A peer-reviewed open-access journal

NeoBiota 56: 3 |—47 (2020)

doi: 10.3897/neobiota.56.47475 @) NeoB 10ta

http:/ / neob iota. pen soft. net Advancing research on alien species and biological invasions

Monitoring the silver carp invasion in Africa: a case
study using environmental DNA (eDNA) in dangerous
watersheds

Steven Crookes'”, Tej Heer?, Rowshyra A. Castafieda*°, Nicholas E. Mandrak?*,
Daniel D. Heath', Olaf L.E Weyl®*, Hugh J. Maclsaac', Llewellyn C. Foxcroft’®

| Great Lakes Institute for Environmental Research, University of Windsor, Ontario, NOB 3P4, Canada 2 De-
partment of Integrative Biology, University of Guelph, Guelph, Ontario, NIG 2W1, Canada 3 Department
of Physical and Environmental Sciences, University of Toronto Scarborough, Toronto, Ontario, M1C 1A4,
Canada 4 Department of Biological Sciences, University of Toronto Scarborough, Toronto, Ontario, M1C
LA4, Canada § DSI/NRF Research Chair in Inland Fisheries and Freshwater Ecology, South African Institute
Jor Aquatic Biodiversity (SAIAB), Makhanda, Eastern Cape, South Africa, 6139, South Africa 6 Center for
Invasion Biology, SAIAB, Makhanda, Eastern Cape, South Africa, 6139, South Africa 1 Conservation Servi-
ces, South African National Parks, Kruger National Park, Skukuza 1350, South Africa 8 Centre for Invasion
Biology, Department of Botany and Zoology, Stellenbosch University, Matieland 7602, South Africa

Corresponding author: Steven Crookes (scrookes@uoguelph.ca; stecrookes@googlemail.com)

Academic editor: M. Uliano-Silva | Received 20 October 2019 | Accepted 26 February 2020 | Published 29 April 2020

Citation: Crookes S, Heer T, Castafieda RA, Mandrak NE, Heath DD, Weyl OLE, Maclsaac HJ, Foxcroft LC (2020)
Monitoring the silver carp invasion in Africa: a case study using environmental DNA (eDNA) in dangerous watersheds.

NeoBiota 56: 31-47. https://doi.org/10.3897/neobiota.56.47475

Abstract

Biodiverse habitats are increasingly subject to an intensification of anthropogenic stressors that may se-
verely diminish species richness. Invasive species pose a dominant threat to biodiversity and biosecurity,
particularly in biodiversity hotspots like Kruger National Park, South Africa. The invasive silver carp, Hy-
pophthalmichthys molitrix, was introduced into the Olifants River and may experience range spread owing
to favorable environmental conditions. Intensive monitoring protocols are necessary to effectively manage
invasions of species like silver carp. Unfortunately, tropical and sub-tropical aquatic systems are difficult
to monitor using conventional methods (e.g., netting, electrofishing and snorkeling) owing to a range of
factors including the presence of dangerous megafauna. Conservation of such systems may be advanced
by the adoption of novel methods, including environmental DNA (eDNA) detection. Here, we explore
the utility of environmental DNA (eDNA) to conduct safe, reliable and repeatable surveys in dangerous
watersheds using silver carp as a case study. We conducted eDNA surveys at 12 sites in two neighbouring
watersheds, and determined that the species has expanded its range within the Olifants River and to the

Copyright Steven Crookes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

32 Steven Crookes et al. / NeoBiota 56: 31-47 (2020)

south in the Sabie River. Expansion in the former is consistent with the presence of suitable spawning con-
ditions. We discuss the implications of this survey for biodiversity monitoring in similar aquatic systems in

the tropics and advocate an integrative approach to biomonitoring in these ecosystems.

Keywords

Biomonitoring, hazardous sampling, invasive species, Asian carp, species detection

Introduction

Africa is home to some of the most diverse habitats on the planet, encompassing
myriad climatic, geologic and biotic zones (Happold and Lock 2012). This continent
contains 22% of the highest-ranked watersheds supporting human populations, 9%
of biodiverse hotspots, and is second to only central and southeast Asia in global im-
portance for ecosystem services (Luck et al. 2009). In Africa and beyond, watersheds
represent essential components of the socio-economic and cultural landscape, provid-
ing key ecosystem services, and are in need of effective and strategic management
(Flotemersch et al. 2015).

