Estimating the spawning activity of fish species using nuclear and mitochondrial environmental DNA concentrations and their ratios

1. During spawning activity, fish release large amounts of sperm and eggs into the water, which has been assumed to cause an increase in environmental DNA (eDNA) levels and nuclear DNA/mitochondrial DNA ratios. To test whether these assumptions are valid and whether nuclear and mitochondrial eDNA analysis can be used to monitor the spawning activity of freshwater fish, we conducted field eDNA surveys and traditional surveys using common carp (Cyprinus carpio), largemouth bass (Micropterus salmoides) and bluegill sunfish (Lepomis macrochirus) as model species.
2. Fish spawning periods were estimated based on age, as estimated using the body lengths of juveniles collected in the Miharu reservoir in Fukushima, Japan. The results showed that the main spawning periods of largemouth bass and bluegill sunfish were from April to July and from July to August, respectively.
3. Field eDNA surveys were conducted in the Hebisawagawa front reservoir, which is connected to the Miharu reservoir. From March to August 2019 and 2020, weekly eDNA sampling was conducted at three sites, and daily sampling was conducted at six sites from 23 June to 3 July 2020. The eDNA concentrations of the nuclear internal transcribed spacer 1 (ITS1) and mitochondrial cytochrome B (CytB), as well as the ITS1/CytB ratio, were measured for each of the three fish in each water sample. Water temperature had a statistically significant effect on eDNA concentration, probably reflecting the relationship between water temperature and spawning.
4. We created generalised additive mixed models to estimate spawning activity periods based on weekly eDNA data. The estimated periods of spawning activity for common carp, largemouth bass and bluegill sunfish were March to May, May to July, and May to August, respectively. The estimated spawning periods coincided with known fish ecology or the results of traditional methods. This method also has been applied to daily eDNA samples, showing the feasibility of high-resolution estimation of spawning activity.
5. For common carp and bluegill sunfish, we were able to estimate the spawning period using this method. Although the method is affected by biomass and the diffusion and degradation of eDNA, it has the potential to accurately estimating spawning activities. These then can be estimated without conducting laborious traditional surveys, facilitating the monitoring of reproduction by rare, invasive or important fishery species. Further research on the diffusion distance and degradation time of the eDNA concentration peak caused by fish spawning activity may improve the accuracy of monitoring.

     

On-site filtration of water samples for environmental DNA analysis to avoid DNA degradation during transportation

Environmental DNA (eDNA) analysis is an innovative tool for determining the distribution or abundance of aquatic macroorganisms. However, because eDNA degrades rapidly in water, long delays between sampling and analysis may hinder eDNA quantification. In the present study, we developed a portable filtration system that enables on-site (and on-the-road) filtration of water samples. Degradation rates of eDNA within 6 h were compared using water from an outdoor pond that was subjected to (1) on-site filtration, (2) transportation of water on ice, and (3) transportation of water at ambient temperature. Groups 2 and 3 were filtered in the laboratory 6 h after sampling. The concentration of eDNA was determined as the copy number of the mitochondrial cytochrome b gene of two fish species using real-time polymerase chain reaction. The portable filtration system offers the following benefits: (1) the eDNA concentration is preserved as is at the time of sampling, permitting higher accuracy of eDNA quantification, (2) use of a disposable sealed plastic bag reduces the risk of contamination and ensures on-the-road filtration, (3) time is saved because filtration can be accomplished when driving between sampling sites.     

Early detection of a highly invasive bivalve based on environmental DNA (eDNA)

Management of non-indigenous invasive species (NIS) is challenging owing in part to limitations of early detection and identification. The advent of environmental DNA (eDNA) techniques provides an efficient way to detect NIS when their abundance is extremely low. However, eDNA-based methods often suffer from uncertain detection sensitivity, which requires detailed testing before applying these methods in the field. Here we developed an eDNA tool for early detection of the highly invasive golden mussel, Limnoperna fortunei, based on the mitochondrial cytochrome c oxidase subunit I gene (COI). Further, we tested technical issues, including sampling strategy and detection sensitivity, based on a laboratory experiment. We then applied the method to field samples collected from water bodies in China where this mussel has or is expected to colonize. Results showed that the detection limit varied extensively among our newly developed primer pairs, ranging from 4 × 10^?2 to 4 × 10^?6 ng of total genomic DNA. Laboratory detection was affected by the availability of eDNA (i.e., both mussel abundance and incubation time). Detection capacity was higher in laboratory samples containing re-suspended matter from the bottom layer versus that collected from the surface. Among 25 field sites, detection was 100% at sites with high mussel abundance and as low as 40% at sites with low abundance when tested using our most sensitive primer pair. Early detection of NIS present at low abundance in nature requires not only sensitive primers, but also an optimized sampling strategy to reduce the occurrence of false negatives. Careful selection and detailed testing of primer pairs ensures effective eDNA-based species detection in surveillance and management programs.     

