Discussion
The development of a multiplex dPCR assay that allows for detection of four invasive species of snake in Florida is a critical advancement in the ability to analyze eDNA and will significantly strengthen early detection and monitoring efforts in the Florida Everglades. Previous studies have utilized dPCR for analyzing eDNA samples in Florida to monitor for pythons (Hunter et al. 2015, Orzechowski et al. 2019, Hunter et al. 2019). However, methods used were limited to detection of only one species (in that instance P. bivittatus ). With the current Absolute Q dPCR system, used in this study, a total of four reporter dyes are available, allowing for the development of more robust multiplex assays and with the system being automated, the workflow for setting up, running and analyzing samples is significantly reduced. To the authors knowledge, this represents the first tetraplex dPCR assay for snakes. Other systems that have reported utility of this technology include salmonid pathogens (von Ammon et al. 2022), breast and ovarian cancer screening (Oscorbin et al. 2019), lung cancer screening (Oscorbin et al. 2022), and GMOs (genetically modified organisms) detection in food crops (Dobnik et al. 2016). The utilization of this technology in areas such as human medicine, especially with regard to cancer, and large-scale agricultural monitoring highlight the power of this technology and validate the need to adapt this tool to invasive species monitoring where critical ecosystems are at stake. While the tetraplex dPCR assay for invasive snakes represents a new, cutting-edge tool to improve monitoring efforts, the utility and value of tetraplex assays based on standard and quantitative PCR (non-dPCR) has been previously demonstrated and include food allergens (Köppel et al. 2010), eDNA of food animals (Safdar et al. 2014, Hossain et al. 2017, Kaltenbrunner et al. 2018), drug resistant fungus (Arastehfar et al. 2018), paternity cases (Seidl et al. 1996) and influenza viral types (Henritzi et al. 2019). The development of a reliable and time efficient tetraplex dPCR is a necessary tool for wildlife monitoring as the rate of habitat encroachment by humans in Florida is exponentially increasing with Florida having the highest net migration rate from July 2021 to July 2022 (U.S. Census Bureau, 2021).
Cross-amplification was not observed for any of the species analyzed in this study despite the close phylogenetic relationship among constrictor species examined. As invasive constrictor snakes in the families Pythonidae and Boidae share more genetic similarity compared to other families (Colubridae, Elapidae, and Viperidae) of native snakes found within Florida (Burbrink et al. 2020), we do not anticipate issues of cross-amplification with native snakes in our assay should DNA of native snakes be collected inadvertently in environmental samples. Previous dPCR assays, while designed to avoid cross-amplification, were not evaluated against closely related taxa.
Numerous factors are responsible for increased sensitivity and reliability of dPCR results over other protocols (standard PCR and quantitative PCR). The physical partitioning of a sample over approximately 20,000 wells functionally allow for replicating a sample 20,000 times where detection of single target (gene representing a target species) is possible. Additionally, the same partitioning of a sample also significantly reduces impact of environmental inhibitors (substances that can inhibit PCR amplification, and hence detection of eDNA) due to significant reduction/elimination of inhibitor concentration per reaction well. This degree of sensitivity allows for detection of trace amounts of DNA in the environment (either terrestrial or aquatic), which can provide an effective means for early detection monitoring efforts of target invasive species. This may be particularly useful for detection of species that exist at low population densities or when only one individual of a non-native species is reported.
With the capability of multiplex testing, researchers and natural resource managers can reliably survey for multiple target species of interest simultaneously from an individual sample. This can reduce resources required for bio-surveillance programs as well as increase the efficiency of monitoring programs in areas where numerous invasive species are present, which can increase the ability to initiate an effective rapid response effort. Additionally, multiplex dPCR testing can be developed for any combination of target species, fitting the real time needs for detection of invasive species across landscapes with multiple invasions. For instance, the range of Burmese pythons encompasses the distribution of northern African pythons, boa constrictors, and rainbow boas in Miami-Dade County, FL (Figure 4) and having this tetraplex dPCR assay will allow for a more time and cost-effective means to evaluate overlapping ranges and how (and if) these overlaps change over time. The capacity to test for multiple target species can also facilitate rapid and accurate confirmation of a species during EDRR efforts when reported sightings of non-natives are ambiguous concerning species identification. Accurate species identification can aid managers in decisions regarding the appropriate response, or depending on the situation, whether to respond (i.e., a large constrictor is reported but the species identification is confirmed to be a Burmese python occurring in an area known to be heavily infested with pythons), which can inform prioritization of resources.
In addition to early detection efforts, multiplex dPCR testing may be an effective way to assess the outcome of removal efforts. For example, when post-removal surveys for an invasive species are desired to demonstrate a decrease in occupancy or repeated zero detections (maximum control; Godfrey et al. 2023), use of dPCR can provide a reliable surveillance method for target species of interest that is accurate, rapid, and cost effective relative to traditional visual survey methods. This can be especially useful when species of interest for detection are cryptic or rare (i.e., snakes, crocodylians), and otherwise hard to detect using other methodologies. When management outcomes of interest are not based on repeated zero detections of a target species, but on the response of a native species impacted by an invader, multiplex testing may provide an additional advantage. For example, multiplex dPCR assays could be developed to assess the impact of python removal efforts on python occupancy across space and time, while simultaneously monitoring occupancy rates of mammals (i.e., opossums, racoons, and marsh rabbits) known to be impacted by pythons through predation (Dorcas et al. 2012; McCleery et al. 2015). An increase in occupancy of impacted native mammals over space and time may be used as an ecological proxy indicative of ecosystem recovery.
While utilizing multiplex dPCR assays for early detection and monitoring has advantages, these benefits are only advantageous if they are accessible to natural resource managers. As the number of invasive species continues to rise, the need for monitoring programs targeting multiple species increases. Use of dPCR assays can increase the efficacy of eDNA monitoring programs through simultaneous monitoring of multiple target species. The dPCR assay developed herein for detection of four invasive constrictor species increases the capacity for rapid and accurate detection of insipient populations and range expansions as well as long-term monitoring for assessment of control efforts. Data gained from monitoring programs can aid natural resource managers in informed decision-making concerning an appropriate response as well as allow managers to prioritize resources relative to the situation. For example, a manger may prioritize resources to respond to detection of target invasive species near biologically sensitive areas. Additionally, with the ability to detect multiple target species in a sample and a reduced workflow for assays, multiplex dPCR may be a more cost-effective means for implementing eDNA monitoring programs.
This study documents successful development of a novel multiplex assay designed to detect eDNA of four species of invasive constrictors rapidly and accurately. Future work will assess multiplex testing under semi-natural conditions with known presence of target species to validate results obtained in this study in the field, which will facilitate development of a monitoring program for invasive constrictors in Florida. As Florida is a hotspot of biological invasions, additional multiplex assays targeting other established invasive taxa (i.e., lizards, fish, and crocodylians) negatively impacting native ecosystems, as well as non-native species with a high risk of adverse effects if they become established, are needed and should be a focal point of future studies to facilitate multi-species eDNA monitoring programs.