Evaluation of soil bacterial biomass using environmental DNA extracted by slow-stirring method

A simple and rapid method (slow-stirring method) for extracting environmental DNA (eDNA) from soils was constructed by physical mild stirring with chemical treatment. eDNA was extracted efficiently with minimal damage from various kinds of soil. The amount of eDNA and soil bacterial biomass showed a linear proportional relation [Y=(1.70×10⁸)X, r ²=0.96], indicating that bacterial biomass could be evaluated by quantifying levels of eDNA. Consequently, the average bacterial biomass in an agricultural field was calculated as 5.95×10⁹ cells/g sample, approximately 10–100 times higher than that in non- and oil-polluted fields.     

Effects of extracellular DNA and DNA‐binding protein on the development of a Streptococcus intermedius biofilm

Aims

The aim of this study was to clarify the effects of homologous and heterologous extracellular DNAs (eDNAs) and histone‐like DNA‐binding protein (HLP) on Streptococcus intermedius biofilm development and rigidity.

Methods and Results

Formed biofilm mass was measured with 0·1% crystal violet staining method and observed with a scanning electron microscope. The localizations of eDNA and extracellular HLP (eHLP) in formed biofilm were detected by staining with 7‐hydoxyl‐9H‐(1,3‐dichloro‐9,9‐dimethylacridin‐2‐one) and anti‐HLP antibody without fixation, respectively. DNase I treatment (200 U ml^?1) markedly decreased biofilm formation and cell density in biofilms. Colocalization of eHLP and eDNA in biofilm was confirmed. The addition of eDNA (up to 1 μg ml^?1) purified from Strep. intermedius, other Gram‐positive bacteria, Gram‐negative bacteria, or human KB cells into the Strep. intermedius culture increased the biofilm mass of all tested strains of Strep. intermedius, wild‐type, HLP‐downregulated strain and control strains. In contrast, the addition of eDNA (>1 μg ml^?1) decreased the biofilm mass of all Strep. intermedius strains.

Conclusions

These findings demonstrated that eDNA and eHLP play crucial roles in biofilm development and its rigidity.

Significance and Impact of the Study

eDNA‐ and HLP‐targeting strategies may be applicable to novel treatments for bacterial biofilm‐related infectious diseases.     

The Release Rate of Environmental DNA from Juvenile and Adult Fish

The environmental DNA (eDNA) technique is expected to become a powerful, non-invasive tool for estimating the distribution and biomass of organisms. This technique was recently shown to be applicable to aquatic vertebrates by collecting extraorganismal DNA floating in the water or absorbed onto suspended particles. However, basic information on eDNA release rate is lacking, despite it being essential for practical applications. In this series of experiments with bluegill sunfish (Lepomis macrochirus), we examined the effect of fish developmental stage on eDNA release rate. eDNA concentration reached equilibrium 3 days after the individual fish were introduced into the separate containers, enabling calculation of the eDNA release rate (copies h⁻¹) from individual fish on the assumption that the number of eDNA released from the fish per unit time equals total degradation in the container (copies h⁻¹). The eDNA release rate was 3–4 times higher in the adult (body weight: 30–75 g) than in the juvenile group (0.5–2.0 g). Such positive relationship between fish size and eDNA release rate support the possibility of biomass rather than density estimation using eDNA techniques. However, the eDNA release rate per fish body weight (copies h⁻¹ g⁻¹) was slightly higher in the juvenile than the adult group, which is likely because of the ontogenetic reduction in metabolic activity. Therefore, quantitative eDNA data should be carefully interpreted to avoid overestimating biomass when the population is dominated by juveniles, because the age structure of the focal population is often variable and unseen in the field. eDNA degradation rates (copies l⁻¹ h⁻¹), calculated by curve fitting of time-dependent changes in eDNA concentrations after fish removal, were 5.1–15.9% per hour (half-life: 6.3 h). This suggests that quantitative eDNA data should be corrected using a degradation curve attained in the target field.     

