Organisms continuously release DNA into their environments via shed cells, excreta, gametes and decaying material. Analysis of this ‘environmental DNA’ (eDNA) is revolutionizing biodiversity ...monitoring. eDNA outperforms many established survey methods for targeted detection of single species, but few studies have investigated how well eDNA reflects whole communities of organisms in natural environments. We investigated whether eDNA can recover accurate qualitative and quantitative information about fish communities in large lakes, by comparison to the most comprehensive long‐term gill‐net data set available in the UK. Seventy‐eight 2L water samples were collected along depth profile transects, gill‐net sites and from the shoreline in three large, deep lakes (Windermere, Bassenthwaite Lake and Derwent Water) in the English Lake District. Water samples were assayed by eDNA metabarcoding of the mitochondrial 12S and cytochrome b regions. Fourteen of the 16 species historically recorded in Windermere were detected using eDNA, compared to four species in the most recent gill‐net survey, demonstrating eDNA is extremely sensitive for detecting species. A key question for biodiversity monitoring is whether eDNA can accurately estimate abundance. To test this, we used the number of sequence reads per species and the proportion of sampling sites in which a species was detected with eDNA (i.e. site occupancy) as proxies for abundance. eDNA abundance data consistently correlated with rank abundance estimates from established surveys. These results demonstrate that eDNA metabarcoding can describe fish communities in large lakes, both qualitatively and quantitatively, and has great potential as a complementary tool to established monitoring methods.
The lack of standardised methodologies in microplastic research has been addressed in recent years as it hampers the comparison of results across studies. The quantification of microplastics in the ...environment is key to the assessment of the potential eco-toxicological impacts that this new category of emerging pollutants could have on terrestrial and aquatic species. Therefore, the need for protocols that are robust, simple and reliable together with their standardisation are of crucial importance. This study has focused on removal of organic matter with Fenton reagent from wastewater and sludge samples. This step of analysis was optimised by implementing a multi-digestion treatment on these samples that have high concentration of complex mixtures of organic matter, which interfere with microplastic enumeration. Moreover, this study targeted the detection of microplastics in the sub-hundred-micron size range due to the potential higher risks associated with smaller-sized particles and the limited data available from previous wastewater research. To show the validity of the method, triplicate samples of raw sewage, final effluent and sludge were independently spiked with two different sizes and types of microplastic polymers. Due to the various analytical stages required for the isolation of microplastics, time is a limiting factor in sample processing. The sequential digestion with Fenton reagent represents an inexpensive and time-efficient procedure for wastewater research providing effective degradation of organic material. These advantages over other currently available methods mean the method is suitable for analysis of large numbers of samples allowing robust monitoring data sets to be generated.
Microplastics were characterized in eight water treatment works (WTWs) in England and Wales (UK). Sources included river water, groundwater, and an upland reservoir. Water treatment varied from ...disinfection, filtration, sedimentation, and activated carbon techniques. At each WTW, five repeat samples of raw and potable water and two repeat sludge samples were taken over 5 months. Microplastics in water were captured on 10 μm filters and nonplastic materials digested in the laboratory. Microplastics ≥25 μm were analyzed using Fourier-transform infrared microscopy. Blanks revealed consistent polyethylene (PE), poly(ethylene terephthalate) (PET), and polypropylene (PP) contamination. Spike recoveries for 63-90 μm polyamide microplastics demonstrated 101% (standard deviation, SD 27%) and 113% (SD 15%) recovery for raw and potable waters and 52% (SD 13%) for sludge. Only four of the six WTWs sampled for raw water and only two of eight WTWs in their potable water had microplastics above the limit of quantification. Considering only the WTWs with quantifiable microplastics, then on average, 4.9 microplastic particles/L were present in raw water and only 0.00011 microplastic particles/L were present in potable water (99.99% removal). Values in waste sludge were highly variable. PE, PET, and PP were the most common polymers quantified in raw water and sludge, and polystyrene and acrylonitrile butadiene styrene were the most common polymers quantified in potable water.
