Environmental<scp>DNA</scp>surveillance for invertebrate species: advantages and technical limitations to detect invasive crayfish<i><scp>P</scp>rocambarus clarkii</i>in freshwater ponds

<jats:title>Summary</jats:title><jats:p><jats:list><jats:list-item><jats:p>The introduction of non‐native species is a major threat to biodiversity. While eradication programs of well‐established invaders are costly and hazardous for non‐target species, the early detection of a non‐native species at low density is critical for preventing biological invasions in recipient ecosystems. Recent studies reveal that environmental<jats:styled-content style="fixed-case">DNA</jats:styled-content>(e<jats:styled-content style="fixed-case">DNA</jats:styled-content>) is a powerful tool for detecting target species in aquatic ecosystems, but these studies focus mostly on fish and amphibians.</jats:p></jats:list-item><jats:list-item><jats:p>We examine the reliability of using e<jats:styled-content style="fixed-case">DNA</jats:styled-content>to detect the presence of an invasive freshwater crustacean species, the red swamp crayfish<jats:italic><jats:styled-content style="fixed-case">P</jats:styled-content>rocambarus clarkii</jats:italic>. Species‐specific primers and probes were designed; their specificity was tested using<jats:italic>in silico</jats:italic><jats:styled-content style="fixed-case">PCR</jats:styled-content>simulations and against tissues of other crayfish species. Limits of detection and quantification were specified for the target<jats:styled-content style="fixed-case">DNA</jats:styled-content>sequence by means of quantitative<jats:styled-content style="fixed-case">PCR</jats:styled-content>amplifications on dilution series of known amount of<jats:italic><jats:styled-content style="fixed-case">P</jats:styled-content>. clarkii</jats:italic><jats:styled-content style="fixed-case">DNA</jats:styled-content>.</jats:p></jats:list-item><jats:list-item><jats:p>The method was applied to water samples collected in 158 ponds in a<jats:styled-content style="fixed-case">F</jats:styled-content>rench<jats:styled-content style="fixed-case">N</jats:styled-content>ature<jats:styled-content style="fixed-case">P</jats:styled-content>ark, and results were compared to a traditional method using food‐baited funnel traps. Environmental<jats:styled-content style="fixed-case">DNA</jats:styled-content>had a better detection efficiency but predominantly led to divergent results compared with the trapping method. While habitat features partly explained the failure of crayfish detection by trapping, detection by e<jats:styled-content style="fixed-case">DNA</jats:styled-content>was problematic at low crayfish abundances. When<jats:italic><jats:styled-content style="fixed-case">P</jats:styled-content>. clarkii</jats:italic>was detected, the estimated concentrations of crayfish<jats:styled-content style="fixed-case">DNA</jats:styled-content>in water samples were always below the limit of quantification for the target<jats:styled-content style="fixed-case">DNA</jats:styled-content>sequence.</jats:p></jats:list-item><jats:list-item><jats:p><jats:italic>Synthesis and applications</jats:italic>. The combination of environmental<jats:styled-content style="fixed-case">DNA</jats:styled-content>(e<jats:styled-content style="fixed-case">DNA</jats:styled-content>) and conventional trapping methods is recommended to monitor the invasion by<jats:italic><jats:styled-content style="fixed-case">P</jats:styled-content>. clarkii</jats:italic>in small waterbodies such as ponds. However, the risk of mortality for non‐target species, notably amphibians, has to be carefully evaluated before large‐scale deployment of traps. Contrary to fish and amphibians, a low amount of extracellular<jats:styled-content style="fixed-case">DNA</jats:styled-content>in water is suspected to be the major limitation for crayfish detection by molecular approaches. Current advancements in<jats:styled-content style="fixed-case">PCR</jats:styled-content>technology, together with optimization of the water sampling method, promise upcoming developments of e<jats:styled-content style="fixed-case">DNA</jats:styled-content>detection for aquatic invertebrate species.</jats:p></jats:list-item></jats:list></jats:p>