Quantity Of eDNA Research Articles

PRELIMINARY ANALYSIS SUGGESTS FRESHWATER INVERTEBRATE ENVIRONMENTAL DNA IS MORE CONCENTRATED IN SURFACE WATER THAN IN BENTHIC SEDIMENTS

Abstract The collection, identification, and census of freshwater invertebrates helps to increase understanding of the ecological function of lakes and streams. However, this work can be time-consuming and laborious because invertebrate identification often requires considerable taxonomic training and expertise. The collection and analysis of environmental DNA (eDNA), the genetic material that organisms shed into their surrounding environment, represents a potentially revolutionary approach for rapid and accurate invertebrate surveillance in freshwater environments. Previous studies have demonstrated that fish eDNA tends to be more abundant in freshwater lake sediments than the water column above, so we conducted an experiment to examine whether this pattern holds true for freshwater invertebrates. We collected paired samples from benthic sediments and the water column at ten sites around an urban playa lake in Lubbock, Texas. Based on cycle threshold values from quantitative PCR (qPCR) amplification with universal invertebrate primers targeting the COI gene, a paired Wilcoxon signed-rank test and Spearman rank-order correlation suggested that invertebrate eDNA quantities were correlated between the sediment and water column but consistently more concentrated in the water compared to the sediment below, directly contrasting with previous studies of fish eDNA. Future work combining eDNA detection and high-throughput sequencing (i.e., metabarcoding) will increase understanding of how eDNA signals relate to local invertebrate pools and increase the utility of eDNA sampling for freshwater invertebrates.

Exploring the eDNA dynamics of the host-pathogen pair Pacifastacus leniusculus (Decapoda) and Aphanomyces astaci (Saprolegniales) under experimental conditions

The oomycete Aphanomyces astaci causes crayfish plague, a disease threatening native European crayfish. It is carried and transmitted by American crayfish species, which are the original hosts of A. astaci. In recent years, environmental DNA (eDNA) methods have been successfully implemented to monitor the spread of both A. astaci and its hosts. However, still little is known about how population density and other environmental factors influence the detectability of this host-pathogen complex. In a mesocosm experiment, we tested the influence of crayfish density, temperature and food availability on the detectability of eDNA for A. astaci and its host, signal crayfish Pacifastacus leniusculus. We also compared eDNA results with crayfish population density measured by catch per unit effort (CPUE) from two lakes with varying crayfish density and A. astaci prevalence. The mesocosm experiment revealed that a limited set of controlled factors can substantially change the detectable amount of eDNA, even though the physical presence of the target organisms remains the same. In cold, clear water, eDNA quantities of both targets increased far more than in a linear fashion with increased crayfish density. However, the presence of food decreased the detectability of crayfish eDNA, presumably through increased microbial-induced eDNA degradation. For A. astaci, where eDNA typically represents living spores, food did not affect the detectability. However, high water temperature strongly reduced it. The increased complexity and variability of factors influencing eDNA concentration under natural conditions, compared to a controlled experimental environment, suggests that establishing a reliable relationship between eDNA quantities and crayfish density is difficult to achieve. This was also supported by field data, where we found minimal correspondence between eDNA quantity and CPUE data. A comparison between quantitative real-time PCR (qPCR) analysis and droplet-digital PCR (ddPCR) analysis revealed higher detection success of the targets in field samples when using qPCR. Overall, our results support eDNA as an effective tool for presence-absence monitoring, but it seems less suited for biomass quantification and population density estimates. Detection of A. astaci and P. leniusculus is not influenced uniformly by respective environmental factors. Consequently, we recommend a strategy of monitoring both targets, where the detection of one may point towards the presence of the other.

