| This paper focuses on the application of environmental DNA (eDNA) in detecting pathogens in decapoda aquaculture. Bacterial pathogens pose significant threats to aquaculture by causing disease outbreaks with high mortality rates, leading to economic losses and product shortages. Decapod crustaceans, such as tropical rock lobster (TRL) (Panulirus ornatus) can harbour a wide range of pathogens and parasites, compromising the health and biosecurity of aquaculture systems. Disease research in crustacean hosts focuses on employing a combination of conventional and contemporary diagnostic methods. Traditional approaches like histopathology, bacterial counting, and microscopy methods can be invasive, time-consuming and expensive. Molecular techniques, such as PCR, are used to detect challenging-to-culture microorganisms and aid in epidemiological studies. The monitoring of environmental DNA (eDNA) is a new method for identifying targeted nucleic acid signals in the field of conservation and animal health. eDNA combines quantitative PCR with filter discs and passive samplers to detect pathogens in aquaculture environments. eDNA offers early pathogen detection, non-invasive sampling, and potential cost savings compared to traditional methods. The use of eDNA has the potential to be a powerful tool for pathogen detection in aquaculture. The eDNA allows for non-invasive and sensitive monitoring of the aquatic environment and provides early detection of pathogens and disease outbreaks. Among the pathogens, Vibrio spp. and Aquimarina spp. are commonly found in marine environments and aquaculture production systems, and they can cause significant mortality in lobster culture. The toxin-encoding genes present in many Vibrio species can be an effective taxonomic marker for the identification of the genus. Relevant toxins in the Vibrio genus include AB-like toxin (Pir-AB), Zona occludens toxins (ZOT), toxR, haemolysins (Hemo) and Repeats-in-Toxin (RTX). The paper presents a pilot application of eDNA first, to demonstrate proof of concept of the use and optimisation of passive sample collection of eDNA to detect pathogen targets within the aquaculture facility, second to explore the variations in bacterial load throughout different life cycles of TRL, and third to establish a correlation between the presence of bacteria detected using environmental DNA (eDNA) analysis and mortality rates observed in the tanks, providing valuable insights into the potential impact of bacterial presence on the health and survival of the species. Data collection occurred over 9 weeks in a Queensland aquaculture facility, focusing on different culture phases (broodstock, larvae, puerulus, juvenile, adults). The study involved passive sampler filter collection to monitor eDNA and to conduct DNA extraction and PCR of relevant Vibrio spp. toxin, Aquimarina spp., and two positive controls: the 16s present in all bacteria and TRL genes. To validate the eDNA, water samples were collected from the tanks in Marine Agar and TCBS plates in order to perform the bacteria count. The farm provided mortality and diseased animal data in juvenile and larval tanks. In this study, the effectiveness of utilizing P.ornatus 18s and 16s genes as positive controls for eDNA detection was examined. The 18s gene demonstrated a robust 100% positive detection rate for lobsters in tanks, with only one false negative noted after 1 hour of incubation. The 16s gene efficiently targeted bacterial presence, consistently achieving positive detection rates above 94% throughout varying incubation periods. Notably, accuracy improved with longer incubation times, reaching 100% detection after 4, 6, and 24 hours. Both positive controls and Aquimarina were consistently detected during each hour of incubation, with higher detection rates observed over extended incubation. The relationship between nucleic acid binding and sampling duration was evident, as demonstrated by decreasing average Ct values for positive controls and Aquimarina genes over time. Vibrio-associated toxin genes exhibited limited positive detections, corroborating the low bacterial count on TCBS agar. Aquimarina was detected across all lobster life stages, particularly prominent in broodstock, larvae, juveniles, and pond-reared individuals. The intriguing link between Aquimarina and mortality dynamics highlights the potential influence of microbial dysbiosis on aquaculture health. Overall, these findings showcase the utility of 16s and Aquimarina Ct values as indicators for predicting and managing mortality trends in aquaculture. |