TREMATODE SPECIES DETECTION AND QUANTIFICATION BY ENVIRONMENTAL DNA-qPCR ASSAY IN LAKE CHANY, RUSSIA.

Environmental DNA (eDNA) surveys promise to be a sensitive and powerful tool for the detection of trematodes. This can contribute to the limited studies on trematode ecology, specifically in aquatic ecosystems. Here, we developed species-specific primer and probe sets for Moliniella anceps, Opisthioglyphe ranae, and Plagiorchis multiglandularis cercariae and applied a novel eDNA qPCR assay to detect larval trematodes quantitatively. We evaluated the effectiveness of the assays using filtered lake water samples collected from different sites of Lake Fadikha and Kargat River Estuary in Lake Chany, Russia, showing high species specificity and sensitivity in all 3 assays. Further, all 3 assays had high efficiencies ranging from 94.9 to 105.8%. Moliniella anceps, O. ranae, and P. multiglandularis were detected in the environmental water samples through real-time PCR. Thus, we anticipate that our approach will be beneficial for biomonitoring, measuring, and managing ecological systems.

Environmental DNA Isolation, Validation, and Preservation Methods.

Environmental DNA (eDNA) workflows contain many familiar molecular-lab techniques, but also employ several unique methodologies. When working with eDNA, it is essential to avoid contamination from the point of collection through preservation and select a meaningful negative control. As eDNA can be obtained from a variety of samples and habitats (e.g., soil, water, air, or tissue), protocols will vary depending on usage. Samples may require additional steps to dilute, block, or remove inhibitors or physically break up samples or filters. Thereafter, standard DNA isolation techniques (kit-based or phenol:chloroform:isoamyl [PCI]) are employed. Once DNA is extracted, it is typically quantified using a fluorometer. Yields vary greatly, but are important to know prior to amplification of the gene(s) of interest. Long-term storage of both the sampled material and the extracted DNA is encouraged, as it provides a backup for spilled/contaminated samples, lost data, reanalysis, and future studies using newer technology. Storage in a freezer is often ideal; however, some storage buffers (e.g., Longmires) require that filters or swabs are kept at room temperature to prevent precipitation of buffer-related solutes. These baseline methods for eDNA isolation, validation, and preservation are detailed in this protocol chapter. In addition, we outline a cost-effective, homebrew extraction protocol optimized to extract eDNA.

The update and optimization of an eDNA assay to detect the invasive rusty crayfish (Faxonius rusticus).

Environmental DNA (eDNA) is nuclear or mitochondrial DNA shed into the environment, and amplifying this DNA can serve as a reliable, noninvasive way to monitor aquatic systems for the presence of an invasive species. Assays based on the collection of eDNA are becoming increasingly popular, and, when optimized, can aid in effectively and efficiently tracking invasion fronts. We set out to update an eDNA assay to detect the invasive rusty crayfish, Faxonius rusticus. We tested for species specificity compared to other stream crayfish and field tested the assay at sites with known presence (N = 3) and absence (N = 4) in the Juniata River watershed in central Pennsylvania, USA. To maximize sensitivity, we field tested different storage buffers (Longmire's buffer and ethanol), DNA extraction methods (Qiagen's DNEasy and PowerWater kits), and quantitative polymerase chain reaction (qPCR) chemistries (TaqMan and SYBR green). Our assay confirmed the presence data and performed optimally when filter samples were stored in Longmire's buffer, DNA was extracted with DNeasy Blood and Tissue Kit, and TaqMan qPCR chemistry was utilized. With proper sample processing, our assay allows for accurate, noninvasive detection of F. rusticus in streams.

A workflow for the relative quantification of multiple fish species from oceanic water samples using environmental DNA (eDNA) to support large-scale fishery surveys.

