eDNA;fieldwork;optimisation;fish;Misgurnus;Carassius;endangered;conservatio;monitoring;wetlands

Elusive and endangered species are prime candidates for environmental DNA (eDNA) monitoring (Duarte et al. 2023), and the European weatherfish (Misgurnus fossilis) is one such species for which traditional fishing methods may fail to provide conclusive presence/absence data. In contrast, eDNA approaches are less labour-intensive and can require only small water volumes when species-specific qPCR/ddPCR markers are used (Thomsen et al. 2011; Sigsgaard et al. 2015; Kusanke et al. 2020; Brys et al. 2020). Here, we aim to develop a standardised eDNA protocol to complement conventional surveys of Misgurnus, particularly in small, vegetated waterbodies and wetlands in Central Europe where electrofishing, dip-netting, or trapping are often ineffective; these habitats are also among the most neglected despite facing significant threats (Šmejkal et al. 2025). We are currently testing field sampling procedures and laboratory workflows that are robust across all known M. fossilis haplotypes and adapted to muddy-water conditions, addressing issues such as sampling away from the shoreline without disturbing sediment, rapid filter clogging, and qPCR inhibition, as well as logistical constraints related to sample storage and transport in large-scale monitoring. While non-frozen samples can be preserved using fixatives, these increase contamination risk, limit sample volume (e.g., 35 ml fixative added to 15 ml water; Ficetola et al. 2008), and may reduce DNA yield (Kusanke et al. 2020); alternative reagents such as lysis buffers may improve preservation even under extreme field conditions (Schenekar et al. 2024). The resulting protocol is intended to be cost-effective and accessible for citizen science applications, with species-specific markers designed for multiplexing to enhance detection probability (Brys et al. 2023), enable discrimination of co-occurring species such as the endangered crucian carp (Carassius carassius) (Šmejkal et al. 2025), reduce analytical costs and handling effort (Jo et al. 2019), and maximise the number of tests per sample when DNA yields are low (Wozney and Wilson 2017). Multiplexing also facilitates the inclusion of inhibition-sensitive internal positive controls and additional markers targeting invasive Misgurnus or Carassius species, thereby improving monitoring of local community composition. As eDNA is particularly effective for detecting invaders at low densities (Jo et al. 2019; Jerde 2019), this approach may support early detection of invasive Asian weatherfish, whose spread is of increasing concern in Europe; early detection is critical because established populations are extremely difficult to eradicate, as individuals can survive buried in sediment for months even after waterbodies are drained (Cano-Barbacil et al. 2025).

Elusive and endangered species are prime candidates for environmental DNA (eDNA) monitoring (Duarte et al. 2023), and the European weatherfish (Misgurnus fossilis) is one such species for which traditional fishing methods may fail to provide conclusive presence/absence data. In contrast, eDNA approaches are less labour-intensive and can require only small water volumes when species-specific qPCR/ddPCR markers are used (Thomsen et al. 2011; Sigsgaard et al. 2015; Kusanke et al. 2020; Brys et al. 2020). Here, we aim to develop a standardised eDNA protocol to complement conventional surveys of Misgurnus, particularly in small, vegetated waterbodies and wetlands in Central Europe where electrofishing, dip-netting, or trapping are often ineffective; these habitats are also among the most neglected despite facing significant threats (Šmejkal et al. 2025). We are currently testing field sampling procedures and laboratory workflows that are robust across all known M. fossilis haplotypes and adapted to muddy-water conditions, addressing issues such as sampling away from the shoreline without disturbing sediment, rapid filter clogging, and qPCR inhibition, as well as logistical constraints related to sample storage and transport in large-scale monitoring. While non-frozen samples can be preserved using fixatives, these increase contamination risk, limit sample volume (e.g., 35 ml fixative added to 15 ml water; Ficetola et al. 2008), and may reduce DNA yield (Kusanke et al. 2020); alternative reagents such as lysis buffers may improve preservation even under extreme field conditions (Schenekar et al. 2024). The resulting protocol is intended to be cost-effective and accessible for citizen science applications, with species-specific markers designed for multiplexing to enhance detection probability (Brys et al. 2023), enable discrimination of co-occurring species such as the endangered crucian carp (Carassius carassius) (Šmejkal et al. 2025), reduce analytical costs and handling effort (Jo et al. 2019), and maximise the number of tests per sample when DNA yields are low (Wozney and Wilson 2017). Multiplexing also facilitates the inclusion of inhibition-sensitive internal positive controls and additional markers targeting invasive Misgurnus or Carassius species, thereby improving monitoring of local community composition. As eDNA is particularly effective for detecting invaders at low densities (Jo et al. 2019; Jerde 2019), this approach may support early detection of invasive Asian weatherfish, whose spread is of increasing concern in Europe; early detection is critical because established populations are extremely difficult to eradicate, as individuals can survive buried in sediment for months even after waterbodies are drained (Cano-Barbacil et al. 2025).