River systems are threatened by direct modification through channelization
(Emerson 1971), channel rerouting, construction of dams, weirs, locks and arte-
rial canal networks, changes in drainage and overflow within the drainage basin
(Johnson et al. 2009), and by the translocation of native species and introduction
of non-indigenous species (Zhang et al. 2015). Poor watershed management can
adversely affect nutrient and habitat availability, species distribution and abundance,
population viability, and isolation, erosion or total loss of genetic variation (Davis
et al. 2018). Poor management of fisheries and other biotic resources has resulted in
introductions of detrimental non-indigenous taxa (i.e. aquatic invasive species; AIS)
(Cucherousset and Olden 2011).

Monitoring the biotic component of river systems is essential to effective manage-
ment of watersheds. For fishes, conventional monitoring of lotic habitats has tradition-
ally relied upon methods including nets (e.g. seine, fyke, gill) or traps, angling, direct
observation (SCUBA-diving or snorkeling), electrofishing, and telemetry and acoustic
monitoring (Portt et al. 2006). In certain circumstances, however, these methods can
be ineffective or too dangerous to deploy. Strong currents can preclude use of these
methods or limit their utility to seasonal windows (Portt et al. 2006). Factors idiosyn-
cratic to each method will also limit the scope and utility of each (e.g. low visibility
will impact direct observation (Mueller et al. 2006)). In large areas of Africa, as well
as Australia, southeast Asia and South America, the presence of large semi- or fully
obligate aquatic mammals or reptiles represent a real danger to river researchers and
affect surveys by destroying or interfering with equipment (World Health Organiza-
tion 2003). In addition, the accidental bycatch and mortality of aquatic mammals and
reptiles during fish surveys is a growing conservation concern (Ellender et al. 2016;
Carrizo et al. 2017).

eDNA detection of silver carp in Africa 33

The Olifants River in southern Africa is home to dangerous aquatic megafauna
animals including the common hippopotamus (Hippopotamus amphibius (Linnaeus,
1758)) and Nile crocodile (Crocodylus niloticus (Laurenti, 1768)) (Carrizo et al. 2017).
Safe monitoring of river ecosystems containing all or some of this megafauna is highly
problematic using conventional sampling tools. Environmental DNA (eDNA) detec-
tion was developed to indirectly detect a target species by collecting cellular and free
aqueous DNA shed by the target species into the water column without the need for
actual observation or collection of the organism itself (Ficetola et al. 2008; Thomsen
and Willerslev 2015). By collecting water, the time and personnel required to conduct
sampling are reduced (Thomas et al. 2019), thereby enhancing safety to both survey-
ing personnel and co-occurring wildlife. In addition, accruing evidence indicates that
eDNA detection may be more sensitive at detecting rare species than conventional
methods (e.g. Dejean et al. 2012; Biggs et al. 2015).

Silver carp (Hypophthalmichthys molitrix (Valenciennes, 1844)) was first intro-
duced to South Africa in 1975, when individuals from a German population were
donated to the Marble Hall experimental fish farm adjacent to the Olifants River
(Liibcker et al. 2014). It was suspected to have spread into the wider Olifants sys-
tem, including the Olifants River, Lake Flag Boshielo (an impoundment on the
Olifants River), and the Massingir dam, Mozambique (Sara et al. 2018), although
the extent of its overall distribution remains uncertain. Liibcker et al. (2014) pro-
posed that most of northeastern South Africa was suitable for colonization by this
species. Here, we use eDNA to assess presence of silver carp in the Olifants River in
Kruger National Park (South Africa) and Limpopo National Park (Mozambique) in
southern Africa. We determined the ability to detect silver carp in the system using
eDNA and comment on the utility of this method in the field to reduce potential
human-wildlife conflict.

Methods

Study organism and sites

Silver carp is a highly invasive fish extensively introduced from its native range in east-
ern Asia to Europe, North America, and southeast Asia (DeGrandchamp et al. 2007).
It is a highly effective filter feeder, consuming a range of microscopic forage that is
channeled into high growth and fecundity rates (DeGrandchamp et al. 2007). Experi-
mental and modeling data indicate that many populations of native North American
fishes may be extirpated through direct competition or cascading trophic effects result-
ing from silver carp introduction, thus further spread in southern Africa is worrisome
(Zhang et al. 2015).

