Detection of environmental DNA (eDNA) has become a commonly used surveillance method for threatened or invasive vertebrates in both aquatic and terrestrial environments. However, use of eDNA methodologies for the detection of aquatic invertebrates (e.g., crayfish and insects) has been limited. 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 the 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 and field conditions. In this study we examined the persistence and accumulation of eDNA from captive S. hineana larvae in experimental mesocosms at temperatures (5.0°C and 16.0°C) that reflect seasonal variation in their natural habitat, and we evaluated the usefulness of eDNA protocols for studying the distribution and abundance of invertebrates by assessing patterns of eDNA distribution for the Hine’s emerald dragonfly and its symbiont the devil crayfish, (Cambarus [=Lacunicambarus] diogenes) in the field over several months. In mesocosms, S. hineana eDNA persisted longer at 5.0°C but accumulated more readily at 16.0°C. In the field, life-history events affected seasonal variations in eDNA more significantly and consistently than temperature for both species. These data can be used to aid in conservation efforts for S. hineana and similar aquatic invertebrates.

This data set is comprised of one table with sampling information and NCBI BioProject accession numbers for sequence information of this amplicon-based study targeting Labyrinthula, Phytophthora, and Halophytophthora of known pathogenic and non-pathogenic [to eelgrass (Zostera marina)] strains from eDNA samples. eDNA samples included water, sediment, and eelgrass from Notsuke Wan (Cove) Japan, Safety Sound, Izembek Lagoon, Port Moller, Chignik Lagoon, and Frederick Sound, Alaska and cloacal swabs from waterfowl hunted near Cold Bay Alaska. Replicate samples and multiple sampling dates of the same location were included. Highly conserved primers which could differentiate species of interest were developed for four portions of mtDNA genes (5.8S, 18S, ITS, and COI). The reference list and conserved primer sets to identify species present were developed using publicly available data https://www.ncbi.nlm.nih.gov/genbank/

This data set is comprised of one table with sampling information and NCBI BioProject accession numbers for sequence information of this amplicon-based study targeting Labyrinthula, Phytophthora, and Halophytophthora of known pathogenic and non-pathogenic [to eelgrass (Zostera marina)] strains from eDNA samples. eDNA samples included water, sediment, and eelgrass from Notsuke Wan (Cove) Japan, Safety Sound, Izembek Lagoon, Port Moller, Chignik Lagoon, and Frederick Sound, Alaska and cloacal swabs from waterfowl hunted near Cold Bay Alaska. Replicate samples and multiple sampling dates of the same location were included. Highly conserved primers which could differentiate species of interest were developed for four portions of mtDNA genes (5.8S, 18S, ITS, and COI). The reference list and conserved primer sets to identify species present were developed using publicly available data https://www.ncbi.nlm.nih.gov/genbank/

U.S Fish and Wildlife Services (USFWS) Lower Great Lakes Fish and Wildlife Conservation Office (LGLFWCO) Aquatic Invasive Species (AIS) Early Detection and Monitoring (EDM) program began in 2012 following the onset of Great Lakes Restoration Initiative (GLRI) funding. This program is apart of a larger basin-wide Great Lakes AIS EDM effort by the USFWS. The goal of this program is to detect novel AIS species (fish and aquatic invertebrates) by sampling a diversity of habitats with a wide array of gear types. Sampling focuses on harbors, rivers and tributaries of the lower Great Lakes (Erie and Ontario) as determined by a risk-based prioritization framework for AIS in the Great Lakes. The program is composed of three components of AIS surveillance: juvenile/adult fish, ichthyoplankton (larval fish), and aquatic invertebrates (including crayfish). Sampling strategies and protocols are analyzed and designed to capture the maximum species richness at locations with the assumption that capturing an abundance of species, including singletons and doubletons, could lead to detecting rare novel AIS species if present. Protocols vary depending on the component of the program and descriptions can be found within the metadata of each dataset. This program is adaptive in nature and standardization, although important for comparisons, is not the primary intention of this AIS sampling strategy. Any significant AIS detections are reported to partners following an internal communications protocol.

