Environmental DNA最新文献

Insect populations are declining in many parts of the world, but a lack of long-term monitoring data is impeding our ability to understand and mitigate the causes of insect biodiversity loss. Whilst high-throughput sequencing (HTS) approaches, such as DNA metabarcoding, have the potential to revolutionize insect biomonitoring through rapid scalable identification, it is unclear to what extent HTS can be applied to long-term stored insect samples. Archived insect samples could inform forecasting and provide valuable information regarding past changes to biodiversity. Here, we assess the efficacy of DNA metabarcoding to identify archived samples from the longest passive monitoring scheme in the United Kingdom: the Rothamsted Insect Survey (RIS). With a focus on aphids as the target taxa of a national network of suction-traps, we analyze a 16-year time-series of stored samples (2003–2018) using DNA metabarcoding from one of the RIS suction traps as an exemplar. We achieved this by using a non-destructive DNA extraction protocol, ensuring the integrity of archival samples for further studies. We compared the identities of aphids determined by both metabarcoding (as inferred amplicon sequence variants [ASVs]) and morphological identification and found that metabarcoding detected most genera with varying success (mean > 76%). When comparing the two methods objectively (i.e., including taxa not detected morphologically), however, congruence decreased (51%). We show that minimum sequence copy thresholds can minimize metabarcoding false positives, but at the expense of introducing false negatives, highlighting the need for careful data curation. Detectability of taxa identified morphologically and similarity between the two methods did not significantly vary over time, demonstrating the viability of metabarcoding for screening archival samples. We discuss the advantages and challenges of metabarcoding for insect biomonitoring, particularly from archival samples, including improvements to sample handling, processing, and archiving. We highlight the wider potential of HTS approaches for stored samples from insect monitoring schemes, unlocking the immense potential of global historical time series.

Agricultural intensification is a major driver in species declines, with changes in land use resulting in widespread alteration of resource availability. An increase in anthropogenic food resources, alongside decreasing natural resources, has resulted in species undergoing dietary changes that can have important ecological consequences, particularly for declining species. Here we use high-throughput sequencing to analyze the diet of the migrant European turtle dove (Streptopelia turtur), a species that has experienced significant population decline throughout its European range. We analyze the diet of this species on both breeding and wintering grounds to gain an understanding of resource use throughout the annual cycle and compare areas of more and less intensive agriculture in western and eastern Europe, respectively. We examine associations with body condition, spatiotemporal variation and the source of food (wild or cultivated). We identified 121 taxonomic units in the diet, with significant variation across sampling seasons, and very little overlap between the breeding and wintering seasons, as well as high levels of cultivated food resources in the diet of turtle doves in both breeding and wintering grounds, with the highest proportion of wild seeds in the diet occurring in birds caught in Hungary, where agricultural intensity was lowest. We detected no association between body condition and the consumption of cultivated food resources. We demonstrate the importance of wild resources in birds on the wintering grounds as they approach migration, where body condition increased as the season progressed, concurrent with an increased consumption of wild seeds. These findings indicate the importance of habitats rich in wild seeds and the need to consider food availability on the wintering grounds, as well as the breeding grounds in turtle dove conservation strategies.

North American freshwater mussels are of special conservation concern due to their high endemism and the multiple anthropogenic stressors affecting them. Of the over 300 species in North America, nearly one third of these species are federally listed as threatened or endangered. Environmental DNA (eDNA) analysis has been successful in detecting freshwater mussels and could aid in monitoring their populations. Production and degradation rates of eDNA for the species of interest are needed to inform interpretation of eDNA detections, allow possible modeling of relative abundance and population location, and aid in mussel conservation through population identification. Here, we designed and tested qPCR assays for three freshwater mussel species, mucket (Ortmanniana ligamentina), fatmucket (Lampsilis siliquoidea), and the federally endangered spectaclecase (Cumberlandia monodonta). We performed laboratory experiments under controlled conditions to measure eDNA shedding and degradation rates for each species. Different biomasses, temperatures, and food regimens were tested independently to determine if these factors influence the amount of DNA produced by the mussels. Degradation rates of eDNA were measured from experimental tank water after mussels were removed. Overall, we observed low eDNA shedding rates for freshwater mussels compared to previous studies of fish eDNA shedding rates. Furthermore, temperature and feeding showed limited or no significant effects in the species studied. Environmental DNA degradation rates were consistent with those reported in the literature for other taxa. Collectively, our results will be useful for designing eDNA monitoring studies, modeling eDNA dispersal, and interpreting eDNA results to help inform freshwater mussel conservation efforts.

