Factors influencing detection of eDNA from a stream‐dwelling amphibian

Environmental DNA (eDNA) methods for detecting and estimating abundance of aquatic species are emerging rapidly, but little is known about how processes such as secretion rate, environmental degradation, and time since colonization or extirpation from a given site affect eDNA measurements. Using stream‐dwelling salamanders and quantitative PCR (qPCR) analysis, we conducted three experiments to assess eDNA: (i) production rate; (ii) persistence time under different temperature and light conditions; and (iii) detectability and concentration through time following experimental introduction and removal of salamanders into previously unoccupied streams. We found that 44–50 g individuals held in aquaria produced 77 ng eDNA/h for 2 h, after which production either slowed considerably or began to equilibrate with degradation. eDNA in both full‐sun and shaded treatments degraded exponentially to <1% of the original concentration after 3 days. eDNA was no longer detectable in full‐sun samples after 8 days, whereas eDNA was detected in 20% of shaded samples after 11 days and 100% of refrigerated control samples after 18 days. When translocated into unoccupied streams, salamanders were detectable after 6 h, but only when densities were relatively high (0.2481 individuals/m²) and when samples were collected within 5 m of the animals. Concentrations of eDNA detected were very low and increased steadily from 6–24 h after introduction, reaching 0.0022 ng/L. Within 1 h of removing salamanders from the stream, eDNA was no longer detectable. These results suggest that eDNA detectability and concentration depend on production rates of individuals, environmental conditions, density of animals, and their residence time.     

A framework for estimating the sensitivity of eDNA surveys

Imperfect sensitivity, or imperfect detection, is a feature of all survey methods that needs to be accounted for when interpreting survey results. Detection of environmental DNA (eDNA) is increasingly being used to infer species distributions, yet the sensitivity of the technique has not been fully evaluated. Sensitivity, or the probability of detecting target DNA given it is present at a site, will depend on both the survey method and the concentration and dispersion of target DNA molecules at a site. We present a model to estimate target DNA concentration and dispersion at survey sites and to estimate the sensitivity of an eDNA survey method. We fitted this model to data from a species‐specific eDNA survey for Oriental weatherloach, Misgurnus anguillicaudatus, at three sites sampled in both autumn and spring. The concentration of target DNA molecules was similar at all three sites in autumn but much higher at two sites in spring. Our analysis showed the survey method had ≥95% sensitivity at sites where target DNA concentrations were ≥11 molecules per litre. We show how these data can be used to compare sampling schemes that differ in the number of field samples collected per site and number of PCR replicates per sample to achieve ≥95% sensitivity at a given target DNA concentration. These models allow researchers to quantify the sensitivity of eDNA survey methods to optimize the probability of detecting target species, and to compare DNA concentrations spatially and temporarily.     

Seasonal temperature patterns in an arctic and two subarctic Alaskan (USA) headwater streams

We investigated the thermal ecology of three Alaskan streams. Monument Creek (MC) and Little Poker Creek (LPC) are subarctic streams in interior Alaska; LPC is in a permafrost-dominated valley. Imnavait Creek (IC) is an arctic tundra beaded stream in the northern foothills of the Brooks Range. Water temperatures were recorded with automated dataloggers hourly (LPC) or bi-hourly (MC and IC). Records for MC extend through almost three entire years, while data from IC (three years) and LPC (one year) represent the majority of the ice-free season. We also collected winter water/ice temperatures from IC (1989–1990). Mean annual water temperatures were 1.1 °C (LPC), 2.3 °C (MC), and 2.9 °C (IC), while maxima were 5.8 °C (LPC), 13.0 °C (MC), and 21.4 °C (IC). Water temperature rose in the spring about twice as fast (both mean and maximum daily increase) in MC as in LPC, and again about twice as fast in IC as in MC. A similar pattern was observed during the autumnal decline in water temperature. Maximum daily amplitude followed a similar pattern, with MC (6.6 °C) intermediate between LPC (4.1°) and IC (11.6°). LPC accumulated approximately 400 degree-days above 0 °C, MC approximately 950 degree-days, and IC approximately 1000 degree-days. Although it is about 450 km north of the other streams, the tundra stream (IC) accumulated more degree-days, had higher maximum and mean temperatures, greater daily temperature amplitude, and steeper slopes of vernal temperature rise and autumnal temperature decline than the subarctic streams (LPC and MC). The absence of a canopy of riparian plants, channel morphology, and continuous sunlight during the arctic mid-summer accounted for these higher temperatures. Beaded tundra streams provide a highly seasonal (< 120 d ice-free) and spatially and temporally complex thermal environment.     

