Behavior and season affect crayfish detection and density inference using environmental DNA.

Although the presence/absence of aquatic invertebrates using environmental DNA (eDNA) has been established for several species, inferring population densities has remained problematic. The invasive American signal crayfish, (Dana), is the leading cause of decline in the UK's only native crayfish species, (Lereboullet). Methods to detect species at low abundances offer the opportunity for the early detection, and potential eradication, of before population densities reach threatening levels in areas occupied by . Using a factorial experimental design with aquaria, we investigated the impacts of biomass, sex ratio, and fighting behavior on the amount of eDNA released by , with the aim to infer density per aquarium depending on treatments. The amount of target eDNA in water samples from each aquarium was measured using the quantitative Polymerase Chain Reaction. We show that the presence of eggs significantly increases the concentration of crayfish eDNA per unit of mass, and that there is a significant relationship between eDNA concentration and biomass when females are egg-bearing. However, the relationship between crayfish biomass and eDNA concentration is lost in aquaria without ovigerous females. Female-specific tanks had significantly higher eDNA concentrations than male-specific tanks, and the prevention of fighting did not impact the amount of eDNA in the water. These results indicate that detection and estimate of crayfish abundance using eDNA may be more effective while females are ovigerous. This information should guide further research for an accurate estimation of crayfish biomass in the field depending on the season. Our results indicate that detection and quantification of egg-laying aquatic invertebrate species using eDNA could be most successful during periods when eggs are developing in the water. We recommend that practitioners consider the reproductive cycle of target species when attempting to study or detect aquatic species using eDNA in the field.