Environmental DNA (eDNA) is a promising tool for rapid and noninvasive biodiversity monitoring. eDNA density is low in environmental samples, and a capture method, such as filtration, is often ...required to concentrate eDNA for downstream analyses. In this study, six treatments, with differing filter types and pore sizes for eDNA capture, were compared for their efficiency and accuracy to assess fish community structure with known fish abundance and biomass via eDNA metabarcoding. Our results showed that different filters (with the exception of 20‐μm large‐pore filters) were broadly consistent in their DNA capture ability. The 0.45‐μm filters performed the best in terms of total DNA yield, probability of species detection, repeatability within pond and consistency between ponds. However performance of 0.45‐μm filters was only marginally better than for 0.8‐μm filters, while filtration time was significantly longer. Given this trade‐off, the 0.8‐μm filter is the optimal pore size of membrane filter for turbid, eutrophic and high fish density ponds analysed here. The 0.45‐μm Sterivex enclosed filters performed reasonably well and are suitable in situations where on‐site filtration is required. Finally, prefilters are applied only if absolutely essential for reducing the filtration time or increasing the throughput volume of the capture filters. In summary, we found encouraging similarity in the results obtained from different filtration methods, but the optimal pore size of filter or filter type might strongly depend on the water type under study.
Environmental DNA (eDNA) analysis is a rapid, non-invasive, cost-efficient biodiversity monitoring tool with enormous potential to inform aquatic conservation and management. Development is ongoing, ...with strong commercial interest, and new uses are continually being discovered. General applications of eDNA and guidelines for best practice in freshwater systems have been established, but habitat-specific assessments are lacking. Ponds are highly diverse, yet understudied systems that could benefit from eDNA monitoring. However, eDNA applications in ponds and methodological constraints specific to these environments remain unaddressed. Following a stakeholder workshop in 2017, researchers combined knowledge and expertise to review these applications and challenges that must be addressed for the future and consistency of eDNA monitoring in ponds. The greatest challenges for pond eDNA surveys are representative sampling, eDNA capture, and potential PCR inhibition. We provide recommendations for sampling, eDNA capture, inhibition testing, and laboratory practice, which should aid new and ongoing eDNA projects in ponds. If implemented, these recommendations will contribute towards an eventual broad standardisation of eDNA research and practice, with room to tailor workflows for optimal analysis and different applications. Such standardisation will provide more robust, comparable, and ecologically meaningful data to enable effective conservation and management of pond biodiversity.
Summary
Earthworms are globally distributed and perform essential roles for soil health and microbial structure. We have investigated the effect of an anthropogenic contamination gradient on the ...bacterial community of the keystone ecological species Lumbricus rubellus through utilizing 16S rRNA pyrosequencing for the first time to establish the microbiome of the host and surrounding soil.
The earthworm‐associated microbiome differs from the surrounding environment which appears to be a result of both filtering and stimulation likely linked to the altered environment associated with the gut micro‐habitat (neutral pH, anoxia and increased carbon substrates). We identified a core earthworm community comprising Proteobacteria (∼50%) and Actinobacteria (∼30%), with lower abundances of Bacteroidetes (∼6%) and Acidobacteria (∼3%). In addition to the known earthworm symbiont (Verminephrobacter sp.), we identified a potential host‐associated Gammaproteobacteria species (Serratia sp.) that was absent from soil yet observed in most earthworms.
Although a distinct bacterial community defines these earthworms, clear family‐ and species‐level modification were observed along an arsenic and iron contamination gradient. Several taxa observed in uncontaminated control microbiomes are suppressed by metal/metalloid field exposure, including eradication of the hereto ubiquitously associated Verminephrobacter symbiont, which raises implications to its functional role in the earthworm microbiome.
There are concerns that antimicrobial usage (AMU) is driving an increase in multi-drug resistant (MDR) bacteria so treatment of microbial infections is becoming harder in humans and animals. The aim ...of this study was to evaluate factors, including usage, that affect antimicrobial resistance (AMR) on farm over time.
A population of 14 cattle, sheep and pig farms within a defined area of England were sampled three times over a year to collect data on AMR in faecal Enterobacterales flora; AMU; and husbandry or management practices. Ten pooled samples were collected at each visit, with each comprising of 10 pinches of fresh faeces. Up to 14 isolates per visit were whole genome sequenced to determine presence of AMR genes.
Sheep farms had very low AMU in comparison to the other species and very few sheep isolates were genotypically resistant at any time point. AMR genes were detected persistently across pig farms at all visits, even on farms with low AMU, whereas AMR bacteria was consistently lower on cattle farms than pigs, even for those with comparably high AMU. MDR bacteria was also more commonly detected on pig farms than any other livestock species.
The results may be explained by a complex combination of factors on pig farms including historic AMU; co-selection of AMR bacteria; variation in amounts of antimicrobials used between visits; potential persistence in environmental reservoirs of AMR bacteria; or importation of pigs with AMR microbiota from supplying farms. Pig farms may also be at increased risk of AMR due to the greater use of oral routes of group antimicrobial treatment, which were less targeted than cattle treatments; the latter mostly administered to individual animals. Also, farms which exhibited either increasing or decreasing trends of AMR across the study did not have corresponding trends in their AMU. Therefore, our results suggest that factors other than AMU on individual farms are important for persistence of AMR bacteria on farms, which may be operating at the farm and livestock species level.