Effects of nutrients and warming on Planktothrix dynamics and diversity: a palaeolimnological view based on sedimentary DNA and RNA

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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.     

Moving eDNA surveys onto land: Strategies for active eDNA aggregation to detect invasive forest insects

The use of environmental DNA (eDNA) surveys to monitor terrestrial species has been relatively limited, with successful implementations still confined to sampling DNA from natural or artificial water bodies and soil. Sampling water for eDNA depends on proximity to or availability of water, whereas eDNA from soil is limited in its spatial scale due to the large quantities necessary for processing and difficulty in doing so. These challenges limit the widespread use of eDNA in several systems, such as surveying forests for invasive insects. We developed two new eDNA aggregation approaches that overcome the challenges of above‐ground terrestrial sampling and eliminate the dependency on creating or utilizing pre‐existing water bodies to conduct eDNA sampling. The first, “spray aggregation,” uses spray action to remove eDNA from surface substrates and was developed for shrubs and other understorey vegetation, while the second, “tree rolling,” uses physical transfer via a roller to remove eDNA from the surface of tree trunks and large branches. We tested these approaches by surveying for spotted lanternfly, Lycorma delicatula, a recent invasive pest of northeastern USA that is considered a significant ecological and economic threat to forests and agriculture. We found that our terrestrial eDNA surveys matched visual surveys, but also detected L. delicatula presence ahead of visual surveys, indicating increased sensitivity of terrestrial eDNA surveys over currently used methodology. The terrestrial eDNA approaches we describe can be adapted for use in surveying a variety of forest insects and represent a novel strategy for surveying terrestrial biodiversity.     

DNA amplification in the field: move over PCR,here comes LAMP

It would not be an exaggeration to say that among molecular technologies, it is PCR (polymerase chain reaction) that underpins the discipline of molecular ecology as we know it today. With PCR, it has been possible to target the amplification of particular fragments of DNA, which can then be analysed in a multitude of ways. The capability of PCR to amplify DNA from a mere handful of copies further means that conservationists and ecologists are able to sample DNA unobtrusively and with minimal disturbance to the environment and the organisms of interest. However, a key disadvantage of PCR‐based methods has been the necessity for a generally non‐portable, laboratory setting to undertake the time‐consuming thermocycling protocols. LAMP (loop‐mediated isothermal amplification) offers a logistically simpler protocol: a relatively rapid DNA amplification reaction occurs at one temperature, and the products are visualized with a colour change within the reaction tubes. In the first field application of LAMP for an ecological study, Centeno‐Cuadros et al. ( 2016 ) demonstrates how LAMP can be used to determine the sex of three raptor species. By enabling DNA amplification in situ and in ‘real‐time’, LAMP promises to revolutionize how molecular ecology is practised in the field.     

Is telomere length a molecular marker of past thermal stress in wild fish?

Telomeres protect eukaryotic chromosomes; variation in telomere length has been linked (primarily in homoeothermic animals) to variation in stress, cellular ageing and disease risk. Moreover, telomeres have been suggested to function as biomarker for quantifying past environmental stress, but studies in wild animals remain rare. Environmental stress, such as extreme environmental temperatures in poikilothermic animals, may result in oxidative stress that accelerates telomere attrition. However, growth, which may depend on temperature, can also contribute to telomere attrition. To test for associations between multitissue telomere length and past water temperature while accounting for the previous individual growth, we used quantitative PCR to analyse samples from 112 young‐of‐the‐year brown trout from 10 natural rivers with average water temperature differences of up to 6°C (and an absolute maximum of 23°C). We found negative associations between relative telomere length (RTL) and both average river temperature and individual body size. We found no indication of RTL–temperature association differences among six tissues, but we did find indications for differences among the tissues for associations between RTL and body size; size trends, albeit nonsignificant in their differences, were strongest in muscle and weakest in fin. Although causal relationships among temperature, growth, oxidative stress, and cross‐sectional telomere length remain largely unknown, our results indicate that telomere‐length variation in a poikilothermic wild animal is associated with both past temperature and growth.     