Various biofilm matrices of the emerging pathogen Staphylococcus lugdunensis: exopolysaccharides,proteins, eDNA and their correlation with biofilm mass

Abstract

Staphylococcus lugdunensis is an emerging high-virulent pathogen causative of hospital-acquired infections. Biofilm formation is a complex pathogenic process that leads to well-established bacterial communities. There is a paucity of data on the composition of the biofilm matrix among S. lugdunensis strains. Here, twenty-two S. lugdunensis clinical isolates, mainly from orthopaedic infections but also from other clinical sources, were sub-grouped by ribotyping and dendrogram analysis. Biofilms were analysed by fluorimetric methods based on FITC-Wheat Germ Agglutinin, SYPRO Ruby and TOTO-1 dyes to detect exopolysaccharides, proteins and extracellular DNA (eDNA), respectively. Biofilm morphology was investigated under confocal laser scanning microscopy (CLSM). Isolates displayed intriguing diversities in biofilm mass and matrix composition. The content of exopolysaccharides was found to be to be strongly associated with the biofilm mass (R² = 0.882), while the content of proteins turned out to be weakly (R² = 0.465) and that of eDNA very weakly associated (R² = 0.202) to the biofilm mass.     

Environmental DNA detects a rare large river crayfish but with little relation to local abundance

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A dual role of extracellular DNA during biofilm formation of Neisseria meningitidis

Major pathogenic clonal complexes (cc) of Neisseria meningitidis differ substantially in their point prevalence among healthy carriers. We show that frequently carried pathogenic cc (e.g. sequence type ST‐41/44 cc and ST‐32 cc) depend on extracellular DNA (eDNA) to initiate in vitro biofilm formation, whereas biofilm formation of cc with low point prevalence (ST‐8 cc and ST‐11 cc) was eDNA‐independent. For initial biofilm formation, a ST‐32 cc type strain, but not a ST‐11 type strain, utilized eDNA. The release of eDNA was mediated by lytic transglycosylase and cytoplasmic N‐acetylmuramyl‐l ‐alanine amidase genes. In late biofilms, outer membrane phospholipase A‐dependent autolysis, which was observed in most cc, but not in ST‐8 and ST‐11 strains, was required for shear force resistance of microcolonies. Taken together, N. meningitidis evolved two different biofilm formation strategies, an eDNA‐dependent one yielding shear force resistant microcolonies, and an eDNA‐independent one. Based on the experimental findings and previous epidemiological observations, we hypothesize that most meningococcal cc display a settler phenotype, which is eDNA‐dependent and results in a stable interaction with the host. On the contrary, spreaders (ST‐11 and ST‐8 cc) are unable to use eDNA for biofilm formation and might compensate for poor colonization properties by high transmission rates.     

Development and application of environmental DNA surveillance for the threatened crucian carp (Carassius carassius)

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Extracellular DNA and lipoteichoic acids interact with exopolysaccharides in the extracellular matrix of Streptococcus mutans biofilms

Streptococcus mutans-derived exopolysaccharides are virulence determinants in the matrix of biofilms that cause caries. Extracellular DNA (eDNA) and lipoteichoic acid (LTA) are found in cariogenic biofilms, but their functions are unclear. Therefore, strains of S. mutans carrying single deletions that would modulate matrix components were used: eDNA – ?lytS and ?lytT; LTA – ?dltA and ?dltD; and insoluble exopolysaccharide – ΔgtfB. Single-species (parental strain S. mutans UA159 or individual mutant strains) and mixed-species (UA159 or mutant strain, Actinomyces naeslundii and Streptococcus gordonii) biofilms were evaluated. Distinct amounts of matrix components were detected, depending on the inactivated gene. eDNA was found to be cooperative with exopolysaccharide in early phases, while LTA played a larger role in the later phases of biofilm development. The architecture of mutant strains biofilms was distinct (vs UA159), demonstrating that eDNA and LTA influence exopolysaccharide distribution and microcolony organization. Thus, eDNA and LTA may shape exopolysaccharide structure, affecting strategies for controlling pathogenic biofilms.     

Riverine distribution of mussel environmental DNA reflects a balance among density,transport, and removal processes

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Methodological considerations for detection of terrestrial small‐body salamander eDNA and implications for biodiversity conservation

