Is it absent or is it present? Detection of a non-native fish to inform management decisions using a new highly-sensitive eDNA protocol

© 2019, The Author(s). Environmental managers require a sensitive and reliable means to prove, with the highest level of confidence possible, where non-native fish species exist and where they do not. Therefore, a nested PCR (nPCR) protocol was developed to detect the environmental DNA (eDNA) of a case-study species, topmouth gudgeon Pseudorasbora parva, which was recently the subject of a national eradication campaign in the UK. The nPCR protocol was tested in the laboratory and in the field in a series of coordinated surveys (eDNA and conventional sampling with traps) at a commercial angling venue in southern England where an initial eDNA survey, based on conventional PCR (cPCR), found P. parva to be present in one of the seven ponds. In the laboratory, the nPCR protocol was on average 100× more sensitive than cPCR, providing a 100% detection rate at DNA concentrations of 3 × 10 −8  ng/µL (8 DNA copies per µL). In the field, nPCR and conventional trapping both detected P. parva in only one of the seven angling ponds, the same infested pond as in the previous cPCR-based study. Following eradication work on the infested pond, no eDNA of P. parva was detected using nPCR in either the formerly-infested pond or the adjacent pond, which had been used to quarantine large commercially-valuable fishes. In management applications where the veracity of negative results may be of equal importance as confirmation of positive detections, nPCR protocols provide a useful addition to the analytical toolkit available to inform decision makers responsible for non-native species management

Combination of biomolecular and stable isotope techniques to determine the origin of organic matter used by bacterial communities: application to sediment

Natural isotopic composition is a good tool to trace organic matter in ecosystems. Recent studies used a combination of molecular and stable isotope techniques to determine the origin of the organic carbon used by bacteria in the water column. In our study, we show that this procedure can be used for analysis of sediment bacterial communities with few modifications. In the water column, bacterial recovery is done before DNA extraction. In the sediment, we tested qualitatively and quantitatively a direct and indirect extraction of DNA. The direct extraction was the most efficient. It recovered between 3.1 and 15.8 mu g DNA g(-1). dry sediment and the contamination of field samples by eucaryotic DNA was less than 13%. In this preliminary study of the salt marsh ecosystem, the delta(13)C values of DNA (-26 to -24 parts per thousand) recovered from the sediment were close to the delta(13)C values of halophytic plants (-26.4 and -25.3 parts per thousand) showing a relationship between plants and microorganisms, Thus, this procedure can be used to trace the flow of carbon through the sediment microbial biomass and to understand the variation of bacterial activity according to the inputs of allocthonous and autochtonous organic matter. [KEYWORDS: DNA; delta C-13 values; bacteria; sediment Direct extraction; carbon; dna; phytoplankton; purification; probes; bloom; soil; rna]

Application of environmental DNA analysis to inform invasive fish eradication operations.

Environmental DNA (eDNA) detection of non-native species has considerable potential to inform management decisions, including identifying the need for population control and/or eradication. An invasive species of European concern is the Asian cyprinid fish, topmouth gudgeon (Pseudorasbora parva). Here, eDNA analyses were applied at a commercial angling venue in southern England to inform operations aiming to eradicate P. parva, which had only ever been observed in one of the venue's seven unconnected angling ponds. Eradication of P. parva was initially attempted by repeated depletion of the population using fish traps (crayfish traps fitted with 5 mm mesh netting) and the introduction of native predators over a 4-year period. The very low number of P. parva captured following these eradication efforts suggested a possible population crash. Conventional PCR analysis of water samples using species-specific primers was applied to all seven ponds to confirm that P. parva was present in only one pond, that the eradication attempt had indeed failed and that the species' distribution in the pond appeared to be restricted to three bankside locations. The continued presence of P. parva at these locations was confirmed by subsequent trapping. Water samples from an adjacent, unconnected stream were also analysed using the eDNA methodology, but no DNA of P. parva was detected. The results suggest that further management action to eradicate P. parva be focused on the pond shown to contain the isolated P. parva population and thereby eliminate the risk of further dispersal. This study is the first to apply eDNA analysis to assess the efficacy of an eradication attempt and to provide evidence that the species was unlikely to be present in the other ponds, thus reducing the resources needed to control the species