Revealing diets of wild-caught ornate spiny lobster, Panulirus ornatus, at puerulus, post-puerulus and juvenile stages using environmental DNA (eDNA) metabarcoding

Diets are a critical factor in the artificial production of lobster larvae, yet knowledge of the diet requirements of spiny lobster especially at its early life stages is rarely investigated. Thus, the present study aimed at finding potential diets of spiny lobster larva by analyzing the stomach content of wild-caught ornate spiny lobster, Panulirus ornatus, at three different life stages: puerulus, post-puerulus, and juvenile using eDNA metabarcoding. The results showed that 10 plankton species were identified at the puerulus stage, and the top five were Oithona sp. (36.30% of the relative quantity of eDNA), Macropthalmus setous (19.18%), Audacallichirus mirim (13.01%), Oithona simplex (5.48%), and Pseudodiaptomus euryhalinus (4.11%). Furthermore, 17 species were identified from the post-puerulus stage, and the five most dominant species were Audacallichirus mirim (28.60%), Oithona sp. (19.36%), Pichia sp. (5.96%), Helice tientsinensis (5.86%), and Oithona simplex (5.36%). At the juvenile stage, 34 diet species were identified, of which the top five most dominant species were Oithona sp. (80.88%), followed by Canthocalanus pauper (5.66%), Acartia bispinosa (4.02%), Longipedia koreana (2.30%), and Oithona davisae (1.92%). In addition, 56 plankton species were identified from the natural habitat including Sicyonia laevigata (33.73%), Oithona simplex (23.70%), Oithona sp. (17.70%), and Acartia tonsa (11.89%). Of the identified species, five were considered highly potential for developing artificially producing lobster seeds which were Oithona sp., Oithona simplex, Acartia bispinosa, Acartia tonsa and Pseudodiaptomus euryhalinus.

Sea lice (Lepeophtherius salmonis) detection and quantification around aquaculture installations using environmental DNA.

The naturally occurring ectoparasite salmon lice (Lepeophtherirus salmonis) poses a great challenge for the salmon farming industry, as well as for wild salmonids in the Northern hemisphere. To better control the infestation pressure and protect the production, there is a need to provide fish farmers with sensitive and efficient tools for rapid early detection and monitoring of the parasitic load. This can be achieved by targeting L. salmonis DNA in environmental samples. Here, we developed and tested a new L. salmonis specific DNA-based assay (qPCR assay) for detection and quantification from seawater samples using an analytical pipeline compatible with the Environmental Sample Processor (ESP) for autonomous water sample analysis of gene targets. Specificity of the L. salmonis qPCR assay was demonstrated through in-silico DNA analyses covering sequences of different L. salmonis isolates. Seawater was spiked with known numbers of nauplii and copepodite free-swimming (planktonic) stages of L. salmonis to investigate the relationship with the number of marker gene copies (MGC). Finally, field samples collected at different times of the year in the vicinity of a salmon production farm in Western Norway were analyzed for L. salmonis detection and quantification. The assay specificity was high and a high correlation between MGC and planktonic stages of L. salmonis was established in the laboratory conditions. In the field, L. salmonis DNA was consequently detected, but with MGC number below that expected for one copepodite or nauplii. We concluded that only L. salmonis tissue or eDNA residues were detected. This novel study opens for a fully automatized L. salmonis DNA quantification using ESP robotic to monitor the parasitic load, but challenges remain to exactly transfer information about eDNA quantities to decisions by the farmers and possible interventions.

Aquatic eDNA can advance monitoring of a small‐bodied terrestrial salamander and amphibian pathogen

