Targeted impact of cyproterone acetate on the sexual reproduction of female rotifers.

Monogonont rotifers constitute, depending on the moment of the year, most of the zooplankton in many freshwater ecosystems. Sexual reproduction is essential in the development cycle of these organisms as it enables them to constitute stocks of cysts which can withstand adverse environmental conditions and hatch when favorable conditions return. However, endocrine disrupting compounds (EDCs) can interfere with the reproduction of organisms. The present work aimed to investigate the effects of cyproterone acetate (CPA, anti-androgen and progestogen synthetic steroid) at 0.5 mg L-1, on the sexual reproduction of Brachionus calyciflorus in a cross-mating experiment. Results show no impact on mixis whereas the fertilization rate and resting egg production were higher in females exposed to CPA (from embryogenesis to adult stage), regardless of the treatment applied to the males with which they were mating (i.e. males hatched from CPA-treated females or from control females). Moreover, neonate females which mothers has been exposed to 0.5 mg L-1 CPA had more oocytes in their germarium than control neonates. Our results suggest that the effects of CPA observed are not related to toxicity but rather are consistent with an endocrine disruption-related impact, probably through disturbance of the mate recognition protein (MRP) production and through interference with a steroid receptor. Moreover, the absence of effect on mixis rate indicates that mixis induction on the one hand and mating process and resting production on the other hand are not controlled by the same hormonal pathways.

About the interest of a zooplankton compartment in pond systems: methodology to study the growth kinetic of Daphnia pulex on Scenedesmus sp.

A reliable characterization of cladocerans' growth kinetic on their substrates is crucial for the estimation of their biochemical conversion rate in pond models. Although many studies reported cladocerans' growth inhibitions by high chlorophyceae contents, their growth kinetics had continued to be described in many pond system models by Monod-type kinetic, which describes growth saturation by high substrate contents, but fails to explain the disappearance of cladocerans observed during chlorophyceae's bloom periods. This study aimed to develop a methodology and assess whether growth-inhibition-type models used to describe microbial growth kinetics can be applicable to cladocerans. Experiments were carried out using Daphnia pulex populations and Scenedesmus sp. First, biomass of D. pulex was measured through digital image processing (DIP) during growth experiments. Then, three candidate models (i.e., Andrews, Edward and Haldane models), along with the Monod model, were fitted to the observed data and compared. The results showed that the DIP technique provided reliable results for estimating the biomass of D. pulex. Our findings show that the candidate growth inhibition-type models satisfactorily described D. pulex's growth kinetic (86% variance accounted for). Scenesdemus sp. were not strong inhibitors of the growth of D. pulex (high inhibition constant and low half-saturation constant found).

Detection of small amounts of human adenoviruses in stools: comparison of a new immuno real-time PCR assay with classical tools.

The detection of low virus concentrations in biological matrices, especially stool samples, is facing significant limitations as far as common diagnostic methods (enzyme-linked-immunosorbent assay (ELISA) or quantitative real-time PCR (qPCR)) are considered. Here the development of a new immuno real-time PCR (iPCR) is described and its performance in the detection of human adenoviruses (HAdVs) in spiked stools is compared with those of ELISA and qPCR assays. For the iPCR, detection of the sandwich formed by the complexation of capture antibody-antigen-detection antibody was performed by qPCR thanks to the substitution of peroxydase by a chimeric DNA. This modification increased the detection sensitivity 200-fold compared to ELISA. The direct qPCR results revealed that only 0.3-9.5% of the spiked HAdV were detectable, resulting from important losses of DNA occurring at the extraction step. This step was not necessary in the iPCR workflow, avoiding this drawback. The losses of viral particles occurred at the elution step from the stool only. The recovery rate of the iPCR was thus better and ranged between 21 and 54%. As a result, iPCR enabled the detection of lower virus concentrations in stool samples compared to those detected by ELISA and qPCR. The iPCR could be considered as a 'hyper sensitive ELISA' for early detection of HAdV infections, especially in the case of immunocompromised patients after haematopoietic stem cell transplant.

