oxygen biostimulation process of aged oil-polluted soils is poorly documented and there is no reference for the long-term monitoring of the unsaturated zone. To assess the potential effect of air supply on hydrocarbon fate and microbial community structure, two treatments (0 and 0.056 mol h⁻¹ molar flow rate of oxygen) were performed in fixed bed reactors containing oil-polluted soil. Microbial activity was monitored continuously over 2 years throughout the oxygen biostimulation process. Microbial community structure before and after treatment for 12 and 24 months was determined using a dual rRNA/rRNA gene approach, allowing us to characterize bacteria that were presumably metabolically active and therefore responsible for the functionality of the community in this polluted soil. Clone library analysis revealed that the microbial community contained many rare phylotypes. These have never been observed in other studied ecosystems. The bacterial community shifted from Gammaproteobacteria to Actinobacteria during the treatment. Without aeration, the samples were dominated by a phylotype linked to the Streptomyces. Members belonging to eight dominant phylotypes were well adapted to the aeration process. Aeration stimulated an Actinobacteria phylotype that might be involved in restoring the ecosystem studied. Phylogenetic analyses suggested that this phylotype is a novel, deep-branching member of the Actinobacteria related to the well-studied genus Acidimicrobium.

Isolation of hydrocarbon-degrading extremely halophilic archaea from an uncontaminated hypersaline pond (Camargue, France).

Little information exists about the ability of halophilic archaea present in hypersaline environments to degrade hydrocarbons. In order to identify the potential actors of hydrocarbon degradation in these environments, enrichment cultures were prepared using samples collected from a shallow crystallizer pond with no known contamination history in Camargue, France, with n-alkanes provided as source of carbon and energy. Five alkane-degrading halophilic archaeal strains were isolated: one (strain MSNC 2) was closely related to Haloarcula and three (strains MSNC 4, MSNC 14, and MSNC 16) to Haloferax. Biodegradation assays showed that depending on the strain, 32 to 95% (0.5 g/l) of heptadecane was degraded after 30 days of incubation at 40 degrees C in 225 g/l NaCl artificial medium. One of the strains (MSNC 14) was also able to degrade phenanthrene. This work clearly shows for the first time the potential role of halophilic archaea belonging to the genera Haloarcula and Haloferax in the degradation of hydrocarbons in both pristine and hydrocarbon-contaminated hypersaline environments.

In vitro propagation of the microsporidian pathogen Brachiola algerae and studies of its chromosome and ribosomal DNA organization in the context of the complete genome sequencing project.

Brachiola algerae has a broad host spectrum from human to mosquitoes. The successful infection of two mosquito cell lines (Mos55: embryonic cells and Sua 4.0: hemocyte-like cells) and a human cell line (HFF) highlights the efficient adaptive capacity of this microsporidian pathogen. The molecular karyotype of this microsporidian species was determined in the context of the B. algerae genome sequencing project, showing that its haploid genome consists of 30 chromosomal-sized DNAs ranging from 160 to 2240 kbp giving an estimated genome size of 23 Mbp. A contig of 12,269 bp including the DNA sequence of the B. algerae ribosomal transcription unit has been built from initial genomic sequences and the secondary structure of the large subunit rRNA constructed. The data obtained indicate that B. algerae should be an excellent parasitic model to understand genome evolution in relation to infectious capacity.

PhylArray: phylogenetic probe design algorithm for microarray.

MOTIVATION: Microbial diversity is still largely unknown in most environments, such as soils. In order to get access to this microbial 'black-box', the development of powerful tools such as microarrays are necessary. However, the reliability of this approach relies on probe efficiency, in particular sensitivity, specificity and explorative power, in order to obtain an image of the microbial communities that is close to reality. RESULTS: We propose a new probe design algorithm that is able to select microarray probes targeting SSU rRNA at any phylogenetic level. This original approach, implemented in a program called 'PhylArray', designs a combination of degenerate and non-degenerate probes for each target taxon. Comparative experimental evaluations indicate that probes designed with PhylArray yield a higher sensitivity and specificity than those designed by conventional approaches. Applying the combined PhyArray/GoArrays strategy helps to optimize the hybridization performance of short probes. Finally, hybridizations with environmental targets have shown that the use of the PhylArray strategy can draw attention to even previously unknown bacteria.

GoArrays: highly dynamic and efficient microarray probe design.

MOTIVATION: The use of oligonucleotide microarray technology requires a very detailed attention to the design of specific probes spotted on the solid phase. These problems are far from being commonplace since they refer to complex physicochemical constraints. Whereas there are more and more publicly available programs for microarray oligonucleotide design, most of them use the same algorithm or criteria to design oligos, with only little variation. RESULTS: We show that classical approaches used in oligo design software may be inefficient under certain experimental conditions, especially when dealing with complex target mixtures. Indeed, our biological model is a human obligate parasite, the microsporidia Encephalitozoon cuniculi. Targets that are extracted from biological samples are composed of a mixture of pathogen transcripts and host cell transcripts. We propose a new approach to design oligonucleotides which combines good specificity with a potentially high sensitivity. This approach is original in the biological point of view as well as in the algorithmic point of view. We also present an experimental validation of this new strategy by comparing results obtained with standard oligos and with our composite oligos. A specific E.cuniculi microarray will overcome the difficulty to discriminate the parasite mRNAs from the host cell mRNAs demonstrating the power of the microarray approach to elucidate the lifestyle of an intracellular pathogen using mix mRNAs.