Comparison between droplet digital PCR and reverse transcription-quantitative PCR methods to measure ecotoxicology biomarkers.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is currently the gold-standard technique for detecting and quantifying messenger RNA. However, without proper validation, the method may produce artefactual and non-reproducible cycle threshold values generating poor-quality data. The newer droplet digital PCR (ddPCR) method allows for the absolute quantification of targeted nucleic acids providing more sensitive and accurate measurements without requiring external standards. This study compared these two PCR-based methods to measure the expression of well-documented genes used in ecotoxicology studies. We exposed Mediterranean mussels (Mytilus galloprovincialis) to copper and analyzed gene expression in gills and digestive glands using RT-qPCR and ddPCR assays. A step-by-step methodology to optimize and compare the two technologies is described. After ten-fold serial complementary DNA dilution, both RT-qPCR and ddPCR exhibited comparable linearity and efficiency and produced statistically similar results. We conclude that ddPCR is a suitable method to assess gene expression in an ecotoxicological context. However, RT-qPCR has a shorter processing time and remains more cost-effective.

Assessing integrated biomarkers of triplefin fish Forsterygion capito inhabiting contaminated marine water - A multivariate approach.

The presence of multiple chemicals in aquatic ecosystems makes evaluation of their real impact on the biota difficult. Integrated biomarkers are therefore needed to evaluate how these chemicals contribute to environmental degradation. The aims of the present study were to evaluate responses to and effects of marine pollution using a series of biomarkers through multivariate analyses. Transcriptional responses of cyp1a (cytochrome P450), mt (metallothionein), vtg (vitellogenin) and cyp19b (cytochrome P450 aromatase); branchial and hepatic histological alterations; and Fulton condition factors (CF) were evaluated, as well as the metals and polycyclic aromatic hydrocarbons present in Forsterygion capito in Auckland, New Zealand. Sites were selected along a contamination gradient: four highly contaminated sites and four less contaminated. Molecular responses with a higher relative expression of the mt and cyp1a genes were detected at a highly contaminated site (Panmure). Several histological lesion types were found in the livers of fish inhabiting both types of sites, but gill lesions were present primarily at highly contaminated sites. In terms of general health status, the lowest CF values were overwhelmingly found in fish from the same site (Panmure). The multivariate approach revealed that telangiectasia and hyperplasia were associated with the presence of chemicals, and these showed negative associations with the CF values, with fish from three highly contaminated sites being most affected. In conclusion, the multivariate approach helped to integrate these biological markers in this blennioid fish, thus providing a more holistic view of the complex chemical mixtures involved. Future studies should implement these analyses.

Bacteria as Emerging Indicators of Soil Condition.

Bacterial communities are important for the health and productivity of soil ecosystems and have great potential as novel indicators of environmental perturbations. To assess how they are affected by anthropogenic activity and to determine their ability to provide alternative metrics of environmental health, we sought to define which soil variables bacteria respond to across multiple soil types and land uses. We determined, through 16S rRNA gene amplicon sequencing, the composition of bacterial communities in soil samples from 110 natural or human-impacted sites, located up to 300 km apart. Overall, soil bacterial communities varied more in response to changing soil environments than in response to changes in climate or increasing geographic distance. We identified strong correlations between the relative abundances of members of Pirellulaceae and soil pH, members of Gaiellaceae and carbon-to-nitrogen ratios, members of Bradyrhizobium and the levels of Olsen P (a measure of plant available phosphorus), and members of Chitinophagaceae and aluminum concentrations. These relationships between specific soil attributes and individual soil taxa not only highlight ecological characteristics of these organisms but also demonstrate the ability of key bacterial taxonomic groups to reflect the impact of specific anthropogenic activities, even in comparisons of samples across large geographic areas and diverse soil types. Overall, we provide strong evidence that there is scope to use relative taxon abundances as biological indicators of soil condition. IMPORTANCE: The impact of land use change and management on soil microbial community composition remains poorly understood. Therefore, we explored the relationship between a wide range of soil factors and soil bacterial community composition. We included variables related to anthropogenic activity and collected samples across a large spatial scale to interrogate the complex relationships between various bacterial community attributes and soil condition. We provide evidence of strong relationships between individual taxa and specific soil attributes even across large spatial scales and soil and land use types. Collectively, we were able to demonstrate the largely untapped potential of microorganisms to indicate the condition of soil and thereby influence the way that we monitor the effects of anthropogenic activity on soil ecosystems into the future.

