Unexpected metabolic disorders induced by endocrine disruptors in Xenopus tropicalis provide new lead for understanding amphibian decline.

Despite numerous studies suggesting that amphibians are highly sensitive to endocrine disruptors (EDs), both their role in the decline of populations and the underlying mechanisms remain unclear. This study showed that frogs exposed throughout their life cycle to ED concentrations low enough to be considered safe for drinking water, developed a prediabetes phenotype and, more commonly, a metabolic syndrome. Female Xenopus tropicalis exposed from tadpole stage to benzo(a)pyrene or triclosan at concentrations of 50 ng⋅L-1 displayed glucose intolerance syndrome, liver steatosis, liver mitochondrial dysfunction, liver transcriptomic signature, and pancreatic insulin hypersecretion, all typical of a prediabetes state. This metabolic syndrome led to progeny whose metamorphosis was delayed and occurred while the individuals were both smaller and lighter, all factors that have been linked to reduced adult recruitment and likelihood of reproduction. We found that F1 animals did indeed have reduced reproductive success, demonstrating a lower fitness in ED-exposed Xenopus Moreover, after 1 year of depuration, Xenopus that had been exposed to benzo(a)pyrene still displayed hepatic disorders and a marked insulin secretory defect resulting in glucose intolerance. Our results demonstrate that amphibians are highly sensitive to EDs at concentrations well below the thresholds reported to induce stress in other vertebrates. This study introduces EDs as a possible key contributing factor to amphibian population decline through metabolism disruption. Overall, our results show that EDs cause metabolic disorders, which is in agreement with epidemiological studies suggesting that environmental EDs might be one of the principal causes of metabolic disease in humans.

Utilization of a Gender-Based Sorting Machine for Crustacean Selection in Bioconcentration Studies with the Freshwater Amphipod Hyalella azteca.

Bioconcentration factors (BCFs) are determined by fish flow-through tests performed according to Organisation for Economic Co-operation and Development test guideline 305. These are time-consuming and expensive and use a large number of animals. An alternative test design using the freshwater amphipod Hyalella azteca for bioconcentration studies has been recently developed and demonstrated a high potential. For bioconcentration studies using H. azteca, male amphipods are preferred compared with female organisms. Manual sexing of male adult amphipods is, however, time-consuming and requires care and skill. A new fully automatic sorting and dispensing machine for H. azteca based on image analysis has recently been developed by the company Life Science Methods. Nevertheless, an anesthesia step is necessary prior to the automatic selection. In the present study, we show that a single-pulse of 90 min of tricaine at the concentration of 1 g/L can be used and is recommended to select H. azteca males manually or automatically using the sorting machine. In the second part, we demonstrate that the machine has the ability to select, sort, and disperse the males of a culture batch of H. azteca as efficiently as manual procedures. In the last part of the study, BCFs of two organic substances were evaluated using the H. azteca bioconcentration test (HYBIT) protocol, with an anesthetizing step and robotic selection compared with manual selection without an anesthetizing step. The different BCF values obtained were in accordance with those indicated in the literature and showed that an anesthetizing step had no effect on the BCF values. Therefore, these data validated the interest in this sorting machine for selecting males to perform bioconcentrations studies with H. azteca. Environ Toxicol Chem 2023;42:1075-1084. © 2023 SETAC.

Isolation identification and biochemical characterization of a novel halo-tolerant lipase from the metagenome of the marine sponge Haliclona simulans.

BACKGROUND: Lipases (EC 3.1.1.3) catalyze the hydrolysis of triacyl glycerol to glycerol and are involved in the synthesis of both short chain and long chain acylglycerols. They are widely used industrially in various applications, such as baking, laundry detergents and as biocatalysts in alternative energy strategies. Marine ecosystems are known to represent a large reservoir of biodiversity with respect to industrially useful enzymes. However the vast majority of microorganisms within these ecosystems are not readily culturable. Functional metagenomic based approaches provide a solution to this problem by facilitating the identification of novel enzymes such as the halo-tolerant lipase identified in this study from a marine sponge metagenome. RESULTS: A metagenomic library was constructed from the marine sponge Haliclona simulans in the pCC1fos vector, containing approximately 48,000 fosmid clones. High throughput plate screening on 1% tributyrin agar resulted in the identification of 58 positive lipase clones. Following sequence analysis of the 10 most highly active fosmid clones the pCC1fos53E1 clone was found to contain a putative lipase gene lpc53E1, encoded by 387 amino acids and with a predicted molecular mass of 41.87 kDa. Sequence analysis of the predicted amino acid sequence of Lpc53E1 revealed that it is a member of the group VIII family of lipases possessing the SXTK motif, related to type C ß-lactamases. Heterologous expression of lpc53E1 in E. coli and the subsequent biochemical characterization of the recombinant protein, showed an enzyme with the highest substrate specificity for long chain fatty acyl esters. Optimal activity was observed with p- nitrophenyl palmitate (C16) at 40°C, in the presence of 5 M NaCl at pH 7; while in addition the recombinant enzyme displayed activity across broad pH (3-12) and temperature (4 -60°C) ranges and high levels of stability in the presence of various solvents at NaCl concentrations as high as 5 M and at temperatures ranging from 10 to 80°C. A maximum lipase activity of 2,700 U/mg was observed with 10 mM p-nitrophenyl palmitate as substrate, in the presence of 5 mM Ca2+ and 5 M NaCl, and a reaction time of 15 min at pH 7 and 40°C; while KM and Vmax values were calculated to be 1.093 mM-1 and 50 µmol/min, respectively. CONCLUSION: We have isolated a novel halo tolerant lipase following a functional screen of a marine sponge fosmid metagenomic library. The activity and stability profile of the recombinant enzyme over a wide range of salinity, pH and temperature; and in the presence of organic solvent and metal ions suggests a utility for this enzyme in a variety of industrial applications.