While the number of published marine studies using environmental DNA (eDNA) has increased substantially in recent years, marine fish surveys are still scarce. To examine the potential for eDNA to support marine fisheries monitoring surveys, we optimized an eDNA isolation method, developed a multispecies assay and tested it on eDNA samples collected along the Pacific coast of the United States. Four commercial DNA extraction kits that exploit the capability of the nucleic acids binding a solid phase (two using a silica matrix and two magnetic beads) as well an organic separation method were tested. A species-specific multiplex qPCR assay was developed and tested to simultaneously target Pacific hake (Merluccius productus), Pacific lamprey (Entosphenus tridentatus) and eulachon (Thaleichthys pacificus). The specificity of the assay was tested in silico, in vitro and in natura. Environmental DNA isolation using phenol:chloroform:isoamyl purification with a phase lock was optimized and yielded the highest amount of total and target DNA and was used to extract 46 marine water samples for the detection of the three species of interest. The multiplex qPCR assay used in the quantification process was also optimized to provide convenience and accuracy. Pacific hake was present in 44% of the eDNA samples while the other two species were absent. Here, we present a complete workflow for the simultaneous detection and quantification of multiple marine fish species using eDNA. This workflow supports large-scale at-sea sampling efforts with preservation at ambient temperatures and has demonstrated DNA extraction efficiency and reliability. The multiplex qPCR assay is shown to be sensitive and specific for the purposes of simultaneously monitoring the relative abundance of multiple targeted fish species.

Intraspecific genetic variability and diurnal activity affect environmental DNA detection in Japanese eel.

Environmental DNA (eDNA) analysis with species-specific primer/probe sets is promising as a tool to quantify fish abundance and distribution. Nevertheless, several factors could reduce the accuracy of this method. Here, we aimed to analyze whether intraspecific variability and diel activity rhythm affect eDNA detection in Japanese eels (Anguilla japonica). For this purpose, we performed tank experiments focusing on two points. First, we assessed the effects of base pair sequences with probe region polymorphism on eDNA detection. Next, we evaluated the influences of diel rhythm, activity, and individual differences in eDNA release rate on eDNA concentration. We examined the base pair sequences of the probe regions of 20 individuals and found genetic mismatches in two of them. The eDNA concentration was estimated to be much lower in these variants than it was in the other individuals. We conducted a rearing experiment on four non-variant individuals to explore the influences of diel activity and inter-individual differences in eDNA detection. Nocturnal eel activity was reflected in the eDNA detection but the inter-individual differences remained large. The observed weak positive correlations between eDNA concentration and activity suggest that eDNA emission is highly dependent on basal metabolism. The present study suggests that consideration of polymorphic sites at the probe region and diel activity rhythms should improve the accuracy and precision of abundance estimation through eDNA. Such fine-tuning is applicable not only for eels but also for other fishes to be targeted by eDNA technology.

Environmental DNA gives comparable results to morphology-based indices of macroinvertebrates in a large-scale ecological assessment.

Anthropogenic activities are changing the state of ecosystems worldwide, affecting community composition and often resulting in loss of biodiversity. Rivers are among the most impacted ecosystems. Recording their current state with regular biomonitoring is important to assess the future trajectory of biodiversity. Traditional monitoring methods for ecological assessments are costly and time-intensive. Here, we compared monitoring of macroinvertebrates based on environmental DNA (eDNA) sampling with monitoring based on traditional kick-net sampling to assess biodiversity patterns at 92 river sites covering all major Swiss river catchments. From the kick-net community data, a biotic index (IBCH) based on 145 indicator taxa had been established. The index was matched by the taxonomically annotated eDNA data by using a machine learning approach. Our comparison of diversity patterns only uses the zero-radius Operational Taxonomic Units assigned to the indicator taxa. Overall, we found a strong congruence between both methods for the assessment of the total indicator community composition (gamma diversity). However, when assessing biodiversity at the site level (alpha diversity), the methods were less consistent and gave complementary data on composition. Specifically, environmental DNA retrieved significantly fewer indicator taxa per site than the kick-net approach. Importantly, however, the subsequent ecological classification of rivers based on the detected indicators resulted in similar biotic index scores for the kick-net and the eDNA data that was classified using a random forest approach. The majority of the predictions (72%) from the random forest classification resulted in the same river status categories as the kick-net approach. Thus, environmental DNA validly detected indicator communities and, combined with machine learning, provided reliable classifications of the ecological state of rivers. Overall, while environmental DNA gives complementary data on the macroinvertebrate community composition compared to the kick-net approach, the subsequently calculated indices for the ecological classification of river sites are nevertheless directly comparable and consistent.

Persistence and accumulation of environmental DNA from an endangered dragonfly.