Environmental DNA (eDNA) can be used as an assessment tool to detect populations of threatened species and provide fine‐scale data required to make management decisions. The objectives of this project were to use quantitative PCR (qPCR) to: (i) detect spiked salamander DNA in soil, (ii) quantify eDNA degradation over time, (iii) determine detectability of salamander eDNA in a terrestrial environment using soil, faeces, and skin swabs, (iv) detect salamander eDNA in a mesocosm experiment. Salamander eDNA was positively detected in 100% of skin swabs and 66% of faecal samples and concentrations did not differ between the two sources. However, eDNA was not detected in soil samples collected from directly underneath wild‐caught living salamanders. Salamander genomic DNA (gDNA) was detected in all qPCR reactions when spiked into soil at 10.0, 5.0, and 1.0 ng/g soil and spike concentration had a significant effect on detected concentrations. Only 33% of samples showed recoverable eDNA when spiked with 0.25 ng/g soil, which was the low end of eDNA detection. To determine the rate of eDNA degradation, gDNA (1 ng/g soil) was spiked into soil and quantified over seven days. Salamander eDNA concentrations decreased across days, but eDNA was still amplifiable at day 7. Salamander eDNA was detected in two of 182 mesocosm soil samples over 12 weeks (n = 52 control samples; n = 65 presence samples; n = 65 eviction samples). The discrepancy in detection success between experiments indicates the potential challenges for this method to be used as a monitoring technique for small‐bodied wild terrestrial salamander populations.     

Environmental DNA analysis for estimating the abundance and biomass of stream fish

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Presence of Extracellular DNA in the <Emphasis Type="Italic">Candida albicans</Emphasis> Biofilm Matrix and its Contribution to Biofilms

DNA has been described as a structural component of the extracellular matrix (ECM) in bacterial biofilms. In Candida albicans, there is a scarce knowledge concerning the contribution of extracellular DNA (eDNA) to biofilm matrix and overall structure. This work examined the presence and quantified the amount of eDNA in C. albicans biofilm ECM and the effect of DNase treatment and the addition of exogenous DNA on C. albicans biofilm development as indicators of a role for eDNA in biofilm development. We were able to detect the accumulation of eDNA in biofilm ECM extracted from C. albicans biofilms formed under conditions of flow, although the quantity of eDNA detected differed according to growth conditions, in particular with regards to the medium used to grow the biofilms. Experiments with C. albicans biofilms formed statically using a microtiter plate model indicated that the addition of exogenous DNA (>160 ng/ml) increases biofilm biomass and, conversely, DNase treatment (>0.03 mg/ml) decreases biofilm biomass at later time points of biofilm development. We present evidence for the role of eDNA in C. albicans biofilm structure and formation, consistent with eDNA being a key element of the ECM in mature C. albicans biofilms and playing a predominant role in biofilm structural integrity and maintenance.     

Biofilm characteristics and evaluation of the sanitation procedures of thermophilic Aeribacillus pallidus E334 biofilms

The ability of Aeribacillus pallidus E334 to produce pellicle and form a biofilm was studied. Optimal biofilm formation occurred at 60 °C, pH 7.5 and 1.5% NaCl. Extra polymeric substances (EPS) were composed of proteins and eDNA (21.4 kb). E334 formed biofilm on many surfaces, but mostly preferred polypropylene and glass. Using CLSM analysis, the network-like structure of the EPS was observed. The A. pallidus biofilm had a novel eDNA content. DNaseI susceptibility (86.8% removal) of eDNA revealed its importance in mature biofilms, but the purified eDNA was resistant to DNaseI, probably due to its extended folding outside the matrix. Among 15 cleaning agents, biofilms could be removed with alkaline protease and sodium dodecyl sulphate (SDS). The removal of cells from polypropylene and biomass on glass was achieved with combined SDS/alkaline protease treatment. Strong A. pallidus biofilms could cause risks for industrial processes and abiotic surfaces must be taken into consideration in terms of sanitation procedures.     

Assessing Environmental DNA Detection in Controlled Lentic Systems

Little consideration has been given to environmental DNA (eDNA) sampling strategies for rare species. The certainty of species detection relies on understanding false positive and false negative error rates. We used artificial ponds together with logistic regression models to assess the detection of African jewelfish eDNA at varying fish densities (0, 0.32, 1.75, and 5.25 fish/m³). Our objectives were to determine the most effective water stratum for eDNA detection, estimate true and false positive eDNA detection rates, and assess the number of water samples necessary to minimize the risk of false negatives. There were 28 eDNA detections in 324, 1-L, water samples collected from four experimental ponds. The best-approximating model indicated that the per-L-sample probability of eDNA detection was 4.86 times more likely for every 2.53 fish/m³ (1 SD) increase in fish density and 1.67 times less likely for every 1.02 C (1 SD) increase in water temperature. The best section of the water column to detect eDNA was the surface and to a lesser extent the bottom. Although no false positives were detected, the estimated likely number of false positives in samples from ponds that contained fish averaged 3.62. At high densities of African jewelfish, 3–5 L of water provided a >95% probability for the presence/absence of its eDNA. Conversely, at moderate and low densities, the number of water samples necessary to achieve a >95% probability of eDNA detection approximated 42–73 and >100 L, respectively. Potential biases associated with incomplete detection of eDNA could be alleviated via formal estimation of eDNA detection probabilities under an occupancy modeling framework; alternatively, the filtration of hundreds of liters of water may be required to achieve a high (e.g., 95%) level of certainty that African jewelfish eDNA will be detected at low densities (i.e., <0.32 fish/m³ or 1.75 g/m³).     

