Environmental DNA and Hydroacoustic Surveys for Monitoring the Spread of the Invasive European Catfish (Silurus glanis Linnaeus, 1758) in the Guadalquivir River Basin, Spain

Academic Editor: Kenji Saitoh Received: 26 September 2024 Revised: 20 December 2024 Accepted: 22 December 2024 Published: 20 January 2025 Citation: Coya, R.; Rodríguez-Ruiz, A.; Fueyo, Á.; Orduna, C.; Miralles, L.; de Meo, I.; Pérez, T.; Cid, J.R.; Fernández-Delgado, C.; Encina, L.; et al. Environmental DNA and Hydroacoustic Surveys for Monitoring the Spread of the Invasive European Catfish (Silurus glanis Linnaeus, 1758) in the Guadalquivir River Basin, Spain. Animals 2025,15, 285. https://doi.org/ 10.3390/ani15020285 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). Article Environmental DNA and Hydroacoustic Surveys for Monitoring the Spread of the Invasive European Catfish (Silurus glanis Linnaeus, 1758) in the Guadalquivir River Basin, Spain Ruth Coya 1, Amadora Rodríguez-Ruiz 2, Álvaro Fueyo 1,3, Carlos Orduna 4, Laura Miralles 1, Ilaria de Meo 4, Trinidad Pérez 1, Juan Ramón Cid 4, Carlos Fernández-Delgado 5, Lourdes Encina 2,*, Yaisel J. Borrell 1,* and Carlos Granado-Lorencio 2 1Department of Functional Biology, Genetics, University of Oviedo, 33006 Oviedo, Spain; [email protected] (R.C.); [email protected] (Á.F.); [email protected] (L.M.); [email protected] (T.P.) 2Department of Plant Biology and Ecology, University of Sevilla, 41012 Sevilla, Spain; [email protected] (A.R.-R.); [email protected] (C.G.-L.) 3Environment and Sustainability Area, Taxus Medio Ambiente, 33006 Oviedo, Spain 4EcoFishUS Research S.L.L., 41009 Sevilla, Spain; [email protected] (I.d.M.); [email protected] (J.R.C.) 5Department of Zoology, University of Cordoba, 14071 Cordoba, Spain; [email protected] *Correspondence: [email protected] (L.E.); [email protected] (Y.J.B.) Simple Summary: Introduced into the Ebro Basin in Spain in 1974, the invasive European catfish is now present in almost all Spanish river basins. This includes the Guadalquivir River, where it threatens native species and the ecological biodiversity of the Doñana National Park. This study provides valuable information on its current distribution using non-invasive methods such as hydroacoustics and environmental DNA and highlights the need for urgent control measures. Abstract: The European catfish (Silurus glanis Linnaeus, 1758) was introduced into the Ebro Basin in Spain in 1974 for recreational fishing. Since then, the species has spread throughout the country’s river basins, reaching the Iznájar Reservoir (Guadalquivir River Basin) in 2011. This area is of great ecological and economic relevance, especially as it includes the Doñana National Park, one of the most important nature reserves in Europe. Recently, the presence of catfish has been reported in the lower reaches of the river. In this work, we used non-invasive vertical and horizontal hydroacoustic surveys and environmental DNA river sampling to unravel the actual distribution and dispersal pattern of the species in the lower Guadalquivir River. The hydroacoustic profiles and the species-specific detections by real-time PCR (qPCR) and droplet digital PCR (ddPCR) showed that these non-invasive methods allow the detection and quantification of catfish and provide valuable information on the species’ presence. We have confirmed the presence of catfish in most of the study area, including downstream areas of the Guadalquivir Basin. The results suggest the possibility of other isolated introductions and/or human-mediated movements of specimens, and imply that a coordinated catfish prevention and mitigation strategy is therefore urgently needed. Keywords: early detection; monitoring; echosounder; eDNA; Doñana National Park Animals 2025,15, 285 https://doi.org/10.3390/ani15020285 Animals 2025,15, 285 2 of 20 1. Introduction Aquatic invasive species (AIS) are increasingly threatening ecosystems globally, causing biodiversity imbalances and having a substantial impact on economic enterprises [ 1 ]. Among the ecological impacts, AIS can significantly affect local biodiversity, potentially leading to the extinction of native species [ 2 ]. These impacts can arise from direct biotic interactions with the native fauna, such as competition and predation, as well as indirect changes in habitat conditions. In aquatic environments, organisms are characterised by strong trophic linkages that can be altered by introduced species. Invasive species in aquatic environments have strong and relatively constant ecological impacts [3]. Among invasive species in continental freshwater and low salinity (estuarine) bodies of water, the European catfish (Silurus glanis, Linnaeus, 1758) stands out as the largest fish in Europe excluding the diadromous sturgeon species [ 4 ]. Due to its piscivorous diet, the catfish poses a risk to native species and ecosystems [ 5 – 8 ]. Catfish were intentionally introduced in the Ebro Basin in Spain in 1974 and have since spread throughout the country’s river basins, primarily in the context of recreational fishing [ 6 ]. In July 2011, an individual of this species was captured in the Iznájar Reservoir, marking the first record of the European catfish introduced into the Guadalquivir River Basin (Figure 1) [ 9 ]. In 2015, the species was cited in the Rivera de Huelva River, a tributary on the right bank of the Guadalquivir River located more than 200 km downstream of the Iznájar Reservoir site [10]. Since then, the species has progressively appeared in different areas of the lower Guadalquivir River. The lower area of the Guadalquivir River is home to a large number of aquatic species of great biological and economic value, making it the most important aquatic biodiversity hotspot in Andalusia, Spain [ 11 – 15 ]. The site covers 50,720 hectares of one of the largest wetlands in Europe and is the last area of largely undisturbed marshland within this ecosystem [ 16 , 17 ]. This region includes the lower Guadalquivir estuary, the southernmost river–estuary–delta system in continental Europe. Increasing human pressure and limited understanding of its dynamics have exacerbated socio-economic and environmental conflicts in recent years [ 18 ]. In addition, the biodiversity of the Doñana National Park and its associated ecosystems is threatened by intensive agriculture, which causes anthropogenic eutrophication of its waters [ 19 ], as well as by high groundwater use, pollution, hydrological infrastructure, climate change, and invasive species, which have significantly altered the ecological functioning of these systems [ 15 ]. As an invasive species, the catfish threatens biodiversity, as well as local economies and culture [ 20 – 22 ]. For this reason, it is important to study catfish presence, distribution, and dispersal patterns in ecologically sensitive areas such as the lower Guadalquivir. This has been the objective of the ’STOPSILURO’ (www.stopsiluro.es, accessed on 20 December 2024) project, launched in 2023 and targeting the development of control programs for this species that minimise its impact and prevent its spread in these areas of high ecological or socio-economic value. Traditionally, aquatic species monitoring has depended on visual detection and often the capture of organisms. However, these methods are costly and labour-intensive [ 23 ]. In recent years, there have been significant advances in technologies for remote and early detection of invasive species. Hydroacoustics has proven to be a highly suitable tool for the quantitative characterisation of fish populations and their spatio-temporal distribution, as it allows large volumes of water to be sampled with great efficiency and high speed of data acquisition, which is why its use has increased in recent decades [ 8 , 24 , 25 ]. Regarding early detection, environmental DNA (eDNA) is considered one of the most promising analysis and management tools due to its high sensitivity in species detection [26–28]. Animals 2025,15, 285 3 of 20 Animals 2025, 15, x FOR PEER REVIEW 9 of 20 catfish detection in 13 sites (76.5%), while we obtained 11 positive sites with ddPCR (64.7%) from the 17 sample sites (Figure S5). A comparison of the results shows that 76.5% of the sites were concordant in terms of the presence of catfish eDNA between the two techniques tested. The qPCR results showed an average of 5.5 × 105 catfish eDNA copies (ranging from 108.2 to 3.4 × 106). Using the ddPCR methodology, the values for accepted generated droplets were on average 17,375 and ranged from 12,284 to 19,592. From this, an average of 454.5 positive catfish eDNA ddPCR copies were estimated (range: 60 to 1260 copies). Statistical analyses revealed a significant positive correlation between the logarithmically transformed copy number data obtained by qPCR and ddPCR for catfish environmental DNA (p-value < 0.01) (Figure S6). Figure 1. Detection and quantification of Silurus glanis using eDNA and hydroacoustic surveys in the Guadalquivir River Basin. (A1) Iberian Peninsula showing the Andalusian region. (B1) The Andalusian region, showing the Guadalquivir River Basin and including the Doñana National Park location. (C1) Detailed map of the Guadalquivir River Basin in the province of Sevilla showing the locations sampled for hydroacoustics and catfish eDNA (qPCR and ddPCR). (C1.1) Detail of the El