Potential increases of phytoplankton concentrations in river systems due to global warming and changing climate could pose a serious threat to the anthropogenic use of surface waters. Nevertheless, ...the extent of the effect of climatic alterations on phytoplankton concentrations in river systems has not yet been analysed in detail. In this study, we assess the impact of a change in precipitation and temperature on river phytoplankton concentration by means of a physically-based model. A scenario-neutral methodology has been employed to evaluate the effects of climate alterations on flow, phosphorus concentration and phytoplankton concentration of the River Thames (southern England). In particular, five groups of phytoplankton are considered, representing a range of size classes and pigment phenotypes, under three different land-use/land-management scenarios to assess their impact on phytoplankton population levels. The model results are evaluated within the framework of future climate projections, using the UK Climate Projections 09 (UKCP09) for the 2030s. The results of the model demonstrate that an increase in average phytoplankton concentration due to climate change is highly likely to occur, with the magnitude varying depending on the location along the River Thames. Cyanobacteria show significant increases under future climate change and land use change. An expansion of intensive agriculture accentuates the growth in phytoplankton, especially in the upper reaches of the River Thames. However, an optimal phosphorus removal mitigation strategy, which combines reduction of fertiliser application and phosphorus removal from wastewater, can help to reduce this increase in phytoplankton concentration, and in some cases, compensate for the effect of rising temperature.
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•Both climate and land-management changes have an impact on phytoplankton.•The phytoplankton response to climatic and land-management changes was estimated.•An increase in phytoplankton concentration due to climate change is likely to occur.•Cyanobacteria are more sensitive to climatic changes than other phytoplankton groups.•P reduction can help alleviate the effects of climate change on phytoplankton.
In order to assess risks to the natural environment from microplastics, it is necessary to have reliable information on all potential inputs and discharges. This relies on stringent quality control ...measures to ensure accurate reporting. Here we focus on wastewater treatment works (WwTWs) and the complex sample matrices these provide. Composite samples of both influent and effluent were collected over a 24 h period on two separate occasions from eight different WwTWs across the UK. Sludge samples were taken on five occasions from five WwTWs. The WwTW treatments included activated sludge, trickling filter and biological aerated flooded filter with or without tertiary treatment. Using micro-FTIR analysis, microplastics ≥25 μm were identified and quantified. Procedural blanks were used to derive limits of detection (LOD) and limits of quantification (LOQ). Where values were above the LOQ, microplastics in the influent ranged from 955 to 17,214 microplastic particles/L and in the effluent from 2 to 54 microplastic particles/L, giving an average removal rate of 99.8%. Microplastics could be quantified in sludge at concentrations of 301–10,380 microplastics/g dry weight, this analytical method therefore revealing higher concentrations than reported in previous studies. The most common polymers present overall were polyethylene (PE), polypropylene (PP) and polyethylene terephthalate (PET). We also report on critical considerations for blank corrections and quality control measures to ensure reliable microplastic analysis across different sample types.
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•Water treatment removed 99.8% microplastics from effluent, but largely to sludge.•Activated sludge plants with tertiary treatment showed high removal performance.•PP, PE and PET were abundant throughout influent, effluent and sludge.•Sludge concentrations were higher than previously reported.•Use of LOD and LOQ allowed more rigorous reporting and limited false positives.
This study describes robust, time efficient methods, used here to analyse microplastics in complex matrices including wastewater influent, effluent and sludge.
Summary
Studying fungal biodiversity using data generated from Illumina MiSeq sequencing platforms poses a number of bioinformatic challenges with the analysis typically involving a large number of ...tools for each analytical step from quality filtering to generating identified operational taxonomic unit (OTU) abundance tables.
Here, we introduce PIPITS, an open‐source stand‐alone suite of software for automated processing of Illumina MiSeq sequences for fungal community analysis. PIPITS exploits a number of state of the art applications to process paired‐end reads from quality filtering to producing OTU abundance tables.
We provide detailed descriptions of the pipeline and show its utility in the analysis of 9 396 092 sequences generated on the MiSeq platform from Illumina MiSeq.
PIPITS is the first automated bioinformatics pipeline dedicated for fungal ITS sequences which incorporates ITSx to extract subregions of ITS and exploits the latest RDP Classifier to classify sequences against the curated UNITE fungal data set.
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