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).     

Use of environmental DNA to detect Eastern Sand Darter (Ammocrypta pellucida Putnam, 1863) in large Laurentian Great Lakes embayments

A single-species environmental DNA (eDNA) method was developed to sample for a small, benthic rare species, Eastern Sand Darter (Ammocrypta pellucida Putnam, 1863) in two large Lake Ontario embayments. Summer water sampling allowed for: (a) surveys of habitats (Wellers Bay) where traditional fish sampling gear could not be used; and, (b) a comparison between eDNA and seining-based detection probabilities at known occupied habitats (West Lake). In 2018, replicate (n = 3) 1 L water samples were collected from 90 Wellers Bay sites and 71 West Lake sites. A site-occupancy model, a hierarchical logistic regression model, was fitted to determine site occupancy, sample occupancy (presence of Eastern Sand Darter DNA in a water sample) and probability of detection (p) based on replicate quantitative polymerase chain reaction (qPCR) results for each water sample. Eastern Sand Darter was detected at 10 West Lake sites, but not from Wellers Bay. Mean site occupancy was 0.31 (0.12–0.70; 95% CLs), mean sample occupancy was 0.28 (0.09–0.58; 95% CLs), and mean detection probability in a subsample (i.e., successful qPCR amplification) given it was present was 0.40 (0.25–0.55; 95% CLs). While the eDNA method successfully detected Eastern Sand Darter from known occupied areas in West Lake, it was not more effective for assessing local site occupancy than traditional sampling methods, such as the seine.     

Using Environmental DNA to Estimate the Distribution of an Invasive Fish Species in Ponds

Knowledge of the presence of an invasive species is critical to monitoring the sustainability of communities and ecosystems. Environmental DNA (eDNA), DNA fragments that are likely to be bound to organic matters in the water or in shed cells, has been used to monitor the presence of aquatic animals. Using an eDNA-based method, we estimated the presence of the invasive bluegill sunfish, Lepomis macrochirus, in 70 ponds located in seven locales on the Japanese mainland and on surrounding islands. We quantified the concentration of DNA copies in a 1 L water sample using quantitative real-time polymerase chain reaction (qPCR) with a primer/probe set. In addition, we visually observed the bluegill presence in the ponds from the shoreline. We detected bluegill eDNA in all the ponds where bluegills were observed visually and some where bluegills were not observed. Bluegills were also less prevalent on the islands than the mainland, likely owing to limited dispersal and introduction by humans. Our eDNA method simply and rapidly detects the presence of this invasive fish species with less disturbance to the environment during field surveys than traditional methods.     

Improved environmental DNA sampling scheme for Alabama sturgeon provides new insight into a species once presumed extinct

Detection of rare species can be challenging and time-consuming using conventional methods, but environmental DNA (eDNA) is becoming a commonly used tool for detection in conservation and management of species. This study demonstrates the utility of the precipitation method (precipitated and preserved in 3 M sodium acetate and 95% ethanol) for collection of eDNA to detect the seasonal distribution of the critically endangered Alabama sturgeon (Scaphirhynchus suttkusi). Surface and benthic water samples were collected across a wider geographic area than previously published for Alabama sturgeon eDNA. Surface and benthic samples both yielded detections and resulted in a similar proportion of positive detections to previous work. However, by sampling a greater portion of the distribution of the Alabama sturgeon, further insight was provided on potential sturgeon movement. The results of the precipitation method show that Alabama sturgeon detections increase during spawning months, and that the fish may be overwintering in the Tombigbee River. High detections from winter benthic samples suggest that habitat choice may play a role in detectability and highlight the need to consider natural history when designing environmental DNA studies. When designing environmental DNA collection for rare species, sampling design should factor in species ecology, habitat use, site characteristics, and specific questions driving the research.     

