Environmental DNA (eDNA) monitoring has been used for great crested newt (Triturus cristatus) survey in the UK since the publication of a Defra-funded trial in 2014. If eDNA results are to be used in support of a great crested newt licence, surveys must be performed during a 76-day survey window (15 April-30 June) to coincide with peak great crested newt activity, and must follow the approved ethanol precipitation protocol. However, eDNA detection is possible in other months and filtration may be a more effective method of eDNA capture. We investigated whether the great crested newt eDNA survey season could be extended and filtration could be used for great crested newt eDNA capture by reviewing the available evidence and conducting a field study from April to October 2022. Paired water samples for ethanol precipitation and filtration were collected from 25 ponds once a month, resulting in 124 samples of each type. All samples (N = 248) were analysed with the approved great crested newt quantitative PCR assay. Our results indicate that great crested newts can be reliably detected using both eDNA capture methods from April to August, with detection rates decreasing in September and October. Great crested newt eDNA detection was comparable or higher with filtration than ethanol precipitation. Practical implication. Acceptance of filtration for great crested newt eDNA surveys could allow more water to be processed for robust and reliable estimates of great crested newt presence. Extending the great crested newt eDNA survey season to August could allow more waterbodies to be surveyed for great crested newt presence (but not absence), and identification of sites that provide important habitat for great crested newts outside of the breeding season. This would also remove logistical challenges and costs associated with completing sampling within 11 weeks and laboratory analysis within 10 working days from sample receipt. Furthermore, great crested newt eDNA surveys could be more frequently carried out alongside monitoring for other species, which are typically surveyed from April to September/October or year-round with conventional methods or eDNA surveys using filtration. This could enable infrastructure projects to develop more effective mitigation measures as well as reduce time required from surveyors and survey costs.

Abstract Cost‐effective and accurate quantification of biodiversity is important for biodiversity conservation, resource management, and forecasting. Traditional monitoring approaches have relied on direct observations, remote sensing, and mark‐recapture techniques, providing insights into species ecology and the impact of pollution and climate change on indicator species. However, these techniques are typically low throughput, expensive, and can be invasive. In addition, they cannot detect cryptic diversity and are biased toward species that leave identifiable remains. DNA‐based methods, such as metabarcoding or single marker gene assays, have enabled high throughput screening of a wide range of taxonomic groups, including ones without well‐preserved remains. When compared with traditional techniques, these approaches have high throughput, can resolve cryptic diversity, do not require taxonomic specialist skills, and are non‐invasive. However, although they are comparatively cheaper than traditional approaches, they are expensive when applied at the community‐level as single marker assays are amplified and sequenced independently. Multilocus approaches in which multiple gene markers are amplified in a single reaction are desirable to deliver community‐level assessments in a cost‐effective manner. Yet, they are uncommon because of technical challenges that may lead to biases in downstream analyses, such as index hopping and unbalanced representation of taxonomic groups. Here, we developed a highly multiplexed protocol that combines the early pooling of marker genes that target broad taxonomic groups and taxon‐specific markers in a single tube reaction. This step is followed by the pooling of up to 384 samples per locus (N = 15,636 samples) with unique dual‐indexed sequencing adapters in a single sequencing run. This approach dramatically reduces the costs of community‐level biodiversity quantification and lowers the need for input DNA without compromising output quality. We optimized the multiplex assay on lake freshwater sediment samples and benchmarked the assay on samples from other environmental matrices, demonstrating its direct application to the river and marine communities.