Novel quantitative TaqMan real-time PCR assays for detection of Cryptosporidium at the genus level and genotyping of major human and cattle-infecting species.

AIMS: Development of TaqMan MGB real-time PCR assays for quantitative typing of major cattle and human-pathogenic Cryptosporidium species. METHODS AND RESULTS: Three specific TaqMan MGB real-time PCRs, based on the SSU rRNA gene, were directed towards livestock-restricted Cryptosporidium andersoni and Cryptosporidium bovis as well as both human-pathogenic Cryptosporidium parvum and Cryptosporidium hominis. A generic TaqMan assay further identified all known Cryptosporidium species and simultaneously monitored PCR inhibition through an external amplification control. The generic and specific assays were highly reproducible, and all displayed a detection limit of one oocyst per reaction. The specific TaqMan protocols also proved valuable for specifically detecting and quantifying target DNA in the presence of non-target DNA in environmental samples. CONCLUSIONS: All TaqMan MGB real-time PCR assays fulfilled the required specificity and sensitivity criteria, both on laboratory strains and on a surface water matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: No molecular-based method was yet available for the quantitative detection of C. andersoni and the cluster formed by C. bovis, Cryptosporidium ryanae and Cryptosporidium xiaoi. This work provides a novel tool to evaluate the parasite load from domestic ruminants and humans, and to improve assessment and management of microbial risk through better appraisal of the origin and fate of faecal pollutions.

Genetic diversity of noroviruses from outbreaks, sporadic cases and wastewater in Luxembourg 2008-2009.

The genetic diversity of norovirus strains obtained from gastroenteritis outbreaks, sporadic case surveillance and wastewater plants was compared in Luxembourg from October 2008 until June 2009. Except for GI.6 and GIV.1 strains detected exclusively in wastewater, all other genotypes were also found in human samples. Of the nine NoV genotypes detected, only three (GII.4, GIIb/II.3 and GIIc/II.12) were associated with institutional outbreaks. The majority of sequences from all sources belonged to genotype GII.4, including two potentially new sub-variants. Strains collected in the context of outbreaks may significantly under-represent the overall genetic diversity of NoVs circulating in a country.

Seasonal depth-related gradients in virioplankton: standing stock and relationships with microbial communities in Lake Pavin (France).

This study presents a depth-related survey of virioplankton abundance in Lake Pavin (Massif Central, France), in relation to the abundances of heterotrophic prokaryotes, picocyanobacteria (Pcy), autotrophic picoeukaryotes (Peu), and of autotrophic (ANF) and heterotrophic (HNF) nanoflagellates. The sampling strategy was designed to be representative of the physico-chemical gradients of the whole water column of the lake, and the seasonal variability as well. In mixolimnic surface waters, all communities were present and viral abundance peaked in summer and autumn. Viral abundance was significantly correlated (p < 0.001) with Pcy, Peu, and ANF, indicating that cyanophages and perhaps other phytoplankton viruses represent a significant pool of viral standing stocks in the mixolimnion of Lake Pavin. Microautotrophs were absent in the deep monimolimnic water masses, where viruses and heterotrophic prokaryotes exhibited highest seasonal abundances in summer and/or autumn and were significantly correlated (p < 0.001) to each other. This indicates that the anoxic monimolimnion of Lake Pavin is an exclusive habitat for viruses and heterotrophic prokaryotes. We conclude that in this habitat, host availability is prevalent over other factors (temperature, oxygen, nutrients, grazers) in favoring viral proliferation.

Uptake visualization of deltamethrin by NanoSIMS and acute toxicity to the water flea Daphnia magna.