Gergal Reservoir in Sevilla. (D1) Detailed map of the Iznájar Reservoir in the Guadalquivir River Basin, province of Córdoba. In this figure, the squares represent catfish eDNA qPCR quantification results while the circles around them represent catfish eDNA ddPCR quantification results. For both molecular methods, a heatmap from color white to color red dots gradient indicates the Figure 1. Detection and quantification of Silurus glanis using eDNA and hydroacoustic surveys in the Guadalquivir River Basin. (A1) Iberian Peninsula showing the Andalusian region. (B1) The Andalusian region, showing the Guadalquivir River Basin and including the Doñana National Park location. (C1) Detailed map of the Guadalquivir River Basin in the province of Sevilla showing the locations sampled for hydroacoustics and catfish eDNA (qPCR and ddPCR). (C1.1) Detail of the El Gergal Reservoir in Sevilla. (D1) Detailed map of the Iznájar Reservoir in the Guadalquivir River Basin, province of Córdoba. In this figure, the squares represent catfish eDNA qPCR quantification results while the circles around them represent catfish eDNA ddPCR quantification results. For both molecular methods, a heatmap from color white to color red dots gradient indicates the number of copies obtained. Hydroacoustic data (aggregate counts) is also represented by a heatmap from white to green indicating the numerical density of individuals greater than 1 m, while the number of individuals per stretch is given numerically in a yellow circle. In (C1.1) each individual is displayed as a yellow dot (some of them overlap in the middle) and white dots indicate the location of the two negative water samples taken for eDNA analyses. The potential of hydroacoustic methodology as a useful tool for fish species management and control programmes, including exotic species, has been shown [ 8 ], although hydroacoustic equipment currently lacks the ability to identify species other than by body size or habitat distribution. However, in ecosystems where there is a clear differentiation of species based on their size, or where fish communities are monospecific or there is clear Animals 2025,15, 285 4 of 20 habitat segregation according to species, modern hydroacoustic techniques have been used as useful evaluation tools for fish species [ 24 , 25 , 29 ]. Regarding the European catfish, the possibility of using hydroacoustic techniques to evaluate and monitor its presence and abundance is of great relevance given the difficulty of catching this species with traditional fishing methods, which often leads to its underestimation [ 30 ]. Catfish are characterised by a high growth rate, especially during the first year of life [ 31 , 32 ]. Thus, the large size reached by this species at an early age, combined with the smaller size of the other species present in the study area, enables adult catfish specimens to be differentiated from the rest of the species using hydroacoustic methods. Previous studies have successfully applied the environmental DNA (eDNA) methodology for the specific detection of catfish, demonstrating its sensitivity [ 33 , 34 ]. Parrondo et al. [ 35 ] proposed the combination of comprehensive reviews of citizen alerts with verifications based on specific molecular techniques as a rapid, cost-effective, and truly accessible strategy for the early detection of catfish in Spain. This could serve as a preliminary step before more comprehensive information phases in management plans addressing invasive species [ 35 ]. Currently, it is feasible to design species-specific primers for rapid detection, even when DNA is partially degraded. DNA degradation can pose a challenge in natural environments or environmental samples, but advances in primer design techniques and DNA amplification overcome these difficulties [ 36 ]. By reducing the time and resources needed for analyses, the implementation of conservation and natural resource management strategies is facilitated, along with the study of biological populations and communities [36,37]. Environmental DNA allows not only detection but also quantification of the species studied through two molecular techniques: the quantitative polymerase chain reaction (qPCR) and the droplet digital PCR (ddPCR) [ 38 ]. The qPCR measures the copy number of the amplified target fragment using a calibration curve derived from serial dilutions of a sample with a known concentration. The threshold fluorescence level is computed from initial cycles, and the cycle number (CT value) correlates with the sample’s template copy number [ 39 ]. Therefore, quantification via qPCR relies on an indirect measure from a calibration curve, limiting its