AbstractSuccessful conservation efforts for threatened species depend on accurate characterization of their distribution, habitat use, and threats. However, surveillance can be challenging for species with small size, cryptic coloring, or elusive behavior. Environmental DNA (eDNA) monitoring can provide a sensitive and noninvasive alternative to traditional surveillance techniques by detecting trace DNA shed by a target species into their environment. We evaluated the efficacy of eDNA monitoring for the four‐toed salamander (Hemidactylium scutatum), a terrestrial salamander threatened throughout much of its range. Additionally, we integrated eDNA monitoring for ranavirus, a widespread pathogen of ectothermic vertebrates, to efficiently evaluate a potential disease threat to H. scutatum. We designed a novel species‐specific quantitative PCR (qPCR) assay for H. scutatum, multiplexed the assay with a previously developed ranavirus qPCR assay, and validated the multiplexed tests in silico, in vitro, and in situ. We collected aquatic eDNA weekly for 8 weeks from pools with historical reports of H. scutatum (n = 6) and contemporary reports of H. scutatum on associated uplands (n = 4). We identified H. scutatum eDNA at low quantities (< 12 copies/reaction) on multiple sampling days in two pools with historic species presence. Ranavirus eDNA was widespread; nine pools tested positive including the pools where H. scutatum eDNA was detected. Ranavirus eDNA quantities increased, peaked, then decreased (i.e., were not sporadic in time) in several pools but never rose in abundance to levels associated with ranavirosis in other studies. Our results suggest that H. scutatum eDNA is distributed sporadically at low quantities in pools; we recommend that future surveillance efforts prioritize increased frequency and quantity of sample collection per site visit and include repeat surveys of candidate sites. Further work to characterize Ranavirus eDNA movement, temporal trends, relationship to infectious virion, and decay is needed to help advance this tool.

Development of species‐specific multiplex real‐time PCR assays for tracing the small pelagic fishes of North Pacific with environmental DNA

AbstractEnvironmental DNA (eDNA) has become a popular non‐invasive method to study the presence of organisms in the environment. To study the ecological behavior and migration routes of small pelagic fishes that are difficult to trace with traditional capturing methods, we developed multiplex real‐time PCR assays to measure the eDNA of chub mackerel, blue mackerel, Japanese jack mackerel, Japanese anchovy, Japanese sardine, and Pacific saury. To test the validity, eDNA samples collected from the water near fisherman set nets in Northeast Japan between 2018 and 2019 were analyzed. Warm‐climate species including blue mackerel, jack mackerel, and anchovy were prominent in autumn, while cold‐climate species such as chub mackerel and sardine were richly present in winter. Pacific saury is an off‐coast species and was not detected in the coastal set net area. The eDNA data were partially correlated to the fishery catch records, suggesting that the eDNA quantities are relevant to the biomass of the fish schools, and our PCR assays can quantify the biological traces left by the small pelagic fish schools. Different patterns of eDNA occurrence were observed between the chub and blue mackerels, indicating that the specificity of the multiplex assay is applicable to field samples to measure the presence of the two closely related species separately. The methods are useful to evaluate the eDNA samples collected from the open water in the North Pacific to elucidate the life cycles and monitor the sustainability of the small pelagic fishes, which not only are important food sources, but also carry a role in the Japanese food culture.

Detecting the brook floater, a freshwater mussel species at risk, using environmental DNA

Abstract The use of environmental DNA (eDNA) is a promising approach for the detection of aquatic species, including species at risk. One freshwater mussel species of interest in Atlantic Canada, the brook floater (Alasmidonta varicosa), is listed as being of Special Concern under the Species at Risk Act in Canada and as Vulnerable on the International Union for Conservation of Nature Red List. Further scientific data regarding species distribution and critical habitat is needed for the protection and conservation of this species. The aim of this study was to design, optimize, and apply a species‐specific quantitative polymerase chain reaction assay for the detection of brook floater from eDNA samples, and to assess temporal variability in brook floater eDNA quantities. Through an eDNA survey performed in New Brunswick rivers in 2017 and 2018, brook floater DNA was found at a total of 16 out of 56 sites sampled. The amount of brook floater DNA detected at all 16 sites was always below the theoretical limit of detection of the assay, and, as such, results were classified as either ‘inconclusive’ or ‘suspected’. The co‐detection of eastern pearlshell (Margaritifera margaritifera), a more abundant freshwater mussel species in Atlantic Canada, was successfully used as a natural positive control. Temporal variability in the amount of eDNA found in the water was also assessed at a site with a known brook floater population and minimal variability in eDNA quantities was observed from May to September. These results provide researchers and managers with a new tool for the detection of the brook floater in support of conservation and monitoring efforts.