The objective of this study was to investigate the uptake of deltamethrin, an insecticide, by Daphnia magna neonates by SIMS and to compare these findings with results based on established toxicity tests. Young daphnids (aged <24 h) were exposed to 0, 50 and 200 microg L(-1) (ppb) deltamethrin. Mobile, immobile and dead animals were enumerated after 24 and 48 h following OECD 202 [OECD 202, 2004. Daphnia sp., acute immobilisation test, guideline for testing of chemicals] guidelines. The animals were embedded in epoxy resin, cut into semi-thin sections (500 nm) and placed on silicon supporters. NanoSIMS 50 (Cameca) images were made from tissues of the intestine for carbon, nitrogen (measured as CN), phosphorus and bromine. To distinguish between relative concentrations of bromine in the guts from different exposure concentrations of deltamethrin, a carbon normalization method was carried out. Both deltamethrin concentrations and time showed a significant effect on immobilization and mortality of the daphnids (P<0.0001). Bromine from deltamethrin could be visualized by NanoSIMS in all exposed gut tissues (gut wall, microvilli layer, perithropic membrane). Highest deltamethrin concentrations following (12)C normalization were found in animals exposed to 200 microg L(-1) deltamethrin, followed by 50 microg L(-1) and the control. NanoSIMS 50 was successfully used as a supplemental technique for elucidating the relation between the uptake and localization of deltamethrin and its toxicity to D. magna. These results highlight the potential usefulness of NanoSIMS to detect marker elements of xenobiotic compounds within exposed organisms, to compare relative exposure concentrations, and to locate these compounds at their original tissue location.

Virioplankton 'pegylation': use of PEG (polyethylene glycol) to concentrate and purify viruses in pelagic ecosystems.

We have described the use of Polyethylene glycol (PEG) for the precipitation of natural communities of aquatic viruses, and its comparison with the usual concentration method based on ultracentrifugation. Experimental samples were obtained from different freshwater ecosystems whose trophic status varied. Based on transmission electron microscope observations and counting of phage-shaped particles, our results showed that the greatest recovery efficiency for all ecosystems was obtained when we used the PEG protocol. On average, this protocol allowed the recovery of >2-fold more viruses, compared to ultracentrifugation. In addition, the diversity of virioplankton, based on genomic size profiling using pulsed field gel electrophoresis, was higher and better discriminated when we used the PEG method. We conclude that pegylation offers a valid, simple and cheaper alternative method to ultracentrifugation, for the concentration and the purification of pelagic viruses.

Occurrence and persistence of bacterial and viral faecal indicators in wastewater biofilms.

Biofilms within wastewater treatment plants can capture enteric microorganisms initially present in the water phase immobilising them either definitively or temporarily. Consequently, fates of microorganisms may totally change depending on whether they interact or not with biofilms. In this study, we assessed the stability of wastewater biofilms comparing the evolution of the concentrations of bacteria (heterotrophic plate count [HPC], thermotolerant coliforms [TC]) and viral (somatic coliphages [SC] and F-specific phages [F +]) indicators in the biofilms and in the corresponding wastewaters at 4 and 20 dgrees C. Additionally, we assessed the monthly occurrence of these bacterial and viral indicators as well as of pathogenic protozoa (Cryptosporidium oocysts and Giardia cysts) in three native wastewater biofilms for four months. Our results show that viral indicators (SC and F + ) persist longer in biofilms than in the corresponding wastewaters at 4 degrees C as well as at 20 degrees C. In contrast, persistence of bacterial indicators (TC and HPC) depends on both the temperature and the matrix. Differences between viral and bacterial persistence are discussed. Monthly analysis of native wastewater biofilms shows that bacterial and viral indicators, as well as Cryptosporidium oocysts and Giardia cysts, attach to wastewater biofilms to a concentration that remains stable in time, probably as a result of a dynamic equilibrium between attachment and detachment processes.

Depth-related gradients of viral activity in Lake Pavin.

High-resolution vertical sampling and determination of viral and prokaryotic parameters in a deep volcanic lake shows that in the absence of thermal stratification but within light, oxygen, and chlorophyll gradients, host availability empirically is prevalent over the physical and chemical environments and favors lytic over lysogenic "viral life cycles."