U.S Fish and Wildlife Services (USFWS) Lower Great Lakes Fish and Wildlife Conservation Office (LGLFWCO) Aquatic Invasive Species (AIS) Early Detection and Monitoring (EDM) program began in 2012 following the onset of Great Lakes Restoration Initiative (GLRI) funding. This program is apart of a larger basin-wide Great Lakes AIS EDM effort by the USFWS. The goal of this program is to detect novel AIS species (fish and aquatic invertebrates) by sampling a diversity of habitats with a wide array of gear types. Sampling focuses on harbors, rivers and tributaries of the lower Great Lakes (Erie and Ontario) as determined by a risk-based prioritization framework for AIS in the Great Lakes. The program is composed of three components of AIS surveillance: juvenile/adult fish, ichthyoplankton (larval fish), and aquatic invertebrates (including crayfish). Sampling strategies and protocols are analyzed and designed to capture the maximum species richness at locations with the assumption that capturing an abundance of species, including singletons and doubletons, could lead to detecting rare novel AIS species if present. Protocols vary depending on the component of the program and descriptions can be found within the metadata of each dataset. This program is adaptive in nature and standardization, although important for comparisons, is not the primary intention of this AIS sampling strategy. Any significant AIS detections are reported to partners following an internal communications protocol.

Benthic invertebrate surveys are a component of the U.S. Fish and Wildlife Services (USFWS) Alpena Fish and Wildlife Conservation Office (FWCO) Aquatic Invasive Species (AIS) Early Detection and Monitoring (EDM) Program. Benthic invertebrate surveys were included in the EDM program starting in 2014 with the goal of detecting novel AIS invertebrate species by sampling a diversity of habitats with a wide array of gear types. Surveys occurred annually from 2013 - 2024. Sampling focuses on harbors, rivers, and streams of Lake Huron and Lake Erie as determined by a risk-based prioritization framework for AIS in the Great Lakes. Benthic invertebrates are collected using a variety of sampling gears with the most utilized including: colonizers (rock bags and Hester-Dendy samplers) and sweep nets. The survey design targets the following three general taxon: Amphipoda, Bivalvia, and Gastropoda. These taxon are targeted due to the potential invasiveness and level of concern of species within those taxa as determined by Ecological Risk Screening Summaries (ERSS). The sampling season is typically between July and October - not all locations are sampled on a similar temporal scale, particularly those locations on the fringes of the programs surveillance range. Sampling strategies are analyzed and designed to capture the maximum species richness at locations with the assumption that capturing an abundance of species, including singletons and doubletons, could lead to detecting rare novel AIS species if present. Samples are preserved using 95% ethanol (EtOH) which allows for tissue preservation. The samples are then picked and sorted in-house (Alpena FWCO) by the three aforementioned general taxon. For several samples, these taxa groupings were screen for Region 3 watch list/target species. Some targeted sampling sites was sent to an external contractor for taxonomic identification a year or more later once funds became available. Given the dynamic and adaptive nature of AIS early detection, the list of watch list species has changed over time. The current watch list of invertebrates is as follows: Signal Crayfish, Spinycheek Crayfish, Yabby, Australian Redclaw Crayfish, Marbled Crayfish, Noble Crayfish, Red Swamp Crayfish, Harris Mud Crab, Bald Urchin Shrimp, Bloody Red Shrimp, Demon Shrimp, Killer Shrimp, Pontogammarus robustoides (no common name), Scud, Banded Mysterysnail, Chinese Mysterysnail, European Ear Snail, European Stream Valvata, Golden Mussel, Gravel Snail, Japanese Mysterysnail, Mud Bithynia (Faucet Snail), New Zealand Mudsnail, Quagga Mussel, and Zebra Mussel. Quagga and Zebra mussels are widespread in Lake Erie and Lake Huron and, with the exception of the St. Marys River, are no longer sorted or counted as bivalves because of the large number and frequency. The information within this dataset is geospatial in nature and documents benthic invertebrate sampling events. Both abiotic and biotic data is collected for each individual sampling event. It is possible that over time, the tools, gears, and instruments used to collect information have changed or been modified. The EDM Program is adaptive in nature and standardization, although important for comparisons, is not the primary intention of this AIS sampling strategy. Therefore, in some instances, the general statements made above regarding sample procedures may not apply. Also, identifying aquatic invertebrates such as amphipods, bivalves, and gastropods are difficult - especially when tasked with discovering novel AIS with limited support from reference materials (keys, voucher specimens, etc.). Because of this, some specimens collected cannot be assigned to the species level. Furthermore, due to these difficulties, identifications within this data set may be inaccurate and those records will remain within this data set unless otherwise detected and removed.