The Effect of Activity, Energy Use, and Species Identity on Environmental DNA Shedding of Freshwater Fish

The quantitative measurement of environmental DNA (eDNA) from field-collected water samples is gaining importance for the monitoring of fish communities and populations. The interpretation of these signal strengths depends, among other factors, on the amount of target eDNA shed into the water. However, shedding rates are presumably associated with species-specific traits such as physiology and behavior. Although such differences between juvenile and adult fish have been previously detected, the general impact of movement and energy use in a resting state on eDNA release into the surrounding water remains hardly addressed. In an aquarium experiment, we compared eDNA shedding between seven fish species occurring in European freshwaters. The investigated salmonids, cyprinids, and sculpin exhibit distinct adaptions to microhabitats, diets, and either solitary or schooling behavior. The fish were housed in aquaria with constant water flow and their activity was measured by snapshots taken every 30 s. Water samples for eDNA analysis were taken every 3 h and energy use was determined in an intermittent flow respirometer. After controlling for the effect of fish mass, our results demonstrate a positive correlation between target eDNA quantities as measured with digital PCR, fish activity, and energy use, as well as species-specific differences. For cyprinids, the model based on data from individual fish was only partly transferable to groups, which showed lower activity and higher energy use. Our findings highlight the importance of fish physiology and behavior for the comparative interpretation of taxon-specific eDNA quantities. Species traits should therefore be incorporated into eDNA-based monitoring and conservation efforts.

Monitoring of spatiotemporal occupancy patterns of fish and amphibian species in a lentic aquatic system using environmental DNA.

To effectively monitor, manage and protect aquatic species and understand their interactions, knowledge of their spatiotemporal distribution is needed. In this study, we used a fine‐scale spatiotemporal water sampling design, followed by environmental DNA (eDNA) 12S metabarcoding, to investigate occupancy patterns of a natural community of fish and amphibian species in a lentic system. In the same system, we experimentally estimated the spatial and temporal dispersion of eDNA by placing a community of different fish and amphibian species in cages at one side of the pond, creating a controlled point of eDNA emission. Analyses of this cage community revealed a sharp spatial decline in detection rates and relative eDNA quantities at a distance of 5–10 m from the source, depending on the species and its abundance. In addition, none of the caged species could be detected 1 week after removal from the system. This indicates high eDNA decay rates and limited spatial eDNA dispersal, facilitating high local resolution for monitoring spatial occupancy patterns of aquatic species. Remarkably, for seven of the nine cage species, the presence of a single individual could be detected by pooling water of subsamples taken across the whole water body, illustrating the high sensitivity of the eDNA sampling and detection method applied. Finally, our work demonstrated that a fine‐scale sampling design in combination with eDNA metabarcoding can cover total biodiversity very precisely and allows the construction of consistent spatiotemporal patterns of relative abundance and local distribution of free‐living fish and amphibian species in a lentic ecosystem.

Quantitative evaluation of intraspecific genetic diversity in a natural fish population using environmental DNA analysis.

Recent advances in environmental DNA (eDNA) analysis using high-throughput sequencing (HTS) enable evaluation of intraspecific genetic diversity in a population. As the intraspecific genetic diversity provides invaluable information for wildlife conservation and management, there is an increasing demand to apply eDNA analysis to population genetics and the phylogeography by quantitative evaluation of intraspecific diversity. However, quantitative evaluations of intraspecific genetic diversity using eDNA is not straightforward because the number of eDNA sequence reads obtained by HTS may not be an index of the quantity of eDNA. In this study, to quantitatively evaluate genetic diversity using eDNA analysis, we applied a quantitative eDNA metabarcoding method using the internal standard DNAs. We targeted Ayu (Plecoglossus altivelis altivelis) and added internal standard DNAs with known copy numbers to each eDNA sample obtained from three rivers during the library preparation process. The sequence reads of each Ayu haplotype were successfully converted to DNA copy numbers based on the relationship between the copy numbers and sequence reads of the internal standard DNAs. In all rivers, the calculated copy number of each haplotype showed a significant positive correlation with the haplotype frequency estimated by a capture-based survey. Furthermore, estimates of genetic indicators such as nucleotide diversity based on the eDNA copy numbers were comparable with those estimated based on a capture-based study. Our results demonstrate that eDNA analysis with internal standard DNAs enables reasonable quantification of intraspecific genetic diversity, and this method could thus be a promising tool in the field of population genetics and phylogeography.