We identified 12 sites in the Olifants watershed (Table 1; Figure 1) as suitable
locations for sampling based upon site access and anecdotal evidence of carp capture,
and to determine if a large hydroelectric dam protected the Upper Olifants from be-

34 Steven Crookes et al. / NeoBiota 56: 31-47 (2020)

-
pa)
ras
=:
Cc
om
ia)

Longitude

Figure |. Map of sampling locations for silver carp eDNA within the continent of Africa (A). All sam-
pled sites within southeast Africa (B) are shown as red circles. The two areas sampled are the Olifants
system (Olifants and Letaba Rivers, grey inset) and the Komati (Sabie River, red inset). Also shown is the
site of introduction and escape of silver carp in South Africa (red star) and the location of the Massingir
dam, Mozambique (yellow star). Dotted line delimits the South Africa - Mozambique border.

Table |. Sampling information and results of the qPCR analysis for the presence of silver carp eDNA at
twelve sites in the Olifants watershed ‘PCR’ column indicates how many duplicate reactions for each of
the three biological replicates were positive. The final column shows the mean Cq values across all positives

(omitting non-amplifications) and their standard deviation (SD).

SiteCode Date Site Description River Geographic Co-Ordinates PCR Mean Cq + SD
Ol 16/06/15 Olifants River Weir Olifants -24.055824, 31.720335 0/0/0 N/A

O2 16/03/15 East of Olifants Camp Olifants -23.982616, 31.775594 2/2/0 34.622 + 0.688
O3 16/03/15 Olifants/Letaba Confluence Olifants -23.989445, 31.826483 2/0/1 31.744 + 0.087
O4 17/03/16 Olifants River Gorge Olifants -23.985550, 31.848714 2/2/2 32.434 + 1.267
O5 17/03/15 South Africa Border Olifants -23.956183, 31.881781 0/1/2 34.884 = 1.737
O6 17/03/15 Letaba River Weir Letaba -23.942911, 31.731429 2/2/2 33.809 + 4.149
O7 19/03/15 Upper Olifants, Above Dam Olifants -23.943567, 31.902952 2/0/2 33.914 + 2.261
O8 19/03/15 Massingir Dam, Pelagic Olifants -23.921727, 31.956548 0/2/2 30.615 42.161
O9 20/03/15 Massingir Dam Wall Olifants -23.873332, 32.145614 1/0/2 33.549 + 0.639
O10 21/03/15 | Coromana, Mozambique Sabie -25.184227, 32.033023 1/2/0 35.038+ 0.091
Oll 21/03/15 Coromana, South Africa Sabie -25.185171, 32.031348 2/2/1 34.238 + 1.619
O12 21/03/15 Upper Sabie, South Africa Sabie -25.183838, 32.030184 2/0/1 36.208 = 2.248

ing invaded from downstream sites. Three sites (O10—O12) were located in the Sabie
River, part of the neighbouring Komati watershed, to determine if the carp has spread
beyond the borders of the Olifants watershed.

Water collection, transportation and filtration

At each site, three 2 L water samples (3 x biological replicates) were collected in sterile
(10% bleach solution (6% w/v sodium hypochloride)) polycarbonate plastic Nalgene

eDNA detection of silver carp in Africa 35

bottles. Unless access was difficult, all sampling was conducted by reaching from the
bank to extract a sample from the top 5 cm of surface water in the littoral zone. Using
single-use gloves for each sampling event, each sterile bottle was swept through the
surface until filled. Each sample was immediately placed in a bleach-sterilized cooler
and held at 4 °C during transportation back to the laboratory. Where direct access to
the riverbank was difficult (e.g., large stretches with high embankments and prolific
scrub vegetation), the site was accessed by boat and water was collected from as close to
the shoreline as possible. At each site, a single Nalgene bottle containing 2 L distilled
water (environment blank control) was opened and exposed to the environment before
the top was resealed.

All water samples were filtered immediately upon return from the field in a central
bleach-sterilized laboratory located in Skukuza, Kruger National Park, or in an ad-
hoc, bleach-sterilized field laboratory near Massingir, Mozambique. Water was vacuum
pumped through 1.2 um pore glass fibre filters (47 mm diameter, VWR 696-filter).
The filtration set-up included a tripartite manifold system of three funnels, each pro-
visioned with a magnetic seal that securely clasped a filter between the funnel and the
pump. Each biological replicate was filtered simultaneously in each of the three fun-
nels (3 x filters per 2 L sample). After each sample was filtered, the entire apparatus
and surrounding area was bleached sterilized, wiped with distilled water, and left to
dry before proceeding with the next sample. After each filtration event, a separate set
of sterile forceps was used to submerge each filter in a 2 ml Eppendorf tube contain-
ing 95% ethanol for storage at -20 °C. All samples were shipped to Canada for eDNA

detection analysis.