Marine metagenomics: new tools for the study and exploitation of marine microbial metabolism.

The marine environment is extremely diverse, with huge variations in pressure and temperature. Nevertheless, life, especially microbial life, thrives throughout the marine biosphere and microbes have adapted to all the divergent environments present. Large scale DNA sequence based approaches have recently been used to investigate the marine environment and these studies have revealed that the oceans harbor unprecedented microbial diversity. Novel gene families with representatives only within such metagenomic datasets represent a large proportion of the ocean metagenome. The presence of so many new gene families from these uncultured and highly diverse microbial populations represents a challenge for the understanding of and exploitation of the biology and biochemistry of the ocean environment. The application of new metagenomic and single cell genomics tools offers new ways to explore the complete metabolic diversity of the marine biome.

Concomitant exposure to benzo[a]pyrene and triclosan at environmentally relevant concentrations induces metabolic syndrome with multigenerational consequences in Silurana (Xenopus) tropicalis.

Numerous studies suggest that amphibians are highly sensitive to endocrine disruptors (ED) but their precise role in population decline remains unknown. This study shows that frogs exposed to a mixture of ED throughout their life cycle, at environmentally relevant concentrations, developed an unexpected metabolic syndrome. Female Silurana (Xenopus) tropicalis exposed to a mixture of benzo[a]pyrene and triclosan (50 ng·L-1 each) from the tadpole stage developed liver steatosis and transcriptomic signature associated with glucose intolerance syndrome, and pancreatic insulin hyper secretion typical of pre-diabetes. These metabolic disorders were associated with delayed metamorphosis and developmental mortality in their progeny, both of which have been linked to reduced adult recruitment and reproductive success. Indeed, F1 females were smaller and lighter and presented reduced reproductive capacities, demonstrating a reduced fitness of ED-exposed Xenopus. Our results confirm that amphibians are highly sensitive to ED even at concentrations considered to be safe for other animals. This study demonstrates that ED might be considered as direct contributing factors to amphibian population decline, due to their disruption of energetic metabolism.

Protein and DNA fingerprinting of a soil bacterial community inoculated into three different sterile soils.

The functional and genetic structures of a soil bacterial community were characterized after inoculation into three different sterile soils using a protein and DNA fingerprinting method, respectively. Principal component analysis (PCA) of profiles revealed that, depending on soil characteristics, bacterial communities with similar genetic structures harbored different functional structures and thus could potentially be of differing ecological significance for soil functioning. Co-inertia analysis between protein fingerprinting data and the corresponding sets of soil physicochemical characteristics demonstrated the correlation between the functional structure of the bacterial community and soil parameters, with pH, clay and CaCO(3) contents being the most discriminating factors.

Fingerprinting and diversity of bacterial copA genes in response to soil types, soil organic status and copper contamination.

A molecular fingerprinting assay was developed to assess the diversity of copA genes, one of the genetic determinants involved in bacterial resistance to copper. Consensus primers of the copA genes were deduced from an alignment of sequences from proteobacterial strains. A PCR detection procedure was optimized for bacterial strains and allowed the description of a novel copA genetic determinant in Pseudomonas fluorescens. The copA DNA fingerprinting procedure was optimized for DNA directly extracted from soils differing in their physico-chemical characteristics and in their organic status (SOS). Particular copA genetic structures were obtained for each studied soil and a coinertia analysis with soil physico-chemical characteristics revealed the strong influence of pH, soil texture and the quality of soil organic matter. The molecular phylogeny of copA gene confirmed that specific copA genes clusters are specific for each SOS. Furthermore, this study demonstrates that this approach was sensitive to short-term responses of copA gene diversity to copper additions to soil samples, suggesting that community adaptation is preferentially controlled by the diversity of the innate copA genes rather than by the bioavailability of the metal.