Benthic invertebrate surveys are a component of the U.S. Fish and Wildlife Services (USFWS) Alpena Fish and Wildlife Conservation Office (FWCO) Aquatic Invasive Species (AIS) Early Detection and Monitoring (EDM) Program. Benthic invertebrate surveys were included in the EDM program starting in 2014 with the goal of detecting novel AIS invertebrate species by sampling a diversity of habitats with a wide array of gear types. Surveys occurred annually from 2013 - 2024. Sampling focuses on harbors, rivers, and streams of Lake Huron and Lake Erie as determined by a risk-based prioritization framework for AIS in the Great Lakes. Benthic invertebrates are collected using a variety of sampling gears with the most utilized including: colonizers (rock bags and Hester-Dendy samplers) and sweep nets. The survey design targets the following three general taxon: Amphipoda, Bivalvia, and Gastropoda. These taxon are targeted due to the potential invasiveness and level of concern of species within those taxa as determined by Ecological Risk Screening Summaries (ERSS). The sampling season is typically between July and October - not all locations are sampled on a similar temporal scale, particularly those locations on the fringes of the programs surveillance range. Sampling strategies are analyzed and designed to capture the maximum species richness at locations with the assumption that capturing an abundance of species, including singletons and doubletons, could lead to detecting rare novel AIS species if present. Samples are preserved using 95% ethanol (EtOH) which allows for tissue preservation. The samples are then picked and sorted in-house (Alpena FWCO) by the three aforementioned general taxon. For several samples, these taxa groupings were screen for Region 3 watch list/target species. Some targeted sampling sites was sent to an external contractor for taxonomic identification a year or more later once funds became available. Given the dynamic and adaptive nature of AIS early detection, the list of watch list species has changed over time. The current watch list of invertebrates is as follows: Signal Crayfish, Spinycheek Crayfish, Yabby, Australian Redclaw Crayfish, Marbled Crayfish, Noble Crayfish, Red Swamp Crayfish, Harris Mud Crab, Bald Urchin Shrimp, Bloody Red Shrimp, Demon Shrimp, Killer Shrimp, Pontogammarus robustoides (no common name), Scud, Banded Mysterysnail, Chinese Mysterysnail, European Ear Snail, European Stream Valvata, Golden Mussel, Gravel Snail, Japanese Mysterysnail, Mud Bithynia (Faucet Snail), New Zealand Mudsnail, Quagga Mussel, and Zebra Mussel. Quagga and Zebra mussels are widespread in Lake Erie and Lake Huron and, with the exception of the St. Marys River, are no longer sorted or counted as bivalves because of the large number and frequency. The information within this dataset is geospatial in nature and documents benthic invertebrate sampling events. Both abiotic and biotic data is collected for each individual sampling event. It is possible that over time, the tools, gears, and instruments used to collect information have changed or been modified. The EDM Program is adaptive in nature and standardization, although important for comparisons, is not the primary intention of this AIS sampling strategy. Therefore, in some instances, the general statements made above regarding sample procedures may not apply. Also, identifying aquatic invertebrates such as amphipods, bivalves, and gastropods are difficult - especially when tasked with discovering novel AIS with limited support from reference materials (keys, voucher specimens, etc.). Because of this, some specimens collected cannot be assigned to the species level. Furthermore, due to these difficulties, identifications within this data set may be inaccurate and those records will remain within this data set unless otherwise detected and removed.