eDNA extraction

eDNA extraction was performed in a dedicated extraction space. Using a protocol
adapted from Dougherty et al. (2016), filters were cut into quarters and placed in
tubes containing 20 ul 1 mm-diameter glass beads and 500 ul of modified (Coyne et
al. 2005) CTAB (2% w/v cetyltrimethylammonium bromide, 2% w/v polyvinylpyr-
rolidone, 1.4M NaCl, 100mM Tris-HCL, 20nM EDTA) buffer pre-warmed to 65
°C in a heat block. Samples were homogenized using an unequal gravity FastPrep
F120 homogenizer (Thermo Savant Instruments, Ltd) at 6.5 m sec’ for two minutes
and then incubated at 65 °C for two hours. 500 ul chloroform-isoamyl alcohol was
added to each tube, mixed over-end for 5 minutes and centrifuged at 13,000 g for 15
minutes. The upper aqueous phase was transferred to a new tube containing 500 ul
ice-cold isopropanol and 250 pl NaCl solution to precipitate out the DNA whilst
incubating at -20 °C overnight. After incubation, the tubes were centrifuged again
at 13,000 g for 15 minutes to allow DNA to pellet at the bottom. ‘The supernatant
was discarded and 200 ul ethanol wash added prior to vortexing and centrifugation
at 13,000 g for three minutes. A second ethanol-washing round followed prior to
carefully discarding the ethanol and drying the pellet in a vacufuge at maximum

spin before being centrifuged for 10-15 minutes at 35 °C. The DNA pellet was re-

36 Steven Crookes et al. / NeoBiota 56: 31-47 (2020)

suspended in 100 ul 1X T/E. buffer and heated to 55 °C for ten minutes. After each
filter had been extracted from the filter paper, the eluted DNA from each quarter
filter paper was pooled into a single tube before decanting into 50 uL aliquots to be
stored long-term at -20 °C.

eDNA quantitative real-time polymerase chain reaction (qPCR) detection

The Asian carp invasion in North America has resulted in development of numerous
eDNA assays to detect all four problem species, including two in the genus Hypoph-
thalmichthys (Jerde et al. 2013). Because only silver carp has been known to have been
introduced into the Olifants River and surrounding systems, we chose the assay with
the highest sensitivity to detect fish in the genus Hypophthalmichthys (Wozney and
Wilson 2017). We chose the CIM primer set (Carp Taqman Multiplex 1), without
using the Taqman probe, to increase sensitivity, designed and optimized in singleplex
reactions (Wozney and Wilson 2017) to amplify both species. The original intent of
this assay was to amplify both target species but discriminate based on differential
probe binding (see Wozney and Wilson 2017 for assay specifications).

All qPCR reactions were performed in a laboratory with no previous history of
Asian carp tissue or DNA samples. For each pooled eDNA extract, two duplicate
technical replicates (PCR reactions) were performed. For all 12 sites, this tallied to 72
reactions in total, six per site/location and two per biological replicate. Alongside the
target reactions (and environmental blank — one per location), two no-template con-
trols were run to control for qPCR reagent/sample contamination. All reactions were
performed on a single reaction plate, thereby eliminating inter-run variance in PCR
results. All reaction volumes were 20 ul, consisting of 200 nM of each primer (0.4 ul),
10 pl of PowerUp SYBR green mastermix (Applied Biosystems, USA) and the remain-
ing volume (9.2 pl) made up of eDNA extract providing the template for the reaction.
To determine whether PCR inhibition may impede positive detection, we reassessed
each sample using a separate internal positive control (IPC) assay that consisted of a
primer and probe set that amplify a unique, and not found in nature, manufactured
DNA sequence. We used the Taqman-probe based IPC developed by Gasparini et
al. (2020) in which 200 copies of the artificial template were added to every reaction
alongside 9.2 uL of the eDNA sample with Taqman Universal Mastermix II (Applied
Biosystems, USA) in lieu of PowerUp SYBR Green. Inhibition was inferred to have
occurred if the PCR Cq values were delayed by more than 1 Cq value relative to the
Cq value of NTC control (i.e. pure water added as template). qPCR was performed on
a 96-well CFX Thermal Cycler (BioRad) and run according to the published protocol
(Wozney and Wilson 2017). PCR data were recorded in two ways: 1) successful ampli-
fication defined as the production of an amplification curve that passed a threshold of
fluorescence; and, 2) of those reactions that amplified successfully, the Cq (quantifica-
tion cycle) value, whose quantity is inversely proportional to the amount of silver carp
DNA in the original sample, was documented.

eDNA detection of silver carp in Africa 37

Statistical analysis

We initially performed a post-hoc power analysis to confirm that the sampling design
was sufficient to correctly reject the null hypothesis of no detection, thus boosting
confidence in any negative finding. We used Olson et al.'s (2012) method:

a Il/_,(—d)

where y = probability of a false negative (upper boundary of 0.05 (alpha)), j = number
of samples to be subject to qPCR, and d is the proportion of samples (out of three
biological replicates) that yield = 1 eDNA positive qPCR detection, to determine the
predicted minimal number of samples necessary to achieve 95% power, predicated on
our observed data.