Field and microcosm experiments to evaluate the effects of agricultural Cu treatment on the density and genetic structure of microbial communities in two different soils.

The effects of Cu amendment on indigenous soil microorganisms were investigated in two soils, a calcareous silty clay (Ep) and a sandy soil (Au), by means of a 1-year field experiment and a two-month microcosm incubation. Cu was added as 'Bordeaux mixture' [CuSO(4), Ca(OH)(2)] at the standard rate used in viticulture (B1=16 kg Cu kg(-1) soil) and at a higher level of contamination (B3=48 kg Cu ha(-1) soil). More extractable Cu was observed in sandy soil (Au) than in silty soil (Ep). Furthermore, total Cu and Cu-EDTA declined with time in Au soil, whereas they remained stable in Ep soil. Quantitative modifications of the microflora were assessed by C-biomass measurements and qualitative modifications were assessed by the characterization of the genetic structure of bacterial and fungal communities from DNA directly extracted from the soil, using B- and F-ARISA (bacterial and fungal automated ribosomal intergenic spacer analysis). In the field study, no significant modifications were observed in C-biomass whereas microcosm incubation showed a decrease in B3 contamination only. ARISA fingerprinting showed slight but significant modifications of bacterial and fungal communities in field and microcosm incubation. These modifications were transient in all cases, suggesting a short-term effect of Cu stress. Microcosm experiments detected the microbial community modifications with greater precision in the short-term, while field experiments showed that the biological effects of Cu contamination may be overcome or hidden by pedo-climatic variations.

Contrasting diversity patterns of crenarchaeal, bacterial and fungal soil communities in an alpine landscape.

BACKGROUND: The advent of molecular techniques in microbial ecology has aroused interest in gaining an understanding about the spatial distribution of regional pools of soil microbes and the main drivers responsible of these spatial patterns. Here, we assessed the distribution of crenarcheal, bacterial and fungal communities in an alpine landscape displaying high turnover in plant species over short distances. Our aim is to determine the relative contribution of plant species composition, environmental conditions, and geographic isolation on microbial community distribution. METHODOLOGY/PRINCIPAL FINDINGS: Eleven types of habitats that best represent the landscape heterogeneity were investigated. Crenarchaeal, bacterial and fungal communities were described by means of Single Strand Conformation Polymorphism. Relationships between microbial beta diversity patterns were examined by using Bray-Curtis dissimilarities and Principal Coordinate Analyses. Distance-based redundancy analyses and variation partitioning were used to estimate the relative contributions of different drivers on microbial beta diversity. Microbial communities tended to be habitat-specific and did not display significant spatial autocorrelation. Microbial beta diversity correlated with soil pH. Fungal beta-diversity was mainly related to soil organic matter. Though the effect of plant species composition was significant for all microbial groups, it was much stronger for Fungi. In contrast, geographic distances did not have any effect on microbial beta diversity. CONCLUSIONS/SIGNIFICANCE: Microbial communities exhibit non-random spatial patterns of diversity in alpine landscapes. Crenarcheal, bacterial and fungal community turnover is high and associated with plant species composition through different set of soil variables, but is not caused by geographical isolation.

Copper dynamics and impact on microbial communities in soils of variable organic status.

The effect of soil organic status on copper impact was investigated by means of a microcosm study carried out on a vineyard soil that had been amended with varying types of organic matter during a previous long-term field experiment. Soil microcosms were contaminated at 250 mg Cu kg(-1) and incubated for 35 days. Copper distribution and dynamics were assessed in the solid matrix by a sequential extraction procedure and in the soil solution by measuring total and free exchangeable copper concentrations. Copper bioavailability was also measured with a whole-cell biosensor. Modifications of microbial communities were assessed by means of biomass-C measurements and characterization of genetic structure using ARISA (automated-ribosomal-intergenic-spacer-analysis). The results showed that copper distribution, speciation, and bioavailability are strongly different between organically amended and nonamended soils. Surprisingly, in solution, bioavailable copper correlated with total copper but not with free copper. Similarly the observed differential copper impact on micro-organisms suggested that organic matter controlled copper toxicity. Bacterial-ARISA modifications also correlated with the estimated metal bioavailability and corresponded to the enrichment of the Actinobacteria. Contrarily, biomass-C and fungal-ARISA measurements did not relate trivially to copper speciation and bioavailability, suggesting that the specific composition of the indigenous-soil communities controls its sensitivity to this metal.

Relationships between soil organic status and microbial community density and genetic structure in two agricultural soils submitted to various types of organic management.