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.     

Long‐term dynamics in microbial eukaryotes communities: a palaeolimnological view based on sedimentary DNA

Assessing the extent to which changes in lacustrine biodiversity are affected by anthropogenic or climatic forces requires extensive palaeolimnological data. We used high‐throughput sequencing to generate time‐series data encompassing over 2200 years of microbial eukaryotes (protists and Fungi) diversity changes from the sedimentary DNA record of two lakes (Lake Bourget in French Alps and Lake Igaliku in Greenland). From 176 samples, we sequenced a large diversity of microbial eukaryotes, with a total 16 386 operational taxonomic units distributed within 50 phylogenetic groups. Thus, microbial groups, such as Chlorophyta, Dinophyceae, Haptophyceae and Ciliophora, that were not previously considered in lacustrine sediment record analyses appeared to be potential biological markers of trophic status changes. Our data suggest that shifts in relative abundance of extant species, including shifts between rare and abundant taxa, drive ecosystem responses to local and global environmental changes. Community structure shift events were concomitant with major climate variations (more particularly in Lake Igaliku). However, this study shows that the impacts of climatic fluctuations may be overpassed by the high‐magnitude eutrophication impacts, as observed in the eutrophicated Lake Bourget. Overall, our data show that DNA preserved in sediment constitutes a precious archive of information on past biodiversity changes.     

Environmental DNA metabarcoding of lake fish communities reflects long‐term data from established survey methods

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 Differential Reaction of Rice Hybrids to Tungro Virus by Phenotyping and PCR Analysis

Twenty popular rice hybrids were used to screen for rice tungro virus (RTV) disease reaction. Virulent green leafhoppers (GLH) were used as vector to introduce RTV to the rice hybrids. Virus symptoms scores were recorded at 14, 21, 34, 41 and 59 days postinoculation (DPI), which suggested that virus symptoms are greatly influenced by growth stage of plants. To confirm the presence of virus, polymerase chain reaction (PCR)‐based detection of Rice tungro bacilliform virus (RTBV) was carried out at 7, 14, 21 and 59 DPI using virus genome‐specific primers. Virus presence was observed in all the rice hybrids and check varieties, particularly at later stages of infection. This study shows that phenotyping for tungro virus resistance in rice hybrids at 21 DPI gives most reliable results based on both virus symptoms and presence of virus. Further, to assess the relative difference in population of RTBV, quantitative PCR was performed in all the genotypes at 21 DPI. Yield data were also recorded from control and virus‐infected plants to estimate yield loss percentage due to tungro disease. This study is important to understand the response of rice hybrids to tungro virus disease. Results obtained in this study emphasize that molecular detection of virus is very important to screen the rice plants accurately for tungro disease reaction.     

Detecting an elusive invasive species: a diagnostic PCR to detect Burmese python in Florida waters and an assessment of persistence of environmental DNA

Recent studies have demonstrated that detection of environmental DNA (eDNA) from aquatic vertebrates in water bodies is possible. The Burmese python, Python bivittatus, is a semi‐aquatic, invasive species in Florida where its elusive nature and cryptic coloration make its detection difficult. Our goal was to develop a diagnostic PCR to detect P. bivittatus from water‐borne eDNA, which could assist managers in monitoring this invasive species. First, we used captive P. bivittatus to determine whether reptilian DNA could be isolated and amplified from water samples. We also evaluated the efficacy of two DNA isolation methods and two DNA extraction kits commonly used in eDNA preparation. A fragment of the mitochondrial cytochrome b gene from P. bivittatus was detected in all water samples isolated with the sodium acetate precipitate and the QIAamp DNA Micro Kit. Next, we designed P. bivittatus‐specific primers and assessed the degradation rate of eDNA in water. Our primers did not amplify DNA from closely related species, and we found that P. bivittatus DNA was consistently detectable up to 96 h. Finally, we sampled water from six field sites in south Florida. Samples from five sites, where P. bivittatus has been observed, tested positive for eDNA. The final site was negative and had no prior documented evidence of P. bivittatus. This study shows P. bivittatus eDNA can be isolated from water samples; thus, this method is a new and promising technique for the management of invasive reptiles.     