The need for widespread occurrence data to inform species conservation has prompted interest in large, national-scale environmental DNA (eDNA) monitoring strategies. However, targeted eDNA assays are seldom validated for use across broad geographic areas. Here, we validated 46 new and previously published probe-based qPCR assays targeting invasive species throughout the continental United States. We drew upon current taxonomies, range maps, publicly available sequences, and tissue archives to evaluate all potentially sympatric confamilial species and genetically similar extrafamilial taxa. Out of 5276 unique assay-nontarget taxon combinations, we were able to test 4206 (80%). We characterized levels of validation and specificity for each of eight federal geographic regions and provided an online tool with state-level information, as well as detailed assay descriptions in an appendix. Specificity testing benefited from extensive use of eDNAssay—a machine learning classifier trained to predict qPCR cross-amplification—which we found to be 96% accurate in 649 unique tests that underwent paired in silico and in vitro testing. Predictions of assay specificity (the true negative rate) were 98–100% accurate, depending on the classification threshold used. This work provides both an immediate resource for invasive species surveillance and demonstrates an enhanced in silico, geographically subdivided validation framework to aid in future large-scale validation efforts.

Environmental DNA (eDNA) has been widely used for species surveillance. However, the lack of adequate quality control in many eDNA research projects and applications can lead to false-negative results, greatly affecting biosecurity surveillance and conservation efforts. Exogenous DNA is routinely added to eDNA samples and used as a positive control, typically after DNA extraction. However, this type of positive control is only able to identify false negatives due to errors at the amplification stage. Therefore, errors in upstream processes, such as sample collection will not be identified by an exogenous control. We designed two independent sets of generic quality control qPCR assays (QCqPCR) targeting abundant endogenous DNA that is obtained during sample collection. Our QCqPCR assays target the chloroplast 16S and 23S ribosomal RNA sequences. In silico analyses indicated these regions were highly conserved among plants, algae and bacteria commonly found in freshwater, marine, or terrestrial environments. These QCqPCR assays were purposely mismatched against the human genome to avoid false positives resulting from human DNA contamination. Both assays remained highly efficient and sensitive under annealing temperatures between 58 and 62°C, allowing them to be multiplexed with most qPCR analyses. We validated our assays by multiplexing with a species-specific Murray cod (Maccullochella peelii) assay on field-collected environmental water samples. Potential false-negative reactions can be identified by the failed or suppressed QCqPCR assay and the negative species-specific assay. We recommend incorporating either one of the QCqPCR assays in qPCR-based eDNA analysis to identify potential false negatives and improve the reliability of eDNA surveys.

Understanding how mesopredators partition their diet and the identity of consumed prey can assist in understanding the ecological role predators and prey play in ecosystem trophodynamics. Here, we assessed the diet of three common coral reef mesopredators; Pseudochromis flavivertex, Pseudochromis fridmani, and Pseudochromis olivaceus from the family Pseudochromidae, commonly known as dottybacks, using a combination of (i) visual stomach content analysis, (ii) stomach content DNA metabarcoding (18S, COI), and (iii) stable isotope analysis (δ¹⁵N, δ¹³C). In addition, P. flavivertex is found in two distinct color morphs in the Red Sea, providing an opportunity to analyze intra-morph differences. These techniques revealed partitioning in the dietary composition and resource use among species. Arthropods comprised the main dietary component of P. flavivertex (18S > 60%; COI > 10%) and P. olivaceus (18S = 57.2%), while P. fridmani ingested predominantly mollusks (18S = 51.3%, COI = 24.6%). Despite being small predators, microplastics were found in the gut content of some of these fishes. Stable isotope analysis showed differences in species' isotopic niche breadth and trophic position. Pseudochromis olivaceus presented the largest isotopic niche (SEA_C = 1.61‰²), while P. fridmani showed the smallest isotopic niche (SEA_C = 0.45‰²) among species. Although the two techniques used for stomach content analysis did not show differences in the diet within color morphs of P. flavivertex, they differed in the isotopic niche and resource use. Despite our limited sampling, our findings provide evidence of species-specific differences in the trophic ecology of dottybacks and demonstrate their important role as predators of cryptic invertebrates and small fishes. This study highlights the importance of combining several approaches (short-term: visual analysis and DNA metabarcoding; and long-term: isotope analysis) when assessing the feeding habits of coral reef fish, as they provide complementary information necessary to delimit their niches and understand the role that small mesopredators play in coral reef ecosystems.

