eDNA detection of corallivorous seastar (Acanthaster cf. solaris) outbreaks on the Great Barrier Reef using digital droplet PCR

Coral loss through consumption by corallivorous crown-of-thorns seastars (CoTS, Acanthaster spp.) is a major contributor to the coral reef crisis in the Indo-Pacific region. The fourth wave of Acanthaster cf. solaris outbreaks since the 1960s started around 2010 on Australia's Great Barrier Reef. Ecological monitoring failed to detect early outbreak stages, thus preventing timely intervention. Here, we develop a digital droplet PCR (ddPCR)-based method to detect environmental DNA (eDNA) of CoTS in 2-l water samples that can be compared with abundances of the species recorded by divers along 200-m(2) transects. Aquarium tests demonstrated that eDNA was readily detectable and increases proportional to the biomass of CoTS (R-2=0.99, p<0.0001). Adaptation from a quantitative PCR technique developed for CoTS larvae (Doyle et al. in Marine Biology 164:176, 2017) to ddPCR improved the limit of quantification (LOQ) by a factor of 45. During field verification on 11 reefs, CoTS eDNA was detectable on all reefs suffering outbreaks. In contrast, CoTS eDNA was absent from post-outbreak' reefs after populations collapsed and from pre-outbreak' reefs. In linear models, CoTS densities explained a high amount of variance of eDNA concentrations, both for water samples taken at the depth of transects (R-2=0.60, p<0.0001) and on the sea surface (R-2=0.46, p=0.0004). The proportion of samples above LOQ was also correlated with CoTS densities, with a similar amount of variance explained as for the concentration (underwater R-2=0.68, p<0.0001; surface R-2=0.49, p=0.0004). We conclude that, after consideration of sampling locations and times, this method is promising for CoTS population monitoring and early detection of outbreaks and might supplement or replace traditional monitoring. Development of automated samplers and possibly on board PCR in the future will further improve early detection.