precision and reproducibility. In recent years, a new quantitative method, ddPCR, has emerged, directly quantifying DNA without reference curves. This involves partitioning the sample into thousands of droplets and independently conducting a PCR in each, detecting fluorescence at the process’s conclusion, as an endpoint measurement. Previous studies have demonstrated the effectiveness of both qPCR and ddPCR techniques in quantifying DNA concentrations of target species in eDNA from mesocosm experiments with known abundances and biomasses [ 40 – 42 ]. Estimates via ddPCR show less variation than qPCR, suggesting they can calculate eDNA concentration more accurately, enhancing the ability to estimate species abundance/biomass in eDNA studies [ 42 , 43 ]. However, not enough studies have been conducted on environmental samples to determine species distributions in natural habitats and the potential problems and benefits of both techniques. Environmental DNA-based technologies, coupled with the use of echosounders, can provide accurate and early detection of invasive species such as catfish, thus contributing to comprehensive monitoring of biodiversity and environmental impacts in different aquatic environments [ 44 ]. In this study, a dual mapping strategy was implemented along the Guadalquivir Basin, using an environmental DNA detection method for monitoring catfish based on the molecular techniques of real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR). The objective of this study was to determine the range of expansion of European catfish in the lower Guadalquivir and also to assess the complementarity and Animals 2025,15, 285 5 of 20 potential synergies between eDNA analysis and hydroacoustic methods for monitoring aquatic invasive species in riverine environments. 2. Materials and Methods 2.1. Hydroacoustic Study 2.1.1. Hydroacoustic Survey The acoustically surveyed area included three main zones, identified based on river morphology. The Alcalá del Río dam represents the final barrier before the Guadalquivir River flows into the sea. Therefore, we considered the section of the river upstream of the Alcalá del Río dam, extending to the Cantillana dam, as a separate area. This section forms the Alcalá del Río Reservoir (Figure S1). Next, we surveyed the section of the Guadalquivir River downstream of the Alcalá del Río dam up to its confluence with the Guadaíra River, covering a total of 45 km. The northern portion of this section, from the Alcalá del Río dam to South Sevilla, is narrower, with a width of approximately 150 m, while the southern portion, from South Sevilla to the confluence with the Guadaíra River, is wider, averaging around 300 m. Finally, we included the El Gergal Reservoir, which connects to the Guadalquivir River through the Rivera de Huelva tributary near North Sevilla. Sampling was conducted using an outboard boat, with navigation always carried out upstream and sailing speed maintained at a steady 4 knots. The sampling was conducted throughout the month of June of 2023, during daylight hours, between 8 a.m. and 9 p.m. While the European catfish is primarily nocturnal, the lack of administrative permissions for night-time navigation, due to the unsafe conditions in the surveyed river area, made sampling during night impractical. Nonetheless, studies have shown that it can be effectively detected during the day because the higher daytime temperatures keep them active [ 8 , 45 , 46 ]. For the hydroacoustic surveys, we used a Simrad EK60 echosounder (Simrad Kongsberg Maritime AS, Horten, Norway) with a split-beam circular transducer operating at 200 kHz (ES200-7 C). The transducer was installed on a stainless-steel frame (utility model number ES1279955, EcoFishUS and University of Sevilla, Sevilla, Spain). The structure was mounted on the side of the boat, providing the equipment with good stability during sailing. The hydroacoustic survey was conducted both vertically and horizontally to cover the entire ecosystem, including the surface and deepest zones. The surface water layer was surveyed using horizontal hydroacoustics, with the transducer positioned 120 cm below and parallel to the water surface. This ensured that the acoustic beam’s opening angle insonified a representative volume of the first 2 m of depth, avoiding the potential effects of engine noise and the near-field effect [ 47 – 49 ]. Zones with a depth greater than 2 m were surveyed using vertical hydroacoustics, with the transducer positioned 40 cm below the water surface and oriented towards the bottom. This configuration ensured that measurements taken below 2 m were reliable and unaffected by engine noise or the near-field