Advancing the use of molecular methods for routine freshwater macroinvertebrate biomonitoring : the need for calibration experiments

Over the last decade, steady advancements have been made in the use of DNA-based methods for detection of species in a wide range of ecosystems. This progress has culminated in molecular monitoring methods being employed for the detection of several species for enforceable management purposes of endangered, invasive, and illegally harvested species worldwide. However, the routine application of DNA-based methods to monitor whole communities (typically a metabarcoding approach) in order to assess the status of ecosystems continues to be limited. In aquatic ecosystems, the limited use is particularly true for macroinvertebrate communities. As part of the DNAqua-Net consortium, a structured discussion was initiated with the aim to identify potential molecular methods for freshwater macroinvertebrate community assessment and identify important knowledge gaps for their routine application. We focus on three complementary DNA sources that can be metabarcoded: 1) DNA from homogenised samples (bulk DNA), 2) DNA extracted from sample preservative (fixative DNA), and 3) environmental DNA (eDNA) from water or sediment. We provide a brief overview of metabarcoding macroinvertebrate communities from each DNA source and identify challenges for their application to routine monitoring. To advance the utilisation of DNA-based monitoring for macroinvertebrates, we propose an experimental design template for a series of methodological calibration tests. The template compares sources of DNA with the goal of identifying the effects of molecular processing steps on precision and accuracy. Furthermore, the same samples will be morphologically analysed, which will enable the benchmarking of molecular to traditional processing approaches. In doing so we hope to highlight pathways for the development of DNA-based methods for the monitoring of freshwater macroinvertebrates

eDNA is a useful tool to evaluate the success of the eradication program of Xenopus laevis in Portugal

Biological invasions are widely recognized as a major driver of global biodiversity loss. The most cost-effective answer is often population eradication, while the number of individuals is still limited. The detection of the invasive species at low densities is essential for eradication success, which can be difficult using traditional methods. Environmental DNA (eDNA) can facilitate the detection and monitoring of invasive aquatic species at low densities and can be more sensitive than traditional sampling. The African clawed frog (Xenopus laevis) is a highly invasive species and has been recorded in several European countries. In Portugal, the species was discovered in 2006 and since 2010, streams of the Lisbon area, suspected or at risk of invasion, are being monitored every year and removal is ongoing. The main method of detection is electrofishing but methods like draining artificial ponds have also been used. This program so far succeeded in containing the spread and reduced frog abundance, although total eradication has not yet been accomplished. To evaluate the success of this program, we collected water samples from fifteen sites. In each site we sampled both pools and riifles to test if lotic microhabitat influences X. laevis eDNA detection and concentration. Surface water velocity in each sampling point was also measured. We filtered the samples, extracted DNA from filters, and assayed the extracted DNA for X. laevis DNA using quantitative polymerase chain reaction (qPCR). From the fifteen sites sampled, five were positive for X. laevis eDNA: four lotic sites and one lentic site. Local eradication success was evident mainly in lentic habitats, and three potential failures were also identified. Finding X. laevis at one of these sites only after X. laevis eDNA detection was an important contribution of this technique for the success of the control and eradication program. No significative differences were found between lotic microhabitat (riffles and pools) regarding X. laevis eDNA concentration and detection but velocity affected the concentration of X. laevis eDNA captured. Our results corroborate other studies that recommend eDNA as a complementary sampling approach to other traditional methods and suggests its suitability as a tool for the detection of invasive amphibians.To be presented as a flash oral presentation at the DNAQUA International Conference - First international conference on the use of DNA for water biomonitoring