Environmental DNA (eDNA) surveys have become important tools for monitoring aquatic biodiversity. Barcode sequencing of eDNA generates community profiles that, while potentially biased in both capture and amplification, can nonetheless yield high information content per unit cost. While factors affecting eDNA capture and amplification have been heavily studied, watershed-scale assessments of fish communities and our confidence in such have been less frequent. We performed an initial watershed-scale characterization of fish eDNA using rapid, low-volume filtering with replicate and control samples scaled for a single Illumina MiSeq flow cell, using the mitochondrial 12S ribosomal RNA locus for taxonomic profiling. Our bioinformatic approach included 1) direct estimation of sequencing error from unambiguous mappings (alignments) and simulation of error in taxonomic assignment under various mapping criteria; 2) binning of species based on inferred assignment error rather than by taxonomic rank; and 3) visualization of mismatch distributions to facilitate discovery of distinct haplotypes attributed to the same reference. Our approach was implemented for the St. Regis River, New York, United States, which supports a valuable recreational fishery and has been a target of restoration activities. We used a large record of St. Regis-specific observations to validate our assignments. We found that 300 mL drawn through 25-mm filters yielded greater than 5 ng/µL DNA at most sites in August and September, which was an approximate threshold for generating strong sequencing libraries in our hands. Using inferred sequence error rates, we binned 12S references for 110 species on a state-level checklist into 85 single-species bins and seven multispecies bins. Of 48 taxonomic bins actually observed in the St. Regis, we detected eDNA consistent with 40, with an additional four detections flagged as potential contaminants post-collection. Sixteen unobserved species detected by eDNA ranged from plausible to implausible based on distributional data, whereas six observed species had no 12S reference sequence.

Environmental DNA (eDNA) surveys have become important tools for monitoring aquatic biodiversity. Barcode sequencing of eDNA generates community profiles that, while potentially biased in both capture and amplification, can nonetheless yield high information content per unit cost. While factors affecting eDNA capture and amplification have been heavily studied, watershed-scale assessments of fish communities and our confidence in such have been less frequent. We performed an initial watershed-scale characterization of fish eDNA using rapid, low-volume filtering with replicate and control samples scaled for a single Illumina MiSeq flow cell, using the mitochondrial 12S ribosomal RNA locus for taxonomic profiling. Our bioinformatic approach included 1) direct estimation of sequencing error from unambiguous mappings (alignments) and simulation of error in taxonomic assignment under various mapping criteria; 2) binning of species based on inferred assignment error rather than by taxonomic rank; and 3) visualization of mismatch distributions to facilitate discovery of distinct haplotypes attributed to the same reference. Our approach was implemented for the St. Regis River, New York, United States, which supports a valuable recreational fishery and has been a target of restoration activities. We used a large record of St. Regis-specific observations to validate our assignments. We found that 300 mL drawn through 25-mm filters yielded greater than 5 ng/µL DNA at most sites in August and September, which was an approximate threshold for generating strong sequencing libraries in our hands. Using inferred sequence error rates, we binned 12S references for 110 species on a state-level checklist into 85 single-species bins and seven multispecies bins. Of 48 taxonomic bins actually observed in the St. Regis, we detected eDNA consistent with 40, with an additional four detections flagged as potential contaminants post-collection. Sixteen unobserved species detected by eDNA ranged from plausible to implausible based on distributional data, whereas six observed species had no 12S reference sequence.

Quantitative PCR results for surveillance of New Zealand mudsnail environmental DNA at the U.S. Fish and Wildlife Service Alchesay National Fish Hatchery.