Introduction
Florida is a hotspot of biological invasions, particularly for reptiles, due to a subtropical climate, peninsular geography, disturbed habitats, thriving exotic pet trade, and multiple major ports of entry to the U.S (Engeman et al. 2011). At least 54 species of non-native reptiles are established (i.e., breeding) in Florida, including 27% of all established non-native reptile species known to occur globally (Capinha et al. 2017). Since establishment, many of these invasive reptiles have increased in abundance and expanded their range, with numerous species becoming sympatric. Among the most concerning are invasive large constrictor snakes, including the Burmese python (Python bivittatus ), northern African python (P. sebae ), and boa constrictor (Boa constrictor ) (Figure 1) due to their large size, high reproductive rate, long life span, and ability to disperse long distances (Guzy et al. 2023). In addition, evidence supports the probable establishment of a fourth species of non-native constrictor snake, the rainbow boa (Epicrates cenchria ) (Figure 1), of which all size classes, including a gravid female, have been captured in the wild (MAM, pers. comm.; EDDMapS 2024). Due to known and potential impacts to native wildlife in Florida, these constrictors are considered priority species by environmental agencies (i.e., Florida Fish and Wildlife Conservation Commission and South Florida Water Management District) targeted for Early Detection and Rapid Response (EDRR) efforts, containment, or long-term management depending on the area invaded and the time since introduction (Florida Python Control Plan 2021).
Documented impacts are known primarily for Burmese pythons, the most studied of the invasive constrictor species in Florida and include severe reductions in populations of mammalian prey species (Dorcas et al. 2012; McCleery et al. 2015), co-introduction of invasive parasites that have spread to infect native wildlife (Miller et al. 2018; Miller et al. 2020), and increased prevalence of zoonotic viruses (Hoyer et al. 2017). Impacts of other invasive constrictors may yield comparable results once fully examined. Of particular concern is how these invaders may affect Everglades restoration efforts, a multi-billion-dollar initiative to restore the quantity, quality, timing, and distribution of water for the benefit and protection of people, habitats, and wildlife (South Florida Ecosystem Restoration Task Force 2022). To address this issue, the Invasive Exotic Species Strategic Action Framework (South Florida Ecosystem Restoration Task Force 2020) was initiated to combat the impacts of invasive species, including constrictor snakes, on these efforts as the establishment of invasive species directly threaten restoration goals.
Substantial resources have been provided to support control efforts of invasive constrictors in Florida with 10.6 million dollars spent by federal and state agencies, and one non-governmental organization during 2004 – 2021 (2021 Florida Python Control Plan) for removal of Burmese pythons, the most abundant and widespread of these invasive snakes (EDDMapS 2024). Despite these unprecedented resources, python removal remains challenging due to their cryptic appearance and propensity for secretive behaviors and large periods of inactivity, which belies our ability to easily detect and remove this species (Guzy et al. 2023, Nafus et al. 2019). The detection probability of Burmese pythons using data from visual encounter surveys and radio telemetry, is estimated to be less than 5% (Nafus et al. 2019) and detection likely decreases with increasing habitat complexity. In addition to low detection, Burmese pythons inhabit vast remote interior locations within the Everglades with dense vegetation and seasonal flooding, often requiring specialized equipment for human access, which can further limit the utility of visual detection methods due to logistics or lack of resources to maintain sustained monitoring efforts. A delay in detection reduces the effectiveness of rapid response and removal efforts, decreasing the likelihood of successful eradication and increasing the probability an introduced species will become established and require long-term control. Therefore, development of methods to increase detection of invasive snakes are paramount for the success of subsequent eradication and control efforts (Hunter et al. 2015).
Environmental DNA (eDNA) sampling can aid in detection of invasive wildlife, improve early detection, inform occupancy modeling, help determine invasion fronts, and provide managers a method to monitor and assess eradication efforts (Hunter et al. 2015, Hunter et al. 2019, Orzechowski et al. 2019, Morisette et al. 2021, Keller et al. 2022; Carim et al. 2019). This method has proven to be a viable tool for increasing detection of cryptic invasive species, including Burmese pythons (Hunter et al. 2015, Orzechowski et al. 2019, Hunter et al. 2019). Using eDNA assays and occupancy modeling, Hunter et al. (2015) estimated the detection probability of pythons to be greater than 91%, demonstrating the utility of eDNA for python detection compared to the less than 5% detection probability of pythons using visual encounter surveys (Nefas et al. 2019). However, despite advantages for increasing detection, particularly of secretive or rare species, eDNA has yet to be widely deployed as a detection tool for invasive species management. A contributing factor may result from a lack of confidence in eDNA detections due to the potential for false positives and false negatives, which can confound management decisions. For example, a positive eDNA detection of an invasive species that cannot be verified by other means of detection may trigger a costly response that may not be warranted (Jerde 2019). The potential for false-negative detections may increase when environmental inhibitors, preventing detection of DNA, are present or when samples contain trace amounts of DNA that may be undetected by the eDNA assay utilized.
Recent advances in digital PCR (dPCR) assays show promise for addressing many of these issues due to increased specificity and accuracy while allowing for multiplex testing for detection of multiple target species within a sample (Gaňová, et al. 2021). Multiplex eDNA assays can be conducted rapidly and efficiently for detection of up to six target species simultaneously with reduced resources compared to traditional eDNA assay methodologies (Gaňová et al. 2021). Development of multiplex dPCR assays is an emerging need to meet demands for early detection and monitoring of multiple target species in systems with multiple invasion events, such as South Florida. Additionally, a streamlined, automated workflow has potential to reduce resources required for implementing multiplex dPCR as a detection tool for large scale application across the landscape, which may increase the accessibility of eDNA for use by natural resource managers for implementing monitoring programs.
Towards this goal, we developed a robust multiplex assay for rapid, accurate, and cost-effective detection of environmental DNA of multiple target species within a sample that meet the needs of natural resource managers. Specifically, our multiplex dPCR assay is designed to detect invasive constrictor snakes (Burmese python, boa constrictor, northern African python, and rainbow boa) in Florida. We provide a detailed account of assay development and discuss potential advantages of this methodology for increasing effectiveness of invasive species monitoring programs in multi-invaded systems.