Detection of environmental DNA (eDNA) has become a commonly used surveillance method for threatened or invasive vertebrates in both aquatic and terrestrial environments. However, most studies in this field favor vertebrate target species. Environmental DNA protocols can be especially useful for endangered invertebrates such as the Hine's emerald dragonfly (Somatochlora hineana) where conservation efforts have been greatly hindered by training, time, overall costs, and environmental impacts associated with conducting surveys in the calcareous fens occupied by this species. An essential step in developing such a protocol is to evaluate the dynamics of eDNA concentration under controlled conditions. We used the quantitative polymerase chain reaction (qPCR) to examine seasonal shifts in the persistence and net-accumulation of eDNA from captive S. hineana larvae in experimental mesocosms at temperatures corresponding with their overwintering (5.0 °C) and active (16.0 °C) seasons. Environmental DNA persisted longer at 5.0 °C but accumulated more readily at 16.0 °C. Differences in the accumulation and persistence of eDNA reflect differences in the longevity of eDNA at different temperatures and seasonal differences in larval S. hineana behavior. This study highlights the importance of considering how seasonal changes in temperature influence not only the speed of eDNA degradation but also the target species' eDNA shedding rates.

The role of internal transcribed spacer 2 secondary structures in classifying mycoparasitic Ampelomyces.

Many fungi require specific growth conditions before they can be identified. Direct environmental DNA sequencing is advantageous, although for some taxa, specific primers need to be used for successful amplification of molecular markers. The internal transcribed spacer region is the preferred DNA barcode for fungi. However, inter- and intra-specific distances in ITS sequences highly vary among some fungal groups; consequently, it is not a solely reliable tool for species delineation. Ampelomyces, mycoparasites of the fungal phytopathogen order Erysiphales, can have ITS genetic differences up to 15%; this may lead to misidentification with other closely related unknown fungi. Indeed, Ampelomyces were initially misidentified as other pycnidial mycoparasites, but subsequent research showed that they differ in pycnidia morphology and culture characteristics. We investigated whether the ITS2 nucleotide content and secondary structure was different between Ampelomyces ITS2 sequences and those unrelated to this genus. To this end, we retrieved all ITS sequences referred to as Ampelomyces from the GenBank database. This analysis revealed that fungal ITS environmental DNA sequences are still being deposited in the database under the name Ampelomyces, but they do not belong to this genus. We also detected variations in the conserved hybridization model of the ITS2 proximal 5.8S and 28S stem from two Ampelomyces strains. Moreover, we suggested for the first time that pseudogenes form in the ITS region of this mycoparasite. A phylogenetic analysis based on ITS2 sequences-structures grouped the environmental sequences of putative Ampelomyces into a different clade from the Ampelomyces-containing clades. Indeed, when conducting ITS2 analysis, resolution of genetic distances between Ampelomyces and those putative Ampelomyces improved. Each clade represented a distinct consensus ITS2 S2, which suggested that different pre-ribosomal RNA (pre-rRNA) processes occur across different lineages. This study recommends the use of ITS2 S2s as an important tool to analyse environmental sequencing and unveiling the underlying evolutionary processes.

Optimising sampling and analysis protocols in environmental DNA studies.

Ecological surveys risk incurring false negative and false positive detections of the target species. With indirect survey methods, such as environmental DNA, such error can occur at two stages: sample collection and laboratory analysis. Here we analyse a large qPCR based eDNA data set using two occupancy models, one of which accounts for false positive error by Griffin et al. (J R Stat Soc Ser C Appl Stat 69: 377-392, 2020), and a second that assumes no false positive error by Stratton et al. (Methods Ecol Evol 11: 1113-1120, 2020). Additionally, we apply the Griffin et al. (2020) model to simulated data to determine optimal levels of replication at both sampling stages. The Stratton et al. (2020) model, which assumes no false positive results, consistently overestimated both overall and individual site occupancy compared to both the Griffin et al. (2020) model and to previous estimates of pond occupancy for the target species. The inclusion of replication at both stages of eDNA analysis (sample collection and in the laboratory) reduces both bias and credible interval width in estimates of both occupancy and detectability. Even the collection of > 1 sample from a site can improve parameter estimates more than having a high number of replicates only within the laboratory analysis.

Environmental DNA detection of an invasive ant species (Linepithema humile) from soil samples.

Alien ant species (Formicidae, Hymenoptera) cause serious damage worldwide. Early detection of invasion and rapid management are significant for controlling these species. However, these attempts are sometimes hindered by the need for direct detection techniques, such as capture, visual observation, or morphological identification. In this study, we demonstrated that environmental DNA (eDNA) analysis can be used as a monitoring tool for alien ants using Linepithema humile (Argentine ant), one of the most invasive ants, as a model species. We designed a new real-time PCR assay specific to L. humile and successfully detected eDNA from the surface soil. The reliability of eDNA analysis was substantiated by comparing eDNA detection results with traditional survey results. Additionally, we examined the relationship between eDNA concentration and distance from nests and trails. Our results support the effectiveness of eDNA for alien ant monitoring and suggest that this new method could improve our ability to detect invasive ant species.