Environmental DNA (eDNA) is often used to determine the presence and absence of species in a specific environment, be it air, water, or soil. Numerous environmental conditions are known to directly alter the rate at which eDNA degrades, including pH, temperature, and UV-B light exposure. Beyond these, many limnological parameters have not been thoroughly examined for their ability to modify the degradation rate of eDNA. Here we used 20 mL microcosms with water collected from 12 lakes from the Kawartha Highlands near Peterborough Ontario, Canada, to study the decay rates of dissolved Yellow perch (Perca flavescens) eDNA. We measured and related rates of eDNA loss to multiple water quality parameters: total dissolved phosphorus, total dissolved nitrogen, size-fractionated carbon, and chlorophyll-a levels. Bioassays were also conducted to examine the bacterial role in eDNA degradation using three treatments under natural system conditions: non-filtered, filtered (0.22 μm), and non-filtered with added phosphorus (50 μg/L). Each microcosm exhibited a unique rate of degradation with eDNA half-life (C_0.5) ranging from 2.5 to 12.9 h. Chlorophyll-a levels exhibited a positive linear relationship to the rate of degradation, while all other parameters showed no effect. The bioassays showed a general trend of the filtered treatments exhibiting the lowest rate of degradation, followed by the phosphorus treatments with the non-filtered treatment containing bacteria exhibiting the highest rate of degradation. Overall, water with an increased level of chlorophyll-a, in conjunction with elevated bacteria (i.e. non-filtered bioassay) will exhibit a faster overall rate of eDNA degradation. These results show the necessity to individualize eDNA survey plans to the water body of interest and to account for environmental conditions relating to the microbial processing of eDNA.

Marine sediments as excellent climate archives, contain among other biomolecules substantial amounts of extracellular DNA. Through mineral binding, some of the DNA remains protected from degradation which aids its preservation. While this pool of DNA represents genomic ecosystem fingerprints spanning over millions of years, the capability of current DNA extraction methods in recovering mineral-bound DNA remains poorly understood. We evaluated current sedimentary DNA extraction approaches and their ability to recover short DNA fragments from artificially created DNA-mineral complexes involving pure clay minerals or quartz, as well as from different types of natural marine sediments. We separately investigated lysis (DNA release) and isolation steps (purification of DNA) comparing five different lysis buffers across two commonly used DNA isolation approaches: silica magnetic beads and liquid-phase organic extraction and purification. The choice of lysis buffer significantly impacted the amount of recovered mineral-bound DNA and facilitated selective desorption of DNA fragments. High molarity EDTA and phosphate lysis buffers recovered on average an order of magnitude more DNA from clay minerals than other tested buffers, while both isolation approaches recovered comparable amounts of DNA. In marine sediments, however, liquid-phase organic extraction caused inhibitory effects in subsequent downstream applications (e.g., PCR), across all assessed DNA extracts, while silica magnetic beads induced inhibition only in half of the tested DNA extracts. Thus, the isolation approach, together with the lysis buffer, played a decisive role in successful library preparation with lysis buffer choice ultimately impacting final library fragment distribution. With this study, we underscore the critical importance of lysis buffer selection to maximize the recovery of mineral-bound DNA and show its profound impact on recovered fragment lengths in sedimentary DNA extractions, a crucial factor alongside existing isolation approaches in facilitating high-quality DNA extracts for downstream analysis related to ancient environmental DNA research.

There is a rapidly growing interest by resource managers to utilize environmental DNA technology (eDNA) as a tool to enhance current management efforts. However, the technology remains relatively specialized, since it requires specific expertise and equipment to perform. To begin to overcome some of the obstacles restricting the widespread, routine adoption of eDNA technology, we evaluated the use of CRISPR Cas12a detection technology for in-the-field eDNA detection using Chinook salmon (Oncorhynchus tshawytscha) as a target species. By targeting a highly variable region in the salmonid mitochondrial DNA D-loop, we were able to demonstrate that CRISPR Cas12a detection technology is both sensitive and specific for Chinook salmon eDNA. Engineering of the technology to work in the field was accomplished by employing rapid eDNA purification and visual readout of results using visual lateral flow or fluorescent detection methods. The technology was piloted on the fall Chinook salmon run in the Snake River of Washington State, USA, and proved to be a viable approach for streamside eDNA monitoring. With the improvement of the technology, CRISPR eDNA detection methods hold great promise in expanding the reach of eDNA as a commonly used resource management tool.

Eastwood, N., Kissane, S., Campbell, L., Briscoe, A. G., Egeter, B., & Orsini, L. (2024). Single metabarcoding multiplex captures community-level freshwater biodiversity and beyond. Environmental DNA, 6, e515.

In the abstract, the text “384 samples per locus (N = 15,636 samples)” was incorrect. This should read “384 samples per locus (N = 1536 samples).”

We apologize for this error.