Experimental assessment of optimal lotic eDNA sampling and assay multiplexing for a critically endangered fish

AbstractDesigning eDNA tools to detect and quantify rare species includes inherent assumptions about the spatial distribution of the organism, spatial nature of eDNA dynamics, and the real‐world performance of alternate assays under field conditions. Here, we use cage experiments with small numbers of Atlantic salmon (Salmo salar), to reveal that eDNA detection rates and eDNA quantities follow a predictable, but nonlinear relationship with distance from a point source. In contrast to the common assumption of consistent eDNA degradation moving away from a source, eDNA detections and concentrations increased up to roughly 70 m downstream before declining steadily. We apply our eDNA distance functions to selection of stream sampling intervals for detecting fish without known locations and find that even a single juvenile salmon can be reliably detected with intervals up to 400 m spacing. Finally, we show that two different qPCR eDNA assays provide very different detection probabilities in nature despite similar efficiency in laboratory testing, demonstrating the importance of experimentally assessing assay efficiencies in the wild as well as the capacity for multiplexing as a strategy to ensure high detection efficiency when monitoring rare species.

Environmental DNA concentrations are correlated with regional biomass of Atlantic cod in oceanic waters

Environmental DNA (eDNA) has emerged as a powerful approach for studying marine fisheries and has the potential to negate some of the drawbacks of trawl surveys. However, successful applications in oceanic waters have to date been largely focused on qualitative descriptions of species inventories. Here we conducted a quantitative eDNA survey of Atlantic cod (Gadus morhua) in oceanic waters and compared it with results obtained from a standardized demersal trawl survey. Detection of eDNA originating from Atlantic cod was highly concordant (80%) with trawl catches. We observed significantly positive correlations between the regional integrals of Atlantic cod biomass (kg) and eDNA quantities (copies) (R2 = 0.79, P = 0.003) and between sampling effort-normalised Catch Per Unit Effort (kg hr−1) and eDNA concentrations (copies L−1) (R2 = 0.71, P = 0.008). These findings extend the potential application of environmental DNA to regional biomass assessments of commercially important fish stocks in the ocean.

Extracellular DNA facilitates bacterial adhesion during Burkholderia pseudomallei biofilm formation.

The biofilm-forming ability of Burkholderia pseudomallei is crucial for its survival in unsuitable environments and is correlated with antibiotic resistance and relapsing cases of melioidosis. Extracellular DNA (eDNA) is an essential component for biofilm development and maturation in many bacteria. The aim of this study was to investigate the eDNA released by B. pseudomallei during biofilm formation using DNase treatment. The extent of biofilm formation and quantity of eDNA were assessed by crystal-violet staining and fluorescent dye-based quantification, respectively, and visualized by confocal laser scanning microscopy (CLSM). Variation in B. pseudomallei biofilm formation and eDNA quantity was demonstrated among isolates. CLSM images of biofilms stained with FITC-ConA (biofilm) and TOTO-3 (eDNA) revealed the localization of eDNA in the biofilm matrix. A positive correlation of biofilm biomass with quantity of eDNA during the 2-day biofilm-formation observation period was found. The increasing eDNA quantity over time, despite constant living/dead ratios of bacterial cells during the experiment suggests that eDNA is delivered from living bacterial cells. CLSM images demonstrated that depletion of eDNA by DNase I significantly lessened bacterial attachment (if DNase added at 0 h) and biofilm developing stages (if added at 24 h) but had no effect on mature biofilm (if added at 45 h). Collectively, our results reveal that eDNA is released from living B. pseudomallei and is correlated with biofilm formation. It was also apparent that eDNA is essential during bacterial cell attachment and biofilm-forming steps. The depletion of eDNA by DNase may provide an option for the prevention or dispersal of B. pseudomallei biofilm.