Following the adoption of eDNA as a proxy of occupancy in habitat occupancy
models (e.g. Schmidt et al. 2013), we performed a simple estimation of detection
probability (p), site occupancy (WV) and the probability of detecting silver carp eDNA
per sampling event (8). As eDNA sampling is inherently hierarchical, Bayesian models
predicting the three detection parameters are less vulnerable to increasing variability in
the data than frequentist methods (Doll and Lauer 2014). Although occupancy mod-
eling is most useful when co-estimating variables that may influence the detection of
the organism by its proxy (i.e. via eDNA molecules), hierarchical models that estimate
base values of each of the above parameters would result in more accurate values than
would a naive calculation based on naked data alone (Dorazio and Erickson 2018). To
estimate these parameters, we performed the analysis using the R package ‘“eDNAoccu-
pancy’ (Dorazio and Erickson 2018) employing 500,000 Markov Chain Monte Carlo
iterations after which model parameters were fitted (discarding first 10% as burn-in).
Finally, an analysis of variance of Cq value across sites and by river system, which were
encoded as categorical variables, was performed using the ‘aov’ function in R.

To assess if the Olifants River is suitable for silver carp spawning, a preliminary
assessment was completed using methodology developed by Heer et al. (2019). The
assessment determined whether a tributary is suitable to potential Asian carp spawn-
ing using a decision tree that categorized the river into four categories (not suitable,
minimally suitable, moderately suitable, and highly suitable). It was based on growing
degree-days (base 15 °C; GDD15) accumulated in a 12-month period spanning 2017—
2018, an estimated hatching distance, and water temperature and velocity thresholds
for spawning. ‘There were five thresholds: 633 GDD15 within a year to achieve matu-
ration, minimum water temperature of 17 °C to initiate spawning run, estimated dis-
tance to hatch is less than the unimpounded length of the river, flow velocity spike of
0.7 m/s to initiate spawning, and 900 GDD15 to trigger mass spawning. To complete
the assessment, mean daily water temperature was obtained for the Mamba weir and
mean daily velocity was estimated at the Balule weir based on weir dimensions and
measured stage and discharge data. One year of temperature and velocity data were
available, July 1, 2017 to June 30, 2018.

38 Steven Crookes et al. / NeoBiota 56: 31-47 (2020)

Results

Molecular eDNA detection

Every site except for Ol was positive for silver carp eDNA in at least one technical
replicate across three biological replicates (Table 1). Apart from site O1, all locations
were positive for silver carp eDNA and had detection levels > 50 % (at least three
out of six technical PCR replicates). Sites O4 and O6 had 100% detection across the
board, although they did not have the lowest mean Cq value, which occurred at site
O8 (Mean Cq = 30.62 = 2.16). Overall mean Cq across all successful PCRs and all
sites was 33.63 = 2.39. All samples, apart from O3, O9 and O10, did not show qPCR
inhibition as all IPC Cq values were within the margin of acceptability (i.e. all samples
were within 1 Cq of the mean NTC IPC value of 30.63). In these cases, a PCR nega-
tive could be correctly interpreted as a lack of target CDNA molecules in the template
volume. However, for sites O03, O9 and O10, the IPC did not amplify at all (Cq = 0),
indicating complete inhibition of the IPC.