Needle in a haystack? A comparison of eDNA metabarcoding and targeted qPCR for detection of the great crested newt (Triturus cristatus)

Environmental DNA (eDNA) analysis is a rapid, cost‐effective, non‐invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species‐specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and “metabarcoding” have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real‐time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using high‐throughput sequencing technology. With qPCR and a detection threshold of 1 of 12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4 of 12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can be initially surveyed to best inform use of funding and time for species‐specific surveys.     

Optimizing techniques to capture and extract environmental DNA for detection and quantification of fish

Few studies have examined capture and extraction methods for environmental DNA (eDNA) to identify techniques optimal for detection and quantification. In this study, precipitation, centrifugation and filtration eDNA capture methods and six commercially available DNA extraction kits were evaluated for their ability to detect and quantify common carp (Cyprinus carpio) mitochondrial DNA using quantitative PCR in a series of laboratory experiments. Filtration methods yielded the most carp eDNA, and a glass fibre (GF) filter performed better than a similar pore size polycarbonate (PC) filter. Smaller pore sized filters had higher regression slopes of biomass to eDNA, indicating that they were potentially more sensitive to changes in biomass. Comparison of DNA extraction kits showed that the MP Biomedicals FastDNA SPIN Kit yielded the most carp eDNA and was the most sensitive for detection purposes, despite minor inhibition. The MoBio PowerSoil DNA Isolation Kit had the lowest coefficient of variation in extraction efficiency between lake and well water and had no detectable inhibition, making it most suitable for comparisons across aquatic environments. Of the methods tested, we recommend using a 1.5 μm GF filter, followed by extraction with the MP Biomedicals FastDNA SPIN Kit for detection. For quantification of eDNA, filtration through a 0.2–0.6 μm pore size PC filter, followed by extraction with MoBio PowerSoil DNA Isolation Kit was optimal. These results are broadly applicable for laboratory studies on carps and potentially other cyprinids. The recommendations can also be used to inform choice of methodology for field studies.     

Estimating fish abundance and biomass from eDNA concentrations: variability among capture methods and environmental conditions

Environmental DNA (eDNA) promises to ease noninvasive quantification of fish biomass or abundance, but its integration within conservation and fisheries management is currently limited by a lack of understanding of the influence of eDNA collection method and environmental conditions on eDNA concentrations in water samples. Water temperature is known to influence the metabolism of fish and consequently could strongly affect eDNA release rate. As water temperature varies in temperate regions (both seasonally and geographically), the unknown effect of water temperature on eDNA concentrations poses practical limitations on quantifying fish populations using eDNA from water samples. This study aimed to clarify how water temperature and the eDNA capture method alter the relationships between eDNA concentration and fish abundance/biomass. Water samples (1 L) were collected from 30 aquaria including triplicate of 0, 5, 10, 15 and 20 Brook Charr specimens at two different temperatures (7 °C and 14 °C). Water samples were filtered with five different types of filters. The eDNA concentration obtained by quantitative PCR (qPCR) varied significantly with fish abundance and biomass and types of filters (mixed‐design ANOVA, P < 0.001). Results also show that fish released more eDNA in warm water than in cold water and that eDNA concentration better reflects fish abundance/biomass at high temperature. From a technical standpoint, higher levels of eDNA were captured with glass fibre (GF) filters than with mixed cellulose ester (MCE) filters and support the importance of adequate filters to quantify fish abundance based on the eDNA method. This study supports the importance of including water temperature in fish abundance/biomass prediction models based on eDNA.     