Aquatic environmental DNA is increasingly used for biodiversity monitoring, such as surveying threatened and invasive species. Mainstreaming these methods in practical applications, however, still requires significant standardization and optimisation, namely regarding DNA capture methods. Here we evaluated how filter type (standard disc filters vs high-capacity capsules), number of sampling sites, volume of water filtered and environmental factors affected amphibian detection in Mediterranean temporary ponds. The study involved water filtering until clogging at one (capsules) and five (discs) sites from 16 small and shallow ponds, where three urodele and seven anuran species were recorded through sweep-netting and adult observations. Detection probabilities were estimated from site occupancy models based on replicate sampling and from an adaptation of time-to-detection models relating detection probability to volume of water filtered. Discs filtered relatively small volumes (15-1250 mL), with detection probabilities of the two abundant species (Pelobates cultripes, Hyla meridionalis) increasing rapidly with sample size and water volume, reaching almost perfect detection (0.95) at four and seven discs, and 420 mL and 1860 mL, respectively. However, reaching high detection probabilities for rare species (Pelodytes atlanticus, Pleurodeles waltl, Triturus pygmaeus) would require larger sampling effort than that used in our study. Despite filtering much larger volumes (600-5300 mL), filtering with capsules at a single site per pond provided lower detection probabilities for abundant species than filtering with discs at five sites. Rarer species showed no difference between methods, which may be due to small sample sizes and reduced statistical power for species with few detections. The effect of conductivity on species detectability was largely negative, while the influence of water clarity varied across species, and pH had no effects. Overall, our results suggest that eDNA amphibian surveys in Mediterranean temporary ponds need to consider filter clogging, heterogeneous DNA distribution, and highly conductive waters.

Various nutrient recycling technologies are currently under development in order to alleviate the dependency of non-renewable raw material for the production of mineral phosphorus fertilizers commonly used in agriculture. The resulting products, such as struvites and ashes, need to be assessed for their application as so-called recycling-derived fertilizers (RDFs) in the agricultural sector prior to commercialization. Here, we conducted a short-term (54 days) trial to investigate the impact of different phosphorus fertilizers on plant growth and the soil P cycling microbiota. Lolium perenne was grown with application of superphosphate (SP) as inorganic fertilizer, two ashes (poultry litter ash (PLA) and sewage sludge ash (SSA)), and two struvites (municipal wastewater struvite (MWS) and commercial CrystalGreen® (CGS)) applied at 20 and 60 kg P ha-1 in four replicates. A P-free control (SP0) was also included in the trial. Struvite application increased plant dry weights, and available P acid phosphatase activity was significantly improved for struvites at the high P application rate. The ash RDFs showed a liming effect at 60 kg P ha-1, and PLA60 negatively affected acid phosphatase activity, while PLA20 had significantly lower phoD copy numbers. P mobilization from phosphonates and phytates was not affected. TCP solubilization was negatively affected by mineral SP fertilizer application at both P concentrations. The bacterial (16S and phoD) communities were only marginally affected by the tested P fertilizers. Overall, struvites appeared to be a suitable substitute for superphosphate fertilization for Irish L. perenne pastures.

Mosquito surveillance programmes are essential to assess the risks of local vector-borne disease outbreaks as well as for early detection of mosquito invasion events. Surveys are usually performed with traditional sampling tools (i.e., ovitraps and dipping method for immature stages or light or decoy traps for adults). Over the past decade, numerous studies have highlighted that environmental DNA (eDNA) sampling can enhance invertebrate species detection and provide community composition metrics. However, the usefulness of eDNA for detection of mosquito species has, to date, been largely neglected. Here, we sampled water from potential larval breeding sites along a gradient of anthropogenic perturbations, from the core of an oil palm plantation to the rainforest on São Tomé Island (Gulf of Guinea, Africa). We showed that (i) species of mosquitoes could be detected via metabarcoding mostly when larvae were visible, (ii) larvae species richness was greater using eDNA than visual identification and (iii) new mosquito species were also detected by the eDNA approach. We provide a critical discussion of the pros and cons of eDNA metabarcoding for monitoring mosquito species diversity and recommendations for future research directions that could facilitate the adoption of eDNA as a tool for assessing insect vector communities.

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 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 (msi package) to increase the reliability of nanopore sequencing to detect invasive species, and we test its application using invasive bivalves while comparing it with Illumina-based sequencing. We sampled water from sites with pre-existing bivalve occurrence and abundance data, and contrasting bivalve communities, in Italy and Portugal. Samples were extracted, amplified, and sequenced by the two platforms. The mean agreement between sequencing methods was 69% and the difference between methods was nonsignificant. The lack of detections of some species at some sites could be explained by their known low abundances. This is the first reported use of MinION to detect aquatic invasive species from eDNA samples.