Detection analysis

The percentage of successful biological replicates yielding at least one detection (d)
was 69.40 %. Post-hoc power analysis revealed that the minimal predicted number of
biological replicates per site should be two for this system, assuming 95% power, thus
providing confidence that non-detection of silver carp at site O1 was unlikely due to a
lack of appropriate sampling intensity. An occupancy analysis resulted in a high level of
detection of silver carp in the study area. Estimated global probability of detection (p)
was 0.849, inferred site occupancy (VY) was 0.892, and sample detection probability
(0) was 0.754. An analysis of variance indicated a near-significant effect of watershed
and sampling site upon the levels of silver carp eDNA (F-value = 2.910, p = 0.0657;
and F = 2.005, p = 0.0652, respectively) (Figure 2 and Suppl. material 1: Table S1).
However, two-way ANOVA revealed that watershed was more important in determin-
ing silver carp occupancy than individual site effects (F-value = 3.298, p = 0.0494 and
F-value = 1.682, p = 0.140 for River and Site, respectively). The concentrated aggre-
gate of sampling sites on the more southerly Sabie River tended to have higher mean
Cq values — and thus lower quantity of silver carp eDNA — than most of those in the
northerly Olifants system in South Africa (Figure 2).

Spawning habitat analysis

Preliminary spawning assessment indicates that the Olifants River was a minimally ap-
propriate habitat in 2017—2018 (Figure 3). The river was always above the minimum
17 °C temperature, but never achieved flow sufficient to reach the higher levels of suit-

eDNA detection of silver carp in Africa 39

40.0

”
®

010
011 [sab River

03 Olifants
Watershed
06 (including
O7 Letaba River
were (O6))

GEC
fe)
PS

30.0

27.5
Sampling Location

Figure 2. Levels and variance of qPCR detection (y-axis) of silver carp DNA among sampling sites

within and between rivers in the Olifants system and Sabie River (x-axis).

1000 2000 3000

900 GDD15

Water Temperature (°C)
20 30
0
Growing Degree Days

0 5 10

0.8
0 20 40 60 80
River Length (km)

Water Velocity (m/s)
0.4

0.0

Jul Sep Nov Jan Mar May Jul

Figure 3. Results of a silver carp spawning suitability model for the Olifants River, July 1, 2017-June
30, 2018 A growing degree days (base 15). 633 GDD15 (red dashed line and solid red vertical line) is
required for maturation and 900 GDD15 (blue dashed line and solid blue vertical line) is required for
initiation of mass spawning B mean daily water temperate. Minimum temperature required (red dashed
line) is always exceeded C unimpeded river length required for egg hatching to occur D mean daily water
velocity. Flow spike required for high suitability is not achieved.

40 Steven Crookes et al. / NeoBiota 56: 31-47 (2020)

ability. Based on a July 1 winter start date, 633 GDD15 would have been achieved by
early October and 900 GDD15 by early November. Following these spawning initia-
tion dates, the length of unimpeded river required for egg hatching ranged 15—40 km,
which is readily available in the Olifants River.

Discussion

Silver carp range expansion as inferred from eDNA presence data

Environmental DNA detection confirmed the presence of silver carp eDNA through-
out the sampled areas of the Olifants River and neighbouring systems (where sam-
pled), except for Site O1. Our study adds to the mounting evidence confirming this
technique as a sensitive tool to monitor highly invasive Asian carps generally (Jerde
et al. 2011, 2013; Turner et al. 2015; Wozney and Wilson 2017) and silver carp in
particular (Amberg et al. 2015; Coulter et al. 2019; Stepien et al. 2019). Our study is
the first to use a targeted eDNA approach to monitor for the presence of Asian carps
on the African continent. However, the detection of silver carp was not without im-
pediment, as the IPC results indicate that, in some sections of the Olifants and Sabie
rivers, low levels of DNA were beyond the level of sensitivity of the assay. The IPC was
set at 200 copies per reaction, so even slight inhibition was detected. As many eDNA
species targets are rare and shed low levels of DNA, PCR inhibition is a potentially
huge issue for delineation of the invasion front and detection of rare species where
low abundances should translate into low eDNA levels and, possibly, false negatives.
However, as we observed 3/6 silver carp qPCR detections for each of the IPC inhibited
sites, we can conclude that, although silver carp eDNA levels were lower than at the
other sites (except O1), they often exceed 200 copies per 9.2 wL (qPCR template vol-
ume) and are thus well within the level of detectability of this assay. As PCR inhibitors
are present in the systems of study, it is likely that silver carp Cq values were delayed,
if not outright inhibited, and thus it would be difficult to translate Cq values to copy
numbers. Moreover, silver carp eDNA levels exhibit a nonlinear relationship with carp
density (Coulter et al. 2019).