Molecular detection of giant snakeheads, Channa micropeltes (Cuvier, 1831), one of the most troublesome fish species.

A lack of reliable tools for determining the presence and distribution of fish species can impede understanding of predator-prey interactions and fishery management. Conventional fish survey methods are invasive, and can be size or species selective. Combining netting and electrofishing is a current method used to monitor fish species in Phayao Lake (Kwan Phayao), Thailand. However, the methods are inefficient and time-consuming. Recently, locals who rely on inland fisheries in Kwan Phayao expressed their deep concerns about the giant snakehead, Channa micropeltes (Cuvier, 1831) destroying other fish there. The giant snakehead prey on many commercially important fish species, as the prey species is reduced, negative effects on both biodiversity and the fishery sector could follow. Here, an eDNA-based survey was developed to detect the presence of the giant snakehead. Water samples were collected from six sites within Kwan Phayao and 17 sites in Ing River where water flowed into and out of Kwan Payao. The eDNA of the giant snakehead was detected in water samples from all collection sites using the developed qPCR assay with various concentrations. The eDNA was shown here to be a sensitive and reliable tool for fish surveillance so there will be a better chance for developing an effective management strategy.

Passive eDNA collection enhances aquatic biodiversity analysis.

Environmental DNA (eDNA) metabarcoding is a sensitive and widely used approach for species detection and biodiversity assessment. The most common eDNA collection method in aquatic systems is actively filtering water through a membrane, which is time consuming and requires specialized equipment. Ecological studies investigating species abundance or distribution often require more samples than can be practically collected with current filtration methods. Here we demonstrate how eDNA can be passively collected in both tropical and temperate marine systems by directly submerging filter membranes (positively charged nylon and non-charged cellulose ester) in the water column. Using a universal fish metabarcoding assay, we show that passive eDNA collection can detect fish as effectively as active eDNA filtration methods in temperate systems and can also provide similar estimates of total fish biodiversity. Furthermore, passive eDNA collection enables greater levels of biological sampling, which increases the range of ecological questions that eDNA metabarcoding can address.

Field application of an improved protocol for environmental DNA extraction, purification, and measurement using Sterivex filter.

Environmental DNA (eDNA) is increasingly popular as a useful non-invasive method to monitor and study biodiversity and community structure in freshwater and marine environments. To effectively extract eDNA from the filter surface is a fundamental factor determining the representativeness of the samples. We improved the eDNA extraction efficiency of an established Sterivex method by 12- to 16-fold using a larger volume of lysis buffer mix coupled with backflushing the cartridges. The DNeasy extraction column could be overloaded when the environmental sample input is high, possibly due to a higher nonspecific binding present in environmental samples, thus resulting in a relatively lower quantity measured. Therefore, we included an internal control DNA in the extraction to monitor the extraction and purification efficiencies in field samples, which is crucial for quantification of original eDNA concentration. The use of Takara Probe qPCR Mix supplemented with protein-based additives improved the robustness of the real time PCR assay on inhibitor-rich environmental samples, but prior purification by Qiagen PowerClean Pro Cleanup kit could be essential for inhibitor-rich water samples, even though the recovery rate was unexpectedly low (average 33.0%). The improved extraction and quantification complement the qualitative analyses including metabarcoding and metagenomics in field application.

Using environmental DNA methods to survey for rare groundwater fauna: Detection of an endangered endemic cave crayfish in northern Alabama.

The conservation and management of subterranean biodiversity is hindered by a lack of knowledge on the true distributions for many species, e.g., the Wallacean shortfall. In recent years, several studies have demonstrated the potential of environmental DNA (eDNA) as an effective approach to detect and monitor biodiversity, including rare, threatened, and endangered taxa. However, there are few eDNA studies of groundwater fauna. Here we report the results of the development and implementation of an eDNA assay targeting a short fragment of the mitochondrial CO1 locus of a critically imperiled cave crayfish, the Sweet Home Alabama Cave Crayfish (Cambarus speleocoopi), known from just four cave systems in the Interior Plateau karst region of northern Alabama. We detected C. speleocoopi DNA from water samples collected at 5 of 16 sites sampled (caves and springs), including two historical sites as well as three additional and potentially new sites in Marshall County, Alabama. All three of these sites were within 2 km of historical sites. Our study is the first to detect a groundwater crustacean in the Interior Plateau karst region. Additionally, our study contributes to the growing literature that eDNA is a viable complementary tool for detection and monitoring of a fauna that is difficult to survey and study using traditional approaches.