Modeling the Sensitivity of Field Surveys for Detection of Environmental DNA (eDNA)

The environmental DNA (eDNA) method is the practice of collecting environmental samples and analyzing them for the presence of a genetic marker specific to a target species. Little is known about the sensitivity of the eDNA method. Sensitivity is the probability that the target marker will be detected if it is present in the water body. Methods and tools are needed to assess the sensitivity of sampling protocols, design eDNA surveys, and interpret survey results. In this study, the sensitivity of the eDNA method is modeled as a function of ambient target marker concentration. The model accounts for five steps of sample collection and analysis, including: 1) collection of a filtered water sample from the source; 2) extraction of DNA from the filter and isolation in a purified elution; 3) removal of aliquots from the elution for use in the polymerase chain reaction (PCR) assay; 4) PCR; and 5) genetic sequencing. The model is applicable to any target species. For demonstration purposes, the model is parameterized for bighead carp (Hypophthalmichthys nobilis) and silver carp (H. molitrix) assuming sampling protocols used in the Chicago Area Waterway System (CAWS). Simulation results show that eDNA surveys have a high false negative rate at low concentrations of the genetic marker. This is attributed to processing of water samples and division of the extraction elution in preparation for the PCR assay. Increases in field survey sensitivity can be achieved by increasing sample volume, sample number, and PCR replicates. Increasing sample volume yields the greatest increase in sensitivity. It is recommended that investigators estimate and communicate the sensitivity of eDNA surveys to help facilitate interpretation of eDNA survey results. In the absence of such information, it is difficult to evaluate the results of surveys in which no water samples test positive for the target marker. It is also recommended that invasive species managers articulate concentration-based sensitivity objectives for eDNA surveys. In the absence of such information, it is difficult to design appropriate sampling protocols. The model provides insights into how sampling protocols can be designed or modified to achieve these sensitivity objectives.     

The future of fish-based ecological assessment of European rivers: from traditional EU Water Framework Directive compliant methods to eDNA metabarcoding-based approaches

Most of the present EU Water Framework Directive (WFD) compliant fish-based assessment methods of European rivers are multi-metric indices computed from traditional electrofishing (TEF) samples, but this method has known shortcomings, especially in large rivers. The probability of detecting rare species remains limited, which can alter the sensitivity of the indices. In recent years, environmental (e)DNA metabarcoding techniques have progressed sufficiently to allow applications in various ecological domains as well as eDNA-based ecological assessment methods. A review of the 25 current WFD-compliant methods for river fish shows that 81% of the metrics used in these methods are expressed in richness or relative abundance and thus compatible with eDNA samples. However, more than half of the member states' methods include at least one metric related to age or size structure and would have to adapt their current fish index if reliant solely on eDNA-derived information. Most trait-based metrics expressed in richness are higher when computed from eDNA than when computed from TEF samples. Comparable values are obtained only when the TEF sampling effort increases. Depending on the species trait considered, most trait-based metrics expressed in relative abundance are significantly higher for eDNA than for TEF samples or vice versa due to over-estimation of sub-surface species or under-estimation of benthic and rare species by TEF sampling, respectively. An existing predictive fish index, adapted to make it compatible with eDNA data, delivers an ecological assessment comparable with the current approved method for 22 of the 25 sites tested. Its associated uncertainty is lower than that of current fish indices. Recommendations for the development of future fish eDNA-based indices and the associated eDNA water sampling strategy are discussed.     

How many replicates to accurately estimate fish biodiversity using environmental DNA on coral reefs?

Quantifying fish species diversity in rich tropical marine environments remains challenging. Environmental DNA (eDNA) metabarcoding is a promising tool to face this challenge through the filtering, amplification, and sequencing of DNA traces from water samples. However, because eDNA concentration is low in marine environments, the reliability of eDNA to detect species diversity can be limited. Using an eDNA metabarcoding approach to identify fish Molecular Taxonomic Units (MOTUs) with a single 12S marker, we aimed to assess how the number of sampling replicates and filtered water volume affect biodiversity estimates. We used a paired sampling design of 30 L per replicate on 68 reef transects from 8 sites in 3 tropical regions. We quantified local and regional sampling variability by comparing MOTU richness, compositional turnover, and compositional nestedness. We found strong turnover of MOTUs between replicated pairs of samples undertaken in the same location, time, and conditions. Paired samples contained non‐overlapping assemblages rather than subsets of one another. As a result, non‐saturated localized diversity accumulation curves suggest that even 6 replicates (180 L) in the same location can underestimate local diversity (for an area <1 km). However, sampling regional diversity using ~25 replicates in variable locations (often covering 10 s of km) often saturated biodiversity accumulation curves. Our results demonstrate variability of diversity estimates possibly arising from heterogeneous distribution of eDNA in seawater, highly skewed frequencies of eDNA traces per MOTU, in addition to variability in eDNA processing. This high compositional variability has consequences for using eDNA to monitor temporal and spatial biodiversity changes in local assemblages. Avoiding false‐negative detections in future biomonitoring efforts requires increasing replicates or sampled water volume to better inform management of marine biodiversity using eDNA.     