Knowledge on diet composition allows defining well-targeted conservation measures of large carnivores. Little is known about ecology of critically endangered Asiatic cheetah, especially the overall diet and its possible regional differences. We used cheetah scats, metabarcoding technique and microsatellite markers to assess the individual and overall diet composition of the species across its entire range in Asia. Cheetahs were primarily predating on mouflon; following by ibex, cape hare and goitered gazelle. Despite their high availability, small-sized livestock was never detected. Goitered gazelles were only detected in an area where the habitat is mainly flatlands. In hilly areas, mouflon was the most frequent prey item taken. Ibex was typically taken in rugged terrain, but mouflon was still the most frequently consumed item in these habitats. High consumption of mouflon in comparison to goitered gazelle suggests that human pressure on lowland habitats has possibly forced Asiatic cheetahs to occupy suboptimal habitats where gazelles are less abundant. The protection of flatlands and the removal of livestock from them are needed to ensure the long-term survival of Asiatic cheetah. The laboratory and bioinformatics pipelines used in this study are replicable and can be used to address similar questions in other threatened carnivores.

Abstract Molecular analysis of gut content is one of the widest used methods to investigate diet in arthropods. Stomach content analysis in some arthropods is particularly difficult, e.g., in arachnids, because they have external digestion and a low foraging frequency. Scorpions have a particularly low feeding frequency, and their diet information is scarce. In this work we explore a DNA metabarcoding approach to detect prey DNA in Vietnamese forest scorpions (Heterometrus laoticus) under a controlled diet regime. A different type of prey (crickets, mealworms, and cockroaches) was offered once every 3 weeks for a total of 9 weeks. To assess the most suitable part of the digestive system and extraction method to use for molecular diet analysis, we separately analyzed three different portions of the digestive tract of scorpions (mid‐gut, hepatopancreas, and hindgut) using two different extraction methods (salt‐out method and a customized beads‐based protocol). We calculated the detectability half‐life of the prey DNA for each digestive tract section. We detected all three targeted prey items, showing that in scorpions multiple predation events can be distinguished in the same specimen within its last 9 weeks of foraging activity. The hepatopancreas was the portion of the digestive tract that provided the best prey detection and the longest DNA detectability half‐life (51 days), followed by the mid‐gut (22 days) and the hindgut (16 days). We found no significant difference between the extraction methods used. However, the salt‐out method was less effective in some of the PCRs and is therefore not recommended for molecular diet analysis.

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.

AbstractEnvironmental 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 filters.

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.

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.

Recent developments on ethanol-based DNA (etDNA) metabarcoding have shown that it is possible to extract meaningful information about macroinvertebrate community diversity and composition from the ethanol used to preserve bulk samples. The major advantages of this molecular approach are the reduced processing time and costs, and the possibility to keep specimens intact for other experiments. Yet, organisms with highly sclerotised exoskeleton or that are rare in the sample have been found to release a lower amount of DNA into solution and tend to be consistently missed by etDNA metabarcoding, thereby compromising the viability of the method. Few studies have shown that the first steps of the metabarcoding workflow are crucial for the good performance of etDNA-based assays, such as the decision on storage time before sampling and the ethanol phase to be analysed, the inclusion of pre-treatment strategies (i.e., freezing), and the choice of the DNA extraction protocol. In this study, we aimed to evaluate the combined effect of various technical choices on the performance of etDNA metabarcoding, considering factors such as sample volume, ethanol phase of sorted and unsorted samples, pre-capture treatments (evaporation vs filtration) and bioinformatic pipelines. Through the application of decision-tree models, our preliminary data revealed that the increase of volume (by itself) is enough to improve PCR amplification yields and proportion of families matching the morphological identifications, with great impact on the detection of hard-bodied and cased taxa. Also, no major differences among phases with or without a sorting step nor among bioinformatic pipelines were detected, particularly at higher volumes. Our results suggest that the higher performance (with lower observed variation) in taxonomic detection at higher volumes is likely a consequence of a higher availability of longer fragments of DNA in solution. This study highlights the importance of understanding the impact of technical choices to improve the efficiency of a DNA-based method, and reinstates etDNA metabarcoding as a potential method in the context of biomonitoring.

A protocol for the metabarcoding of DNA samples using nanopore technology, for the purposes of biomonitoring, biodiversity assessment, DNA-based diet analysis, or other related applications. The protocol is being used in our laboratory for detecting aquatic invasive bivalve species and for measuring biodiversity of fish and other vertebrates from water eDNA samples, as well as for species identification of scats. The key steps are a first PCR to amplify metabarcoding fragments, a 2nd PCR to add indexes (barcodes) to allow pooling of multiple samples, and the preparation of the pool for sequencing on a MinION sequencer. The protocol is adapted from PCR barcoding (96) amplicons (SQK-LSK109) by Nanopore.