Effect of extracellular DNA on the formation of Streptococcus mutans biofilm under sucrose environment

To study the effect of extracellular DNA(eDNA) on the formation of Streptococcus mutans(Sm) biofilms during different growth periods in sucrose environment. Sm biofilms were established on smooth glass surfaces under the environment of 1% sucrose and cultured in the condition of 37 ℃, 5% O2, 85% N2 and 10% CO2. Samples were randomly divided into four groups based on fourculture time(6,12, 24 and 48 h), respectively. Each group was further divided into two subgroups: control group(without deoxyribonuclease Ⅰ[DNaseⅠ] treatment) and test group(with DNaseⅠtreatment). DNaseⅠ was added 1 h advance in the treatment group to a final concentration of 100 U/ml. Each sample was stained with mixed SYTO-9/PI fluorescent dye. Confocal laser scanning microscopy was used for biofilm observation and scanning. The total biomass, the thickness and the volume of red fluorescence of each biofilm sample were measured following three-dimensional reconstruction using the softwear of Imaris 7.2.3. Under the environment of 1% sucrose, the Sm bacterial adhesion and distribution density increased over time, the quantity of eDNA and membrane-damaged bacteria which were indicated by red fluorescence also increased within 24 h but dropped later. The biofilm biomasses of Sm biofilm in 6, 12, 24 and 48 h DNaseⅠ treatment group reduced significantly(P<0.05) compared to those in the corresponding control groups by 81.3%, 85.0%, 90.1% and 12.4%, respectively. The biofilm thicknesses in each DNase Ⅰ treatment group (except 6 h group) also reduced significantly(P<0.05) compared to those in the corresponding control group by 34.4%, 45.6% and 23.6%, respectively. The quantities of eDNA and membrane-damaged bacteria reduced in each treatment group except 48 h group compared to that in the corresponding control group. Under the environment of 1% sucrose, eDNA plays an important role in promoting the formation of Sm biofilm.

Quantifying relative fish abundance with eDNA: a promising tool for fisheries management

Summary Assessment and monitoring of exploited fish populations are challenged by costs, logistics and negative impacts on target populations. These factors therefore limit large‐scale effective management strategies. Evidence is growing that the quantity of eDNA may be related not only to species presence/absence, but also to species abundance. In this study, the concentrations of environmental DNA (eDNA) from a highly prized sport fish species, Lake Trout Salvelinus namaycush (Walbaum 1792), were estimated in water samples from 12 natural lakes and compared to abundance and biomass data obtained from standardized gillnet catches as performed routinely for fisheries management purposes. To reduce environmental variability among lakes, all lakes were sampled in spring, between ice melt and water stratification. The eDNA concentration did not vary significantly with water temperature, dissolved oxygen, pH and turbidity, but was significantly positively correlated with relative fish abundance estimated as catch per unit effort (CPUE), whereas the relationship with biomass per unit effort (BPUE) was less pronounced. The value of eDNA to inform about local aquatic species distribution was further supported by the similarity between the spatial heterogeneity of eDNA distribution and spatial variation in CPUE measured by the gillnet method. Synthesis and applications. Large‐scale empirical evidence of the relationship between the eDNA concentration and species abundance allows for the assessment of the potential to integrate eDNA within fisheries management plans. As such, the eDNA quantitative method represents a promising population abundance assessment tool that could significantly reduce the costs associated with sampling and increase the power of detection, the spatial coverage and the frequency of sampling, without any negative impacts on fish populations.