The Relationship between the Distribution of Common Carp and Their Environmental DNA in a Small Lake

Although environmental DNA (eDNA) has been used to infer the presence of rare aquatic species, many facets of this technique remain unresolved. In particular, the relationship between eDNA and fish distribution is not known. We examined the relationship between the distribution of fish and their eDNA (detection rate and concentration) in a lake. A quantitative PCR (qPCR) assay for a region within the cytochrome b gene of the common carp (Cyprinus carpio or ‘carp’), an ubiquitous invasive fish, was developed and used to measure eDNA in Lake Staring (MN, USA), in which both the density of carp and their distribution have been closely monitored for several years. Surface water, sub-surface water, and sediment were sampled from 22 locations in the lake, including areas frequently used by carp. In water, areas of high carp use had a higher rate of detection and concentration of eDNA, but there was no effect of fish use on sediment eDNA. The detection rate and concentration of eDNA in surface and sub-surface water were not significantly different (p≥0.5), indicating that eDNA did not accumulate in surface water. The detection rate followed the trend: high-use water > low-use water > sediment. The concentration of eDNA in sediment samples that were above the limit of detection were several orders of magnitude greater than water on a per mass basis, but a poor limit of detection led to low detection rates. The patchy distribution of eDNA in the water of our study lake suggests that the mechanisms that remove eDNA from the water column, such as decay and sedimentation, are rapid. Taken together, these results indicate that effective eDNA sampling methods should be informed by fish distribution, as eDNA concentration was shown to vary dramatically between samples taken less than 100 m apart.     

Individual haplotyping of whale sharks from seawater environmental DNA

Population genetic data can provide valuable information on the demography of a species. For rare and elusive marine megafauna, samples for generating the data are traditionally obtained from tissue biopsies, which can be logistically difficult and expensive to collect and require invasive sampling techniques. Analysis of environmental DNA (eDNA) offers an alternative, minimally invasive approach to provide important genetic information. Although eDNA approaches have been studied extensively for species detection and biodiversity monitoring in metabarcoding studies, the potential for the technique to address population-level questions remains largely unexplored. Here, we applied “eDNA haplotyping” to obtain estimates of the intraspecific genetic diversity of a whale shark (Rhincodon typus) aggregation at Ningaloo reef, Australia. Over 2 weeks, we collected seawater samples directly behind individual sharks prior to taking a tissue biopsy sample from the same animal. Our data showed a 100% match between mtDNA sequences recovered in the eDNA and tissue sample for all 28 individuals sampled. In the seawater samples, >97% of all reads were assigned to six dominant haplotypes, and a clear dominant signal (~99% of sample reads) was recovered in each sample. Our study demonstrates accurate individual-level haplotyping from seawater eDNA. When DNA from one individual clearly dominates each eDNA sample, it provides many of the same opportunities for population genetic analyses as a tissue sample, potentially removing the need for tissue sampling. Our results show that eDNA approaches for population-level analyses have the potential to supply critical demographic data for the conservation and management of marine megafauna.     

Using occupancy modelling to compare environmental DNA to traditional field methods for regional‐scale monitoring of an endangered aquatic species

Environmental DNA (eDNA) monitoring approaches promise to greatly improve detection of rare, endangered and invasive species in comparison with traditional field approaches. Herein, eDNA approaches and traditional seining methods were applied at 29 research locations to compare method‐specific estimates of detection and occupancy probabilities for endangered tidewater goby (Eucyclogobius newberryi). At each location, multiple paired seine hauls and water samples for eDNA analysis were taken, ranging from two to 23 samples per site, depending upon habitat size. Analysis using a multimethod occupancy modelling framework indicated that the probability of detection using eDNA was nearly double (0.74) the rate of detection for seining (0.39). The higher detection rates afforded by eDNA allowed determination of tidewater goby occupancy at two locations where they have not been previously detected and at one location considered to be locally extirpated. Additionally, eDNA concentration was positively related to tidewater goby catch per unit effort, suggesting eDNA could potentially be used as a proxy for local tidewater goby abundance. Compared to traditional field sampling, eDNA provided improved occupancy parameter estimates and can be applied to increase management efficiency across a broad spatial range and within a diversity of habitats.     