DNA metabarcoding from the ethanol used to store macroinvertebrate bulk samples is a convenient methodological option in molecular biodiversity assessment and biomonitoring of aquatic ecosystems, as it preserves specimens and reduces problems associated with sample sorting. However, this method may be affected by errors and biases, which need to be thoroughly quantified before it can be mainstreamed into biomonitoring programmes. Here, we used 80 unsorted macroinvertebrate samples collected in Portugal under a Water Framework Directive monitoring programme, to compare community diversity and taxonomic composition metrics estimated through morphotaxonomy versus metabarcoding from storage ethanol using three markers (COI-M19BR2, 16S-Inse01 and 18S-Euka02) and a multimarker approach. A preliminary in silico analysis showed that the three markers were adequate for the target taxa, with detection failures related primarily to the lack of adequate barcodes in public databases. Metabarcoding of ethanol samples retrieved far less taxa per site (alpha diversity) than morphotaxonomy, albeit with smaller differences for COI-M19BR2 and the multimarker approach, while estimates of taxa turnover (beta diversity) among sites were similar across methods. Using generalized linear mixed models, we found that after controlling for differences in read coverage across samples, the probability of detection of a taxon was positively related to its proportional abundance, and negatively so to the presence of heavily sclerotized exoskeleton (e.g., Coleoptera). Overall, using our experimental protocol with different template dilutions, the COI marker showed the best performance, but we recommend the use of a multimarker approach to detect a wider range of taxa in freshwater macroinvertebrate samples. Further methodological development and optimization efforts are needed to reduce biases associated with body armouring and rarity in some macroinvertebrate taxa.

Enhancing the role of the soil microbiota in plant phosphorus (P) and sulfur (S) supply through application of organic fertilizer could reduce dependencies on non-sustainable synthetic fertilizers. To compare the effects of organic/inorganic fertilizers on the soil microbiota, soil columns with Lolium perenne (ryegrass) were set up in a greenhouse and amended with an inorganic fertilizer, cattle slurry (organic), or urea (P- and S-free control). Ryegrass rhizosphere of the slurry treatment had significantly higher abundances of bacterial feeding nematodes, mycorrhizal colonization, cultivable heterotrophic bacteria, phosphonate- and sulfonate-utilizing bacteria, arylsulfatase activity, available P, and Variovorax asfA gene copies compared to the inorganic and urea treatments. Phosphomonoesterase activities, and gene abundances involved in organic P and S transformations (phoD, phoC, Burkholderia, and Polaromonas asfA) were similar in all treatments. Grass dry matter yield and shoot uptake of N, P, and S were significantly higher in the inorganic treatment compared to the urea and slurry treatments. Community compositions differed significantly between the three fertilizer treatments and included the bacterial, alkaline phosphomonoesterase-producing bacterial, fungal, AM fungal, and nematode communities. Bacteriodetes were found in higher relative abundance in the organic treatment, while Acidobacteria were more abundant in the urea and inorganic fertilizer treatments. These community shifts correlated significantly with grass dry matter yield, uptake of N, P, and S, mycorrhizal colonization, enzyme activities, abundances of bacteria, and bacterial feeding nematodes. We concluded that organic fertilization promoted soil microbes and nematodes which have the potential to support sustainable plant growth, provided that the overall nutrient requirements are met.

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.

Trophic networks in small isolated islands are in a fragile balance, and their disturbance can easily contribute toward the extinction vortex of species. Here, we show, in a small Atlantic island (Raso) in the Cabo Verde Archipelago, using DNA metabarcoding, the extent of trophic dependence of the Endangered giant wall gecko Tarentola gigas on endemic populations of vertebrates, including one of the rarest bird species of the world, the Critically Endangered Raso lark Alauda razae. We found that the Raso lark (27%), Iago sparrow Passer iagoensis (12%), Bulwer's petrel Bulweria bulwerii (15%), and the Cabo Verde shearwater Calonectris edwardsii (10%) are the most frequent vertebrate signatures found in the feces of the giant wall gecko. This work provides the first integrative assessment of their trophic links, an important issue to be considered for the long-term conservation of these small and isolated island ecosystems.