Assessment of fishery resources using environmental DNA: Small yellow croaker (Larimichthys polyactis) in East China Sea.

Species distribution monitoring and biomass assessment are crucial for fishery management and resource conservation. However, traditional methods such as motor trawling are costly and less effective than the novel environmental DNA (eDNA) approach. This study employs eDNA approach to investigate horizontal and vertical distributions of small yellow croaker (Larimichthys polyactis), an economically important species, in the East China Sea. The analysis of 171 eDNA samples collected from 44 stations using the species-specific primers and Taqman probe suggests a presence of small yellow croaker at 28 sampling layers in 44 stations. Significant differences in croaker eDNA concentrations were revealed among sampling stations and layers, consistent with previous findings through motor-trawl capture offshore and nearshore ichthyoplakton surveys, indicating small yellow croaker exhibits strong regional distribution and layer preference. In addition, we found a high eDNA concentration of small yellow croaker in the surface waters beyond the motor-trawl prohibition line, which confirms spawning grounds have been expanded from nearshore to offshore areas. Such expansion of spawning grounds could be a response by small yellow croaker to stressors such as overfishing, climate change, and nearshore environment contamination. To identify environmental variables potentially associated with small yellow croaker presence and absence, we conducted a correlation analysis between eDNA concentration and environmental variables, and the results provide a guideline for further investigation of fishery resources in the future. In conclusion, this study demonstrates the power of the eDNA approach in monitoring small yellow croaker at extensive geographic scales. The developed protocols and the findings are expected to assist in long-term monitoring and protection programs and benefit sustainable fishery in small yellow croaker.

Natrix: a Snakemake-based workflow for processing, clustering, and taxonomically assigning amplicon sequencing reads.

BACKGROUND: Sequencing of marker genes amplified from environmental samples, known as amplicon sequencing, allows us to resolve some of the hidden diversity and elucidate evolutionary relationships and ecological processes among complex microbial communities. The analysis of large numbers of samples at high sequencing depths generated by high throughput sequencing technologies requires efficient, flexible, and reproducible bioinformatics pipelines. Only a few existing workflows can be run in a user-friendly, scalable, and reproducible manner on different computing devices using an efficient workflow management system. RESULTS: We present Natrix, an open-source bioinformatics workflow for preprocessing raw amplicon sequencing data. The workflow contains all analysis steps from quality assessment, read assembly, dereplication, chimera detection, split-sample merging, sequence representative assignment (OTUs or ASVs) to the taxonomic assignment of sequence representatives. The workflow is written using Snakemake, a workflow management engine for developing data analysis workflows. In addition, Conda is used for version control. Thus, Snakemake ensures reproducibility and Conda offers version control of the utilized programs. The encapsulation of rules and their dependencies support hassle-free sharing of rules between workflows and easy adaptation and extension of existing workflows. Natrix is freely available on GitHub ( https://github.com/MW55/Natrix ) or as a Docker container on DockerHub ( https://hub.docker.com/r/mw55/natrix ). CONCLUSION: Natrix is a user-friendly and highly extensible workflow for processing Illumina amplicon data.

Got Glycogen?: Development and Multispecies Validation of the Novel Preserve, Precipitate, Lyse, Precipitate, Purify (PPLPP) Workflow for Environmental DNA Extraction from Longmire's Preserved Water Samples.