Sec Pathway Influences the Growth of Deinococcus radiodurans

The release of extracellular DNA molecules (eDNA) contributes to various biological processes, such as biofilm formation, virulence, and stress tolerance. The quantity of eDNA released by bacteria is usually regulated by extracellular nucleases that are secreted by different systems. In this study, we show that high concentrations of eDNA inhibit the growth of two strains of Deinococcaceae, Deinococcus radiodurans, and Deinococcus radiopugnans, but have no effect on other selected organisms, such as Escherichia coli. In D. radiodurans, an extracellular nuclease was shown to be secreted through the Sec pathway. Disruption of one member of this pathway, SecD/F, inhibited cell growth, suggesting that the Sec pathway plays an important role in growth rate. However, the Sec pathway mutant exhibited a greater deficiency in growth rate compared with the extracellular nuclease mutant, indicating that the pathway not only secretes the extracellular nuclease, but has other unknown functions as well.

Extracellular DNA in adhesion and biofilm formation of four environmental isolates: a quantitative study

The importance of extracellular DNA (eDNA) in biofilm formation has become increasingly clear from research on clinically relevant bacteria. This study aimed to determine whether the quantity of eDNA produced can be linked to the ability to form biofilm. We systematically quantified eDNA over time during planktonic growth and biofilm formation in Reinheimera sp. F8 and three other environmental isolates belonging to the genera Pseudomonas, Microbacterium and Serratia. eDNA in biofilms was visualised by fluorescence microscopy and quantified by PicoGreen(®) labelling without further sample preparation, whereas eDNA in planktonic cultures was precipitated before labelling and quantification. The effect of eDNA removal was investigated by DNase treatment. eDNA appeared in the early exponential growth phase of planktonic batch cultures and the concentration peaked in the stationary phase. The concentration in biofilms differed substantially between strains and over time during biofilm development. eDNA was important for the initial attachment in all strains, and DNase treatment reduced biofilm formation in three of four strains. The extent to which eDNA accumulated in planktonic cultures or biofilms did not reflect its significance to biofilm formation, and even very low concentrations of eDNA affected biofilm formation strongly. The significance of eDNA for biofilm formation in nature may thus be more widespread than previously anticipated.

Presence of Extracellular DNA in the Candida albicans 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.

Extracellular DNA in Single- and Multiple-Species Unsaturated Biofilms

The extracellular polymeric substances (EPS) of bacterial biofilms form a hydrated barrier between cells and their external environment. Better characterization of EPS could be useful in understanding biofilm physiology. The EPS are chemically complex, changing with both bacterial strain and culture conditions. Previously, we reported that Pseudomonas aeruginosa unsaturated biofilm EPS contains large amounts of extracellular DNA (eDNA) (R. E. Steinberger, A. R. Allen, H. G. Hansma, and P. A. Holden, Microb. Ecol. 43:416-423, 2002). Here, we investigated the compositional similarity of eDNA to cellular DNA, the relative quantity of eDNA, and the terminal restriction fragment length polymorphism (TRFLP) community profile of eDNA in multiple-species biofilms. By randomly amplified polymorphic DNA analysis, cellular DNA and eDNA appear identical for P. aeruginosa biofilms. Significantly more eDNA was produced in P. aeruginosa and Pseudomonas putida biofilms than in Rhodococcus erythropolis or Variovorax paradoxus biofilms. While the amount of eDNA in dual-species biofilms was of the same order of magnitude as that of of single-species biofilms, the amounts were not predictable from single-strain measurements. By the Shannon diversity index and principle components analysis of TRFLP profiles generated from 16S rRNA genes, eDNA of four-species biofilms differed significantly from either cellular or total DNA of the same biofilm. However, total DNA- and cellular DNA-based TRFLP analyses of this biofilm community yielded identical results. We conclude that extracellular DNA production in unsaturated biofilms is species dependent and that the phylogenetic information contained in this DNA pool is quantifiable and distinct from either total or cellular DNA.