Short-Term Impact of Recycling-Derived Fertilizers on Their P Supply for Perennial Ryegrass (Lolium perenne).

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.

Monitoring mosquito richness in an understudied area: can environmental DNA metabarcoding be a complementary approach to adult trapping?

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.

Assessing Asiatic cheetah's individual diet using metabarcoding and its implication for conservation.

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.

Speeding up the detection of invasive bivalve species using environmental DNA: A Nanopore and Illumina sequencing comparison.

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.

Modelling technical and biological biases in macroinvertebrate community assessment from bulk preservative using multiple metabarcoding markers.

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.

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.

Intricate trophic links between threatened vertebrates confined to a small island in the Atlantic Ocean.

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.

Challenges for assessing vertebrate diversity in turbid Saharan water-bodies using environmental DNA.

The Sahara desert is the largest warm desert in the world and a poorly explored area. Small water-bodies occur across the desert and are crucial habitats for vertebrate biodiversity. Environmental DNA (eDNA) is a powerful tool for species detection and is being increasingly used to conduct biodiversity assessments. However, there are a number of difficulties with sampling eDNA from such turbid water-bodies and it is often not feasible to rely on electrical tools in remote desert environments. We trialled a manually powered filtering method in Mauritania, using pre-filtration to circumvent problems posed by turbid water in remote arid areas. From nine vertebrate species expected in the water-bodies, four were detected visually, two via metabarcoding, and one via both methods. Difficulties filtering turbid water led to severe constraints, limiting the sampling protocol to only one sampling point per study site, which alone may largely explain why many of the expected vertebrate species were not detected. The amplification of human DNA using general vertebrate primers is also likely to have contributed to the low number of taxa identified. Here we highlight a number of challenges that need to be overcome to successfully conduct metabarcoding eDNA studies for vertebrates in desert environments in Africa.

Detecting frogs as prey in the diets of introduced mammals: a comparison between morphological and DNA-based diet analyses.

Amphibians are currently the most threatened group of vertebrates worldwide, and introduced fauna play a major role in their decline. The control of introduced predators to protect endangered species is often based on predation rates derived from diet studies of predators, but prey detection probabilities using different techniques are variable. We measured the detectability of frogs as prey, using morphological and DNA-based diet analyses, in the stomachs and faeces of four mammal species that have been introduced to many areas of the world. Frogs (Litoria raniformis) were fed to rats (Rattus norvegicus and R. rattus), mice (Mus musculus) and hedgehogs (Erinaceus europaeus). DNA-based analysis outperformed morphological analysis, increasing the prey detection rate from 2% to 70% in stomachs and from 0% to 53% in faeces. In most cases, utilizing either stomachs or faeces did not affect the success of prey DNA detection; however, using faeces extended the detectability half-life from 7 to 21 h. This study is the first to measure prey DNA detection periods in mammalian stomachs, and the first to compare prey DNA detection periods in the stomachs and faeces of vertebrates. The results indicate that DNA-based diet analysis provides a more reliable approach for detecting amphibians as prey and has the potential to be used to estimate the rate of predation by introduced mammals on endangered amphibians.