Species detection from aquatic eDNA: assessing the importance of capture methods

Original ResearchEnvironmental DNA (eDNA) is increasingly used for biodiversity monitoring, particularly in aquatic systems. However, each step, from sample collection to bioinformatic analysis, can introduce biases and influence the reliability of results. While much effort has been put into the optimization of laboratory methods, less attention has been devoted to estimate the impacts of eDNA capture methods. To address this issue, water samples were collected at nine small ponds and puddles where up to 10 amphibian species occur, using precipitation, disc filters, and capsules. We focused on targeted detection of an amphibian species, Salamandra salamandra, and on the composition of the whole amphibian community. Species detection was performed using a novel qPCR assay for S. salamandra and high-throughput sequencing, combined with stringent versus relaxed PCR replication thresholds. Filtration techniques (disc filters and capsules) outperformed precipitation, generating a higher number of detections of S. salamandra and higher amounts of captured eDNA, while species detection was identical between disc filters and capsules. There were no significant differences between capture methods regarding amphibian community composition. The variation in detection success associated with capture methods was far higher than that associated with PCR replication, regardless of the detection method used. Our results highlight the importance of choosing a suitable capture method for eDNA studies and suggest that the choice of capture method outweighs the choice of detection method used. To the best of our knowledge, this is the first study to compare high-capacity capsules with common eDNA methods for water samples, such as precipitation and standard disc filtersinfo:eu-repo/semantics/publishedVersio

A web-based tool to standardise reporting and interpret results of eDNA qPCR assays

Environmental DNA, or eDNA, methodologies can enable the rapid detection of a target species without either visual or physical confirmation of the species presence. Over the last decade, targeted quantitative PCR (qPCR) assays have become an increasingly useful method employed by government and non-government agencies alike for purposes such as protecting and preserving ecosystems from invasive species, or for the conservation of endangered species. As the application of eDNA to answer ecological questions pushes the limits of qPCR-based detection, there is a pressing need to standardise the way qPCR results are reported and interpreted, as well as the way qPCR assays are evaluated for use outside of the remit of the original study.Natural England is one such government agency who have begun to use eDNA methodologies more widely to answer ecological questions. However, while some qPCR assays available for detecting the presence or absence of species such as the great crested newt (Triturus cristatus) have been specified, validated and quality assured to a high degree (Biggs et al. 2014), existing qPCR assays for other species are generally less well developed and validated. Additionally, for some species there are multiple qPCR assays available, with each being developed and validated to different stages. As such, Natural England identified a need to understand how the data derived from eDNA should be interpreted dependent on the level of qPCR assay development, and ultimately the confidence they can have in the accuracy of resulting data, including the associated risk of false positives or false negatives.NatureMetrics has developed a prototype web-based tool and protocol (European Technical Readiness Level 5) which would enable end users such as Natural England to inform their interpretation of qPCR results. The prototype is currently in the beta testing stage and is expected to be available in the coming months. The web-based tool will simplify qPCR assay evaluation, enabling end users to select the most appropriate qPCR assay for their needs, as well as standardise the reporting and interpretation of their qPCR results by generating a report at both the sample and site level from the inputted qPCR data

Using meta-barcoding tools to monitor primate meat consumption at dedicated establishments in Guinea-Bissau, West Africa

Guinea-Bissau (GB) is a regional stronghold for primate conservation. Ten primates occur in the country, including the Western chimpanzee (Pan troglodytes verus) and two colobus monkeys (Colobus polykomos and Piliocolobus badius temminckii). Primate meat is consumed at households and bushmeat-dedicated establishments, locally named "Abafatório". Such establishments are mentioned to be common in urban areas since the 1980s and to be specialized in serving primate meat while drinking alcoholic beverages. The meat is typically cooked in a stew and eaten with bread. However, as the trade and consumption of primate meat are illegal activities, the location of Abafatório establishments and details of the trade, namely species being consumed, are usually hidden from outsiders. Here, we characterize illicit bushmeat commerce and consumption at six Abafatórios of a small town. Our team visited the establishments every week for 15 months (2015-2017) and collected data on the type and prices of meals and gathered tissue samples taken from carcasses by establishment owners. A meta-barcoding approach (cytb and 12S mitochondrial DNA regions and Illumina MiSeq next-generation sequencing technology) was used to identify tissue samples to the species level. Two types of establishments can be distinguished – “restaurants” and “snack-bars”. Restaurants are similar to the ones found by previous works in the capital city where primate meat is sold as a dish containing few pieces of stewed meat. Snack-bars are smaller and the meat is sold inexpensively and by the piece. In the present study, 249 tissue samples were identified to be from four primates (Cercopithecus campbelli, Chlorocebus sabaeus, Papio papio, and Erythrocebus patas) and four Artiodactyla (Philantomba maxwellii, Tragelaphus scriptus, Potamochoerus porcus and Phacochoerus africanus). Primates represented approximately 92% of all species consumed across establishments, and C. campbelli was the most traded species. Our work suggests that primate meat is monetarily accessible for locals in rural areas and that the trade at Abafatórios may have extensive negative consequences to primate conservation, in particular, the reduction of primates' populations in the southern part of GB. Our work quantifies and identifies the species consumed in Abafatório establishments for the first time and highlights the need to improve regulation and law enforcement in Guinea-Bissau

