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.