eDNA detection levels were high (except Site O1), with at least half of all GPCR
reactions positive for silver carp. Notwithstanding the likely nonlinear relationship be-
tween eDNA levels and population densities, the fact that silver carp was detected in
half of all qPCR reactions at three sites for which the IPC (200 copies per reaction)
was completely inhibited is consistent with a large, established population constantly
shedding a lot of eDNA into the surrounding environs. These results indicate that
silver carp is pervasive in the Olifants watershed, supporting the proposition that this
species is established within South Africa (Ellender and Weyl 2014). The data confirm
the expansion of the silver carp range into Kruger National Park from elsewhere in the
Olifants system, either from upstream at the Flag Boshielo Dam, Mpumalanga (Britts
2009) or from downstream (Schneider 2003). The data also confirm that the Massingir

eDNA detection of silver carp in Africa 4]

population is still extant and healthy and corroborates local reports of the successful
fishing of this species for local consumption. It is unlikely that escapees, or the inter-
mittent use of silver carp as a baitfish, would yield a similar pattern of strong detections.
All field controls were negative, thereby discounting the possibility of spurious results
stemming from contamination as a factor responsible for positive detection results.

One criticism of eDNA utility in lotic systems, however, is the potential system-
ic bias introduced by the directional flow of water. Until recently, the conveyance of
eDNA downstream was thought to operate primarily on relatively small spatial scales
(up to 12 km) varying by hydrology, season and target organism (e.g. Deiner and Alter-
matt 2014; Shogren et al. 2017). Interestingly, Pont et al. (2018) reported eDNA trans-
port distances of over 100 km in the voluminous River Rhone, France. However, if all
positive eDNA tests in the upper Olifants River resulted from advection from a single
upstream source, one would expect the eDNA signal to attenuate (e.g. show rising Cq
values) with increasing distance from that source, albeit at low flow conditions (Jane
et al. 2015). We observed no such trend with our data. The silver carp eDNA signal
was strong above and below Massingir Dam, a large reservoir of the Olifants River on
the border of South Africa and Mozambique. This is consistent with Schneider (2003),
who reported silver carp invading the Massingir Dam in 1996/1997 and dispersing
downstream into the mainstream Limpopo River during floods in 2000 (Schneider
2003). Natural dispersal events may also be supplemented by accidental and intention-
al anthropogenic release of larval and juvenile carp, which are hard to identify during
early stages of ontogeny and which may be used as baitfish. In the Great Lakes region
of North America, juvenile silver carp have been positively identified using qPCR as a
part of the selection of baitfish available to anglers (Stepien et al. 2019).

Affirmation of habitat suitability for establishment of silver carp

Due to the long unimpounded distance of the Olifants River upstream of Massingir
Dam, along with high temperatures, this system is suitable for silver carp spawning.
Temperature data in the river indicate that maturation can occur by early October
and mass spawning by early November based on a July 1 winter start date. However,
silver carp maturity can be reached within approximately three months, indicating that
spawning could potentially occur at any point in the year. The minimum temperature
required for spawning (17 °C) was always met in this system. There exists some un-
certainty with this approach, primarily due to the estimate of velocity based on weir
dimensions and the assumption of linearity in the hatching distance.

Our findings are consistent with those derived from ecological niche modelling, in
which the middle-lower Olifants River was assessed to have a high predicted climatic
suitability for silver carp establishment (>75%) (Liibcker et al. 2014). However, the
Komati watershed lies in an area predicted to have 80-100 suitability for the estab-
lishment of the silver carp. The modelling result is consistent with our observation of
eDNA in the Sabie River, albeit at lower levels than in the Olifants. Interestingly, silver

42 Steven Crookes et al. / NeoBiota 56: 31-47 (2020)

carp has yet to be visually observed here, thus eDNA detection would be the first re-
cord of its occupancy in the Sabie River. The discrepancy in eDNA levels between the
two watersheds is likely a function of the time since invasion. Silver carp has occupied
the Olifants River at least since escaping captivity in the upper reaches of the river in
1992 (Britts 2009). Assuming the eDNA signal to be redolent of occupancy of silver
carp in the Sabie River, it may be that the species has yet to establish and expand its
range, although such an event may be anticipated if invasion follows a stepping-stone
model of expansion (Alharbi and Petrovskii 2019). Indeed, the large floods caused by
Cyclone Eline (UNDP 2000) resulted not only in the flooding of several fish farms
containing silver carp in the lower Limpopo River floodplain in Mozambique, but also

linked the Limpopo and Komati Rivers in March 2000 (Schneider 2003).