Selective and efficient extraction of recombinant proteins from the periplasm of Escherichia coli using low concentrations of chemicals

Experiments were conducted to determine chemicals at low concentrations, which can be utilized for selective release of periplasmic proteins. It was revealed that 80–100 % of the activity of alpha-amylase, beta-lactamase, and Fab D1.3 was retained in the presence of 0.05 and 0.1 % Triton X-100, 0.1 % Tween 20, 0.1 % DOC, 0.01 % BAC, 0.01 % CTAB, 10 mM EDTA, 1 mM and 10 mM DEA, 10 mM NTA, 0.1 and 1 % SHMP, 200 mM urea, 100–500 mM GndCl, and 1 % solvents (hexane, xylene, toluene, benzene, pyridine and isoamyl alcohol). Performance of these chemicals, recognized as generally safe, for selective release of proteins from the periplasm of Escherichia coli was investigated. DOC was a general and very efficient agent, and at concentrations as low as 0.05, 0.1, and 0.025 %, released beta-lactamase, alpha-amylase, and Fab D1.3 selectively with yield factors of 2.7, 2.3, and 3.6 times greater than osmotic shock procedure, respectively. EDTA (1 and 10 mM) discharged Fab D1.3 with efficiency more than osmotic shock (target protein yield of 110 and 138 %, correspondingly). Isoamyl alcohol (10 % v/v) was effective for periplasmic release of alpha-amylase and particularly Fab D1.3, with target protein yields of 75 and 168 %, respectively.     

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.     

Spatial and temporal patterns of environmental DNA detection to inform sampling protocols in lentic and lotic systems

The development of efficient sampling protocols for the capture of environmental DNA (eDNA) could greatly help improve accuracy of occupancy monitoring for species that are difficult to detect. However, the process of developing a protocol in situ is complicated for rare species by the fact that animal locations are often unknown. We tested sampling designs in lake and stream systems to determine the most effective eDNA sampling protocols for two rare species: the Sierra Nevada yellow‐legged frog (Rana sierrae) and the foothill yellow‐legged frog (Rana boylii). We varied water volume, spatial sampling, and seasonal timing in lakes and streams; in lakes we also tested multiple filter types. We found that filtering 2 L versus 1 L increased the odds of detection in streams 5.42X (95% CI: 3.2–9.19X) in our protocol, from a probability of 0.51–0.85 per technical replicate. Lake sample volumes were limited by filter clogging, and we found no effect of volume or filter type. Sampling later in the season increased the odds of detection in streams by 1.96X for every 30 days (95% CI: 1.3–2.97X) but there was no effect for lakes. Spatial autocorrelation of the quantity of yellow‐legged frog eDNA captured in streams ceased between 100 and 200 m, indicating that sampling at close intervals is important.     

The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol–chloroform–isoamyl alcohol DNA extraction

Current research targeting filtered macrobial environmental DNA (eDNA) often relies upon cold ambient temperatures at various stages, including the transport of water samples from the field to the laboratory and the storage of water and/or filtered samples in the laboratory. This poses practical limitations for field collections in locations where refrigeration and frozen storage is difficult or where samples must be transported long distances for further processing and screening. This study demonstrates the successful preservation of eDNA at room temperature (20 °C) in two lysis buffers, CTAB and Longmire's, over a 2‐week period of time. Moreover, the preserved eDNA samples were seamlessly integrated into a phenol–chloroform–isoamyl alcohol (PCI) DNA extraction protocol. The successful application of the eDNA extraction to multiple filter membrane types suggests the methods evaluated here may be broadly applied in future eDNA research. Our results also suggest that for many kinds of studies recently reported on macrobial eDNA, detection probabilities could have been increased, and at a lower cost, by utilizing the Longmire's preservation buffer with a PCI DNA extraction.     

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

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