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.