eDNA detection as a tool for aquatic biomonitoring in dangerous systems

The adoption of eDNA detection methods is especially pertinent in areas in which
conventional monitoring can be inefficient or dangerous. These areas are often in the
tropics, and harbor much of the world’s biodiversity, including charismatic freshwater
megafauna (Carrizo et al. 2017). At the time of our survey (2015), we adopted best
practices for that time. This included taking physical water samples from the surface
of water in close proximity to a range of unpredictable predators (Nile crocodile) and
large herbivores (hippopotamus), which, while not without risk, was safer than deploy-
ing and collecting nets or physically entering the water. ‘This also reduced the risk of
accidental by-catch and accidental mortality of conservation priority taxa such as Nile
crocodile, which are susceptible to entanglement and drowning in sampling gear such
as gill nets (Ellender et al. 2016). As such, using eDNA to successfully detect the silver
carp validates this approach for use in dangerous systems. Using eDNA lowers the
risk of loss of equipment and time to pernicious and unpredictable events in the field.
Dangerous systems may not just be limited to those containing large animals but also
those with high levels of pollutants or pathogens, strong tidal or convectional currents,
or rapids and unpredictable, seasonal flows. In the intervening time period, onsite
eDNA developments have progressed so rapidly that much of the physical workflow is
automated and can be operated remotely, including performing onsite qPCR detection
(Thomas et al. 2018, 2019). Onsite methods also have the advantage of reducing the
risk of laboratory-based contamination and the degradation of rare eDNA molecules
in transit (attenuating both Types I and II error). Thus, eDNA detection methodology
is suitable for highly biodiverse and remote areas such as Kruger National Park.

The success of this pilot project to investigate the utility of using eDNA detec-
tion to identify the presence of the silver carp in two watersheds in southern Africa
cannot be disputed, at least for AIS. However, at-risk species (SAR) show similari-
ties with invasion-front species in that often their a priori distribution is unknown
or merely suspected, and whose populations are fragmented and characterized by
few individuals. Yet, eDNA has been shown to be just as effective at targeting SAR
as it has AIS (e.g. Balasingham et al. 2018; Currier et al. 2018). Furthermore, the

eDNA detection of silver carp in Africa 43

molecular methods used to conduct eDNA monitoring may also be extended. For
example, qPCR-based tools can also be used to screen eggs to confirm spawning
success in suitable habitat identified by a posteriori modelling or previous eDNA
detection (Fritts et al. 2019).

Conclusion

We recommend that eDNA detection be used as part of the conservation biologist’s
toolbox when considering the management of invaders in dangerous aquatic ecosys-
tems in the tropics and elsewhere. Moreover, future investigations should take into
account the complexities of hydrodynamics when monitoring rivers, potentially by
using hydrodynamic models (e.g. Garcia et al. 2013), which could be integrated with
eDNA data (e.g. Carroro et al. 2018). Our results indicate that the Olifants River is
suitable for silver carp spawning and could reach high suitability if it coincided with a
flow spike that exceeds 0.7 m/s. These projections were validated by the eDNA detec-
tion data. We highlight the combined power of eDNA detection and habitat model-
ling tools to predict not only current distribution — and habitat suitability — of a target
organism, but to also forecast which areas are at risk of imminent invasion. Adopting
an integrative methodology, combining aspects of molecular, theoretical and field ecol-
ogy to better effectively manage extremely limited resources is beneficial to optimizing
efforts to conserve important refuges of aquatic biodiversity in the coming decades.

Acknowledgements

We thank Pauli Viljoen for logistical and field support. We thank South African Na-
tional Parks for financial and logistical support. OLFW acknowledges support received
through the National Research Foundation — South African Research Chairs Initiative
of the Department of Science and Innovation (Grant No. 110507). The CIB/DSI
Centre of Excellence for Invasion Biology (CIB) for continued support. LCF acknowl-
edges support from the DSI-NRF Centre of Excellence for Invasion Biology, Dept.
of Botany and Zoology, Stellenbosch University, and the National Research Founda-
tion (of South Africa, Project Numbers IFR2010041400019 and IFR160215158271).
NEM, DDH and HJM were supported by NSERC Discovery grants and the NSERC
CAISN Strategic Network, while HJM also was supported by a Canada Research

Chair in Aquatic Invasive Species.

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Supplementary material |

Table S1

Authors: Steven Crookes, Tej Heer, Rowshyra A. Castafieda, Nicholas E. Mandrak,

Daniel D. Heath, Olaf L.E Weyl, Hugh J. Maclsaac, Llewellyn C. Foxcroft

Data type: analytical table

Explanation note: ANOVA of eDNA detection levels (Cq) across sites and rivers.

Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODDbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.

Link: https://doi.org/10.3897/neobiota.56.47475.suppl1