Behavior and season affect crayfish detection and density inference using environmental DNA

Although the presence/absence of aquatic invertebrates using environmental DNA (eDNA) has been established for several species, inferring population densities has remained problematic. The invasive American signal crayfish, Pacifastacus leniusculus (Dana), is the leading cause of decline in the UK's only native crayfish species, Austropotamobius pallipes (Lereboullet). Methods to detect species at low abundances offer the opportunity for the early detection, and potential eradication, of P. leniusculus before population densities reach threatening levels in areas occupied by A. pallipes. Using a factorial experimental design with aquaria, we investigated the impacts of biomass, sex ratio, and fighting behavior on the amount of eDNA released by P. leniusculus, with the aim to infer density per aquarium depending on treatments. The amount of target eDNA in water samples from each aquarium was measured using the quantitative Polymerase Chain Reaction. We show that the presence of eggs significantly increases the concentration of crayfish eDNA per unit of mass, and that there is a significant relationship between eDNA concentration and biomass when females are egg‐bearing. However, the relationship between crayfish biomass and eDNA concentration is lost in aquaria without ovigerous females. Female‐specific tanks had significantly higher eDNA concentrations than male‐specific tanks, and the prevention of fighting did not impact the amount of eDNA in the water. These results indicate that detection and estimate of crayfish abundance using eDNA may be more effective while females are ovigerous. This information should guide further research for an accurate estimation of crayfish biomass in the field depending on the season. Our results indicate that detection and quantification of egg‐laying aquatic invertebrate species using eDNA could be most successful during periods when eggs are developing in the water. We recommend that practitioners consider the reproductive cycle of target species when attempting to study or detect aquatic species using eDNA in the field.     

Detection of the endangered European weather loach (Misgurnus fossilis) via water and sediment samples: Testing multiple eDNA workflows

The European weather loach (Misgurnus fossilis) is classified as highly endangered in several countries of Central Europe. Populations of M. fossilis are predominantly found in ditches with low water levels and thick sludge layers and are thus hard to detect using conventional fishing methods. Therefore, environmental DNA (eDNA) monitoring appears particularly relevant for this species. In previous studies, M. fossilis was surveyed following eDNA water sampling protocols, which were not optimized for this species. Therefore, we created two full factorial study designs to test six different eDNA workflows for sediment samples and twelve different workflows for water samples. We used qPCR to compare the threshold cycle (C_t) values of the different workflows, which indicate the target DNA amount in the sample, and spectrophotometry to quantify and compare the total DNA amount inside the samples. We analyzed 96 water samples and 48 sediment samples from a pond with a known population of M. fossilis. We tested several method combinations for long‐term sample preservation, DNA capture, and DNA extraction. Additionally, we analyzed the DNA yield of samples from a ditch with a natural M. fossilis population monthly over one year to determine the optimal sampling period. Our results showed that the long‐term water preservation method commonly used for eDNA surveys of M. fossilis did not lead to optimal DNA yields, and we present a valid long‐term sample preservation alternative. A cost‐efficient high salt DNA extraction led to the highest target DNA yields and can be used for sediment and water samples. Furthermore, we were able to show that in a natural habitat of M. fossilis, total and target eDNA were higher between June and September, which implies that this period is favorable for eDNA sampling. Our results will help to improve the reliability of future eDNA surveys of M. fossilis.     

Abundance estimation of riverine macrophyte Egeria densa using environmental DNA: effects of sampling season and location

Invasive macrophytes can have a variety of effects on aquatic ecosystems. The early detection and abundance estimation of an invasive species is important to effectively control it and minimize the ecosystem impacts. It is imperative to develop more efficient sampling methods for the abundance quantification of aquatic plants in large riverine systems. We examined (1) relationships between the environmental DNA (eDNA) concentrations of the invasive macrophyte, Egeria densa, and the upstream coverage area on the multiple life-history stages (dormant, growing, and senescence seasons) in a large riverine system in Japan and (2) if the relationships between the eDNA concentrations and coverage area could vary with the lateral sampling locations (left bank, middle, and right bank). The eDNA concentrations had significant positive relationships with the upstream coverage area of E. densa at multiple spatial scales for the dormant and senescence seasons. These results suggest that the eDNA analysis could be useful to quantify the relative abundance of this aquatic macrophyte in the riverine system; however, the selection of the eDNA sampling season could be important to accurately estimate abundance. Our results also showed that the eDNA concentrations of E. densa did not significantly differ from the lateral sampling location, suggesting that the eDNA samples could reflect the relative abundance of E. densa upstream of the study sites regardless of the lateral sampling location.