Unfiltered and filtered water samples can be used to collect environmental DNA (eDNA). We developed the novel "Preserve, Precipitate, Lyse, Precipitate, Purify" (PPLPP) workflow to efficiently extract eDNA from Longmire's preserved unfiltered and filtered water samples (44-100% recovery). The PPLPP workflow includes initial glycogen-aided isopropanol precipitation, guanidium hypochlorite and Triton X-100-based lysis, terminal glycogen-aided polyethylene glycol precipitation, and inhibitor purification. Three novel eDNA assays that exclusively target species invasive to Australia were also developed: Tilapia_v2_16S concurrently targets Oreochromis mossambicus (Mozambique tilapia) and Tilapia mariae (spotted tilapia) while R.marina_16S and C.caroliniana_matK discretely target Rhinella marina (cane toad) and Cabomba caroliniana (fanwort), respectively. All 3 assays were validated in silico before in vitro and in situ validations using PPLPP workflow extracted samples. PPLPP workflow was concurrently validated in vitro and in situ using all 3 assays. In vitro validations demonstrated that 1) glycogen inclusion increased extracellular DNA recovery by ∼48-fold compared with glycogen exclusion, 2) swinging-bucket centrifugation for 90 min at 3270 g is equivalent to fixed-angle centrifugation for 5-20 min at 6750 g, and 3) Zymo OneStep Inhibitor Removal Kit, Qiagen DNeasy PowerClean Pro Cleanup Kit, and silica-Zymo double purification provide effective inhibitor removal. In situ validation demonstrated 95.8 ± 2.8% (mean ± SEM) detectability across all 3 target species in Longmire's preserved unfiltered and filtered water samples extracted using the PPLPP workflow (without phenol:chloroform:isoamyl alcohol purification) after 39 d of incubation at room temperature and 50°C. PPLPP workflow is recommended for future temperate and tropical eDNA studies that use Longmire's to preserve unfiltered or filtered water samples.

A duplex real-time PCR with probe for simultaneous detection of Geosmithia morbida and its vector Pityophthorus juglandis.

The cultivation of walnuts (Juglans sp.) in Europe retains high economic, social, and environmental value. The recent reporting of the Thousand Cankers Disease (TCD) fungus, Geosmithia morbida, and of its vector, Pityophthorus juglandis, in walnut trees in Italy is alarming the whole of Europe. Although Italy is at present the only foothold of the disease outside North America, given the difficulties inherent in traditional identification of both members of this beetle/fungus complex, a rapid and effective protocol for the early detection and identification of TCD organisms is an absolute priority for Europe. Here we report the development of an effective and sensitive molecular tool based on simplex/duplex qPCR assays for the rapid, accurate and highly specific detection of both the bionectriaceous fungal pathogen and its bark-beetle vector. Our assay performed excellently, detecting minute amounts of target DNA without any non-specific amplification. Detection limits from various and heterogeneous matrices were lower than other reported assays. Our molecular protocol could assist in TCD organism interception at entry points, territory monitoring for the early identification and eradication of outbreaks, delineation of quarantine areas, and tracing back TCD entry and dispersal pathways.

Gene-informed decomposition model predicts lower soil carbon loss due to persistent microbial adaptation to warming.

Soil microbial respiration is an important source of uncertainty in projecting future climate and carbon (C) cycle feedbacks. However, its feedbacks to climate warming and underlying microbial mechanisms are still poorly understood. Here we show that the temperature sensitivity of soil microbial respiration (Q10) in a temperate grassland ecosystem persistently decreases by 12.0 ± 3.7% across 7 years of warming. Also, the shifts of microbial communities play critical roles in regulating thermal adaptation of soil respiration. Incorporating microbial functional gene abundance data into a microbially-enabled ecosystem model significantly improves the modeling performance of soil microbial respiration by 5-19%, and reduces model parametric uncertainty by 55-71%. In addition, modeling analyses show that the microbial thermal adaptation can lead to considerably less heterotrophic respiration (11.6 ± 7.5%), and hence less soil C loss. If such microbially mediated dampening effects occur generally across different spatial and temporal scales, the potential positive feedback of soil microbial respiration in response to climate warming may be less than previously predicted.

Pooled samples and eDNA-based detection can facilitate the "clean trade" of aquatic animals.

The regional and international trade of live animals facilitates the movement, spillover, and emergence of zoonotic and epizootic pathogens around the world. Detecting pathogens in trade is critical for preventing their continued movement and introduction, but screening a sufficient fraction to ensure rare infections are detected is simply infeasible for many taxa and settings because of the vast numbers of animals involved-hundreds of millions of live animals are imported into the U.S.A. alone every year. Batch processing pools of individual samples or using environmental DNA (eDNA)-the genetic material shed into an organism's environment-collected from whole consignments of animals may substantially reduce the time and cost associated with pathogen surveillance. Both approaches, however, lack a framework with which to determine sampling requirements and interpret results. Here I present formulae for pooled individual samples (e.g,. swabs) and eDNA samples collected from finite populations and discuss key assumptions and considerations for their use with a focus on detecting Batrachochytrium salamandrivorans, an emerging pathogen that threatens global salamander diversity. While empirical validation is key, these formulae illustrate the potential for eDNA-based detection in particular to reduce sample sizes and help bring clean trade into reach for a greater number of taxa, places, and contexts.