Data from: Using molecular diet analysis to inform invasive species management: a case study of introduced rats consuming endemic New Zealand frogs

The decline of amphibians has been of international concern for more than two decades and the global spread of introduced fauna is a major factor in this decline. Conservation management decisions to implement control of introduced fauna are often based on diet studies. One of the most common metrics to report in diet studies is Frequency of Occurrence (FO), but this can be difficult to interpret, as it does not include a temporal perspective. Here we examine the potential for FO data derived from molecular diet analysis to inform invasive species management, using invasive ship rats (Rattus rattus) and endemic frogs (Leiopelma spp.) in New Zealand as a case study. Only two endemic frog species persist on the mainland. One of these, Leiopelma archeyi, is Critically Endangered (IUCN, 2017) and ranked as the world´s most evolutionarily distinct and globally endangered amphibian (EDGE, 2018). Ship rat stomach contents were collected by kill-trapping and subjected to three methods of diet analysis (one morphological and two DNA-based). A new primer pair was developed targeting all anuran species that exhibits good coverage, high taxonomic resolution and reasonable specificity. Incorporating a temporal parameter allowed us to calculate the minimum number of ingestion events per rat per night, providing a more intuitive metric than the more commonly reported FO. We are not aware of other DNA-based diet studies that have incorporated a temporal parameter into FO data. The usefulness of such a metric will depend on the study system, in particular the feeding ecology of the predator. Ship rats are consuming both species of native frogs present on mainland New Zealand and this study provides the first detections of remains of these species in mammalian stomach contents

Frog tissue sanger sequences generated by EGETER-2019-16S primer pair

Frog tissue sanger sequences generated by EGETER-2019-16S primer pai

Metabarcoding with MinION: Speeding up the detection of invasive aquatic species using environmental DNA and nanopore sequencing

Traditional detection of aquatic invasive species, via morphological identification is often time-consuming and can require a high level of taxonomic expertise, leading to delayed mitigation responses. Environmental DNA (eDNA) detection approaches of multiple species using Illumina-based sequencing technology have been used to overcome these hindrances, but sample processing is often lengthy. More recently, portable nanopore sequencing technology has become available, which has the potential to make molecular detection of invasive species more widely accessible and to substantially decrease sample turnaround times. However, nanopore-sequenced reads have a much higher error rate than those produced by Illumina platforms, which has so far hindered the adoption of this technology. We provide a detailed laboratory protocol and bioinformatic tools to increase the reliability of nanopore sequencing to detect invasive species, and we test its application using invasive bivalves. We sampled water from sites with pre-existing bivalve occurrence and abundance data, and contrasting bivalve communities, in Italy and Portugal. We extracted, amplified and sequenced eDNA with a turnaround of 3.5 days. The majority of processed reads were ≥ 99 % identical to reference sequences. There were no taxa detected other than those known to occur. The lack of detections of some species at some sites could be explained by their known low abundances. The approach is now being tested on other target taxa such as fish and other vertebrates.