Comparing local‐ and regional‐scale estimations of the diversity of stream fish using eDNA metabarcoding and conventional observation methods

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Invasion genetics from eDNA and thousands of larvae: A targeted metabarcoding assay that distinguishes species and population variation of zebra and quagga mussels

Identifying species and population genetic compositions of biological invasions at early life stages and/or from environmental (e)DNA using targeted high‐throughput sequencing (HTS) metabarcode assays offers powerful and cost‐effective means for early detection, analysis of spread patterns, and evaluating population changes. The present study develops, tests, and applies this method with a targeted sequence assay designed to simultaneously identify and distinguish between the closely related invasive Eurasian zebra and quagga mussels (Dreissena polymorpha and D. rostriformis) and their relatives and discern their respective population genetic patterns. Invasions of these dreissenid mussel species have markedly changed freshwater ecosystems throughout North America and Europe, exerting severe ecological and economic damage. Their planktonic early life stages (eggs and larvae) are morphologically indistinguishable, yet each species exerts differential ecological effects, with the quagga often outcompeting the zebra mussel as adults. Our targeted assay analyzes genetic variation from a diagnostic sequence region of the mitochondrial (mt)DNA cytochrome oxidase I (COI) gene, to assess temporal and spatial inter‐ and intra‐specific genetic variability. The assay facilitates analysis of environmental (e)DNA from water, early life stages from thousands of individuals, and simultaneous analysis of 50–100 tagged field‐collected samples. Experiments evaluated its accuracy and performance using: (a) mock laboratory communities containing known DNA quantities per taxon, (b) aquaria with mixed‐species/haplotype compositions of adults, and (c) field‐collected water and plankton versus traditional sampling of adult communities. Results delineated species compositions, relative abundances, and population‐level diversity differences among ecosystems, habitats, time series, and life stages from two allopatric concurrent invasions in the Great Lakes (Lake Erie) and the Hudson River, which had separate founding histories. Findings demonstrate application of this targeted assay and our approach to accurately and simultaneously discern species‐ and population‐level differences across spatial and temporal scales, facilitating early detection and ecological understanding of biological invasions.     

Re‐building brown trout populations in dredged boreal forest streams: in‐stream restoration combined with stocking of young trout

1. Rivers in boreal forested areas were often dredged to facilitate the transport of timber resulting in channels with simplified bed structure and flow fields and reduced habitat suitability for stream organisms, especially lotic fishes. Currently, many streams are being restored to improve their physical habitat, by replacing boulders and gravel and removing constraining embankments. The most compelling justification behind stream restoration of former floatways has been the enhancement of native fish populations, specifically salmonids. 2. We examined the success of a stream management programme aimed at re‐building diminished brown trout (Salmo trutta) populations by monitoring densities of young‐of‐year and older trout in 18 managed and three reference streams during 2000–2005. Rehabilitation included in‐stream restoration combined with a 5‐year post‐restoration period of stocking young brown trout. Our space‐for‐time substitution design comprised four pre‐management, four under‐management, 10 post‐management and three reference streams. 3. Densities of young‐of‐year brown trout, indicating population establishment, were significantly higher in post‐ compared with pre‐management streams. However, density of young‐of‐year brown trout in post‐management streams was significantly lower compared with near‐pristine reference streams. Furthermore, success of managed brown trout population re‐building varied, indicating stream‐specific responses to management measures. Density of burbot (Lota lota), a native generalist predator, was associated with low recruitment of brown trout. 4. Stream‐specific responses imply that rehabilitation of brown trout populations cannot be precisely predicted thereby limiting application. Our findings support the importance of adaptive stream restoration and management, with focus on identifying factor(s) limiting the establishment of target fish populations.     

Environmental constraints on oviposition of aquatic invertebrates with contrasting life cycles in two human‐modified streams

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