Improving the detection of rare native fish species in environmental DNA metabarcoding surveys

The presence of threatened or endangered species often strongly influences management and conservation decisions. Within the Murray–Darling Basin (MDB), Australia, the presence of threatened native fish affects the management and allocation of water resources. These decisions are currently based on traditional fisheries data and a predictive MaxEnt model. However, it is important to verify the model's predictive power given the implication it may have, but this requires methods with a high detection sensitivity for rare species. Although the use of environmental DNA (eDNA) metabarcoding achieves a higher detection sensitivity compared with traditional methods, earlier surveys in the MDB have shown that the highly abundant and invasive common carp (Cyprinus carpio) can reduce detection probabilities for rare species. Consequently, a polymerase chain reaction (PCR) blocking primer designed to block the amplification of carp eDNA could increase the detection probabilities for rare native species while simultaneously reducing the required sampling effort and survey costs. Although PCR blocking primers are often used in ancient DNA and dietary studies, no aquatic eDNA metabarcoding study to date has evaluated the potential benefits of using PCR blocking primers. A laboratory and field‐based pilot study was used to address this knowledge gap and assess the impact of a blocking primer on the detection probabilities of native species and the minimum sampling effort required. Results showed that the inclusion of the blocking primer increased the detection probabilities for native species by 10–20% and reduced the minimum required sampling effort by 25–50%. These findings provide important insights into possible methods for optimizing eDNA metabarcoding surveys for the detection of rare aquatic species.

Vertebrate prey composition analysis of the Pallas’s cat (Otocolobus manul) in the Gongga Mountain Nature Reserve, based on fecal DNA

Diet analysis is essential to fully understanding the biology of a species and its function within the ecosystem, and is also key in identifying food web interactions and the population dynamics of predators and prey. There is currently little information on the diet of small to mid-sized carnivores due to their elusive behavior. We used a DNA-based method to analyze the vertebrate prey of the Pallas’s cat (Otocolobus manul) in the Gongga Mountain Nature Reserve of southwestern China, a global biodiversity hotspot region. We confirmed 14 scat samples containing the Pallas’s cat DNA from 100 small carnivore scats collected at altitudes between 3200 and 4900 m, and then performed polymerase chain reaction (PCR) amplifications using primers targeting the vertebrate mitochondrial 12S rRNA gene with a blocking oligonucleotide specific to the predator sequence. We identified a total of 18 prey taxa, including nine mammals, eight birds and one fish. Our results show that pikas (in 79% of the scats) and birds (in 50% of the scats) are the predominant prey consumed by the Pallas’s cat in our investigation area. The most important finding in our study is that Cypriniformes species were found in the Pallas’s cat’s diet, exhibiting dietary diversification consistent with the accessible fauna of the surrounding environment. In order to increase the detectability of both typical and rare vertebrate prey items, we constructed a blocking primer to inhibit the amplification of the Pallas’s cat DNA.

A Host-Specific Blocking Primer Combined with Optimal DNA Extraction Improves the Detection Capability of a Metabarcoding Protocol for Canine Vector-Borne Bacteria

Bacterial canine vector-borne diseases are responsible for some of the most life-threatening conditions of dogs in the tropics and are typically poorly researched with some presenting a zoonotic risk to cohabiting people. Next-generation sequencing based methodologies have been demonstrated to accurately characterise a diverse range of vector-borne bacteria in dogs, whilst also proving to be more sensitive than conventional PCR techniques. We report two improvements to a previously developed metabarcoding tool that increased the sensitivity and diversity of vector-borne bacteria detected from canine blood. Firstly, we developed and tested a canine-specific blocking primer that prevents cross-reactivity of bacterial primer amplification on abundant canine mitochondrial sequences. Use of our blocking primer increased the number of canine vector-borne infections detected (five more Ehrlichia canis and three more Anaplasma platys infections) and increased the diversity of bacterial sequences found. Secondly, the DNA extraction kit employed can have a significant effect on the bacterial community characterised. Therefore, we compared four different DNA extraction kits finding the Qiagen DNeasy Blood and Tissue Kit to be superior for detection of blood-borne bacteria, identifying nine more A. platys, two more E. canis, one more Mycoplasma haemocanis infection and more putative bacterial pathogens than the lowest performing kit.