The effects of soil organic management on indigenous microorganisms were studied by comparing mulching straw (S), conifer compost (CC), and conifer bark (CB) as well as grass landing with grass (G), clover (Cl), and fescue (F) in a silty-clay soil (Mâcon), and by incorporating vine shoot (VS) and single and double doses of farmyard manure (FM) and mushroom manure (MM) in a calcareous sandy soil (Chinon). Soil physicochemical and microbial characteristics were assessed at each site at two depths by sampling at 0-5 and 5-20 cm for the Mâcon site and 0-10 and 10-20 cm for the Chinon site. Changes in the quantity of soil organic matter (SOM), through an increase in C(org) and N(org) contents, and in its quality, through modifications in the C/N and humic acid/fulvic acid ratios, were essentially recorded at the surface layer of treated plots with differential magnitudes according to the inputs and soil type. Quantitative modifications in microbial communities were assessed by means of C-biomass measurements and resulted in an increase in microbial densities fitted with the increase of C(org) and N(org) contents. However, the deduced C incorporation in microbial biomass was negatively correlated with the C/N ratio, demonstrating a strong influence of the type of organic management on the rate of microbial processes. Qualitative modifications in microbial communities were evaluated by the characterization of the genetic structure of bacterial and fungal communities from DNA directly extracted from the soil, using bacterial and fungal automated ribosomal intergenic spacer analysis. Organic amendments led to changes in the bacterial and fungal communities of both sites. However, the magnitude and the specificity of these changes were different between sites, organic amendments, and microorganisms targeted, revealing that the impact of organic management is dependent on the soil and organic input types as well as on the particular ecology of microorganisms. A co-inertia analysis was performed to specify the role of the quantity and quality of SOM on the modifications of the genetic structure. A significant costructure was only observed for Mâcon plots at 0-5 cm between the bacterial genetic structure and the SOM characteristics, demonstrating the influence of the relative amount of the different humic substances (humic and fulvic acids) on microbial composition.

Microbial community structure and density under different tree species in an acid forest soil (Morvan, France).

Overexploitation of forests to increase wood production has led to the replacement of native forest by large areas of monospecific tree plantations. In the present study, the effects of different monospecific tree cover plantations on density and composition of the indigenous soil microbial community are described. The experimental site of "Breuil-Chenue" in the Morvan (France) was the site of a comparison of a similar mineral soil under Norway spruce (Picea abies), Douglas fir (Pseudotuga menziesii), oak (Quercus sessiflora), and native forest [mixed stand dominated by oak and beech (Fagus sylvatica)]. Sampling was performed during winter (February) at three depths (0-5, 5-10, and 10-15 cm). Abundance of microorganisms was estimated via microbial biomass measurements, using the fumigation-extraction method. The genetic structure of microbial communities was investigated using the bacterial- and fungal-automated ribosomal intergenic spacer analysis (B-ARISA and F-ARISA, respectively) DNA fingerprint. Only small differences in microbial biomass were observed between tree species, the highest values being recorded under oak forest and the lowest under Douglas fir. B- and F-ARISA community profiles of the different tree covers clustered separately, but noticeable similarities were observed for soils under Douglas fir and oak. A significant stratification was revealed under each tree species by a decrease in microbial biomass with increasing depths and by distinct microbial communities for each soil layer. Differences in density and community composition according to tree species and depth were related to soil physicochemical characteristics and organic matter composition.

Sampling strategy in molecular microbial ecology: influence of soil sample size on DNA fingerprinting analysis of fungal and bacterial communities.

Assessing soil microbial community structure by the use of molecular techniques requires a satisfactory sampling strategy that takes into account the high microbial diversity and the heterogeneous distribution of microorganisms in the soil matrix. The influence of the sample size of three different soil types (sand, silt and clay soils) on the DNA yield and analysis of bacterial and fungal community structure were investigated. Six sample sizes from 0.125 g to 4 g were evaluated. The genetic community structure was assessed by automated ribosomal intergenic spacer analysis (A-RISA fingerprint). Variations between bacterial (B-ARISA) and fungal (F-ARISA) community structure were quantified by using principal component analysis (PCA). DNA yields were positively correlated with the sample size for the sandy and silty soils, suggesting an influence of the sample size on DNA recovery, whereas no correlation was observed in the clay soil. B-ARISA was shown to be consistent between the different sample sizes for each soil type indicating that the sampling procedure has no influence on the assessment of bacterial community structure. On the contrary for F-ARISA profiles, strong variations were observed between replicates of the smaller samples (<1 g). Principal component analysis analysis revealed that sampling aliquots of soil > or =1 g are required to obtain robust and reproducible fingerprinting analysis of the genetic structure of fungal communities. However, the smallest samples could be adequate for the detection of minor populations masked by dominant ones in larger samples. The sampling strategy should therefore be different according to the objectives: rather large soil samples (> or =1 g) for a global description of the genetic community structure, or a large number of small soil samples for a more complete inventory of microbial diversity.