Functional gene composition, diversity and redundancy in microbial stream biofilm communities.

We surveyed the functional gene composition and diversity of microbial biofilm communities in 18 New Zealand streams affected by different types of catchment land use, using a comprehensive functional gene array, GeoChip 3.0. A total of 5,371 nutrient cycling and energy metabolism genes within 65 gene families were detected among all samples (342 to 2,666 genes per stream). Carbon cycling genes were most common, followed by nitrogen cycling genes, with smaller proportions of sulphur, phosphorus cycling and energy metabolism genes. Samples from urban and native forest streams had the most similar functional gene composition, while samples from exotic forest and rural streams exhibited the most variation. There were significant differences between nitrogen and sulphur cycling genes detected in native forest and urban samples compared to exotic forest and rural samples, attributed to contrasting proportions of nitrogen fixation, denitrification, and sulphur reduction genes. Most genes were detected only in one or a few samples, with only a small minority occurring in all samples. Nonetheless, 42 of 65 gene families occurred in every sample and overall proportions of gene families were similar among samples from contrasting streams. This suggests the existence of functional gene redundancy among different stream biofilm communities despite contrasting taxonomic composition.

Climate warming and agricultural stressors interact to determine stream periphyton community composition.

Lack of knowledge about how the various drivers of global climate change will interact with multiple stressors already affecting ecosystems is the basis for great uncertainty in projections of future biological change. Despite concerns about the impacts of changes in land use, eutrophication and climate warming in running waters, the interactive effects of these stressors on stream periphyton are largely unknown. We manipulated nutrients (simulating agricultural runoff), deposited fine sediment (simulating agricultural erosion) (two levels each) and water temperature (eight levels, 0-6 °C above ambient) simultaneously in 128 streamside mesocosms. Our aim was to determine the individual and combined effects of the three stressors on the algal and bacterial constituents of the periphyton. All three stressors had pervasive individual effects, but in combination frequently produced synergisms at the population level and antagonisms at the community level. Depending on sediment and nutrient conditions, the effect of raised temperature frequently produced contrasting response patterns, with stronger or opposing effects when one or both stressors were augmented. Thus, warming tended to interact negatively with nutrients or sediment by weakening or reversing positive temperature effects or strengthening negative ones. Five classes of algal growth morphology were all affected in complex ways by raised temperature, suggesting that these measures may prove unreliable in biomonitoring programs in a warming climate. The evenness and diversity of the most abundant bacterial taxa increased with temperature at ambient but not with enriched nutrient levels, indicating that warming coupled with nutrient limitation may lead to a more evenly distributed bacterial community as temperatures rise. Freshwater management decisions that seek to avoid or mitigate the negative effects of agricultural land use on stream periphyton should be informed by knowledge of the interactive effects of multiple stressors in a warming climate.

Soil phosphorus depletion and shifts in plant communities change bacterial community structure in a long-term grassland management trial.

Agricultural systems rely on healthy soils and their sustainability requires understanding the long-term impacts of agricultural practices on soils, including microbial communities. We examined the impact of 17 years of land management on soil bacterial communities in a New Zealand randomized-block pasture trial. Significant variation in bacterial community structure related to mowing and plant biomass removal, while nitrogen fertilizer had no effect. Changes in soil chemistry and legume abundance described 52% of the observed variation in the bacterial community structure. Legumes (Trifolium species) were absent in unmanaged plots but increased in abundance with management intensity; 11% of the variation in soil bacterial community structure was attributed to this shift in the plant community. Olsen P explained 10% of the observed heterogeneity, which is likely due to persistent biomass removal resulting in P limitation; Olsen P was significantly lower in plots with biomass removed (14 mg kg(-1) ± 1.3SE) compared with plots that were not mown, or where biomass was left after mowing (32 mg kg(-1) ± 1.6SE). Our results suggest that removal of plant biomass and associated phosphorus, as well as shifts in the plant community, have greater long-term impacts on soil bacterial community structure than application of nitrogen fertilizers.