effect [ 47 – 49 ]. The survey was conducted while navigating upstream in zigzag trajectories, except in areas where the river was too narrow for horizontal hydroacoustic surveys to insonify a representative volume. In such cases, parallel transects along the shores were performed, ensuring the insonification of the entire area. Before sampling, the acoustic unit was calibrated using a calibration copper sphere following the standard calibration method [ 50 ]. The pulse duration was 0.256 ms, and the pulse mode was set to the maximum offered by the echosounder for the depths studied. The position of the transducer was geo-referenced in real time using an Emlid Reach RS2 GPS (Emlid Tech Kft., Budapest, Hungary) placed on the transducer structure. Animals 2025,15, 285 6 of 20 2.1.2. Hydroacoustic Data Post-Processing The data stored during the echo surveys were processed using the hydroacoustic analysis software Sonar5 Pro 608.43 [ 51 ]. Prior to processing, the bottom line was set in all sampling files to be able to discriminate between fish and bottom echoes. Subsequently, various sources of noise that could lead to errors in fish detection, such as bubbles, vegetation, patches of phytoplankton, zooplankton aggregations, and other noise sources were removed. 2.1.3. Catfish Detection The acoustic signals were analysed to detect specimens that could potentially be catfish based on their size. To differentiate catfish from other fish species in the study area, a threshold of 1 m in total length was established for detected individuals. Among the species cohabiting the study area, some can reach lengths exceeding 50 cm, including the carp (Cyprinus carpio, Linnaeus, 1758), southern Iberian barbel (Luciobarbus sclateri, Günther, 1868), striped mullet (Mugil cephalus, Linnaeus, 1758), thinlip grey mullet (Chelon ramada, Risso 1826), twaite oliveshad (Alosa fallax, Lacépède, 1803), largemouth bass (Micropterus salmoides, Lacepède, 180), or European eel (Anguilla anguilla, Linnaeus, 1758) (Table S1) [ 6 , 52 – 54 ]. While this size threshold excludes smaller catfish, it minimises potential overlap with other species, ensuring that all detected specimens are unequivocally catfish, which is the primary focus of the survey. For the analysis of transects from vertical surveys, the echo counting method based on single echo detection (SED) was used. The Target Strength (TS)-length relationship equation published by Love [ 55 ] and included in the standard UNE-EN 15910 [ 56 ] was employed for the TS-size conversion. In this case, the equation relates the TS to the total length (TL) of the fish. Size ranges with TS greater than − 25.81 dB correspond to sizes greater than 1 m, so they were counted as catfish. All detections corresponding to individuals within these size ranges were georeferenced using the information stored during simultaneous GPS-acoustic surveying. For the analysis of transects from horizontal surveys, the trace counting method based on fish tracks was employed. The equation used for the TS-size relationship was developed by Kubeˇcka and Duncan [ 57 ], as specified in the UNE-EN 15910 standard. This equation considers the different orientations in which a specimen can be insonified during horizontal surveys. Unlike vertical surveys, where the fish is almost always insonified dorsally, in a horizontal survey, a fish can be insonified in different positions, ranging from head-to-tail to lateral. The head-to-tail position generates a lower TS by exposing less of the surface area of the fish to the acoustic beam than the lateral position, where a larger surface area is exposed. However, both positions correspond to the same size, so it is crucial to consider the orientation of the fish relative to the acoustic beam. In the track analysis, the program assigns the specific size corresponding to the specimen that has generated a particular trajectory based on the angle of the trajectory relative to the acoustic beam. Each track with a size equal to or greater than 1 m was manually checked using the analysis software tools, which provide a visual representation of the tracks across the different spatial axes to verify they originate from a catfish. As in vertical sampling, all catfish detections were georeferenced in the study area. 2.2. The eDNA Procedures and Analyses 2.2.1. Field Sample eDNA Collections Water sampling was conducted in rivers and reservoirs of the Guadalquivir Basin located in the provinces of Sevilla and Córdoba in southern Spain (Figure S2) between 25 and 29 June 2023. The sampling sites were mostly located in areas that are exposed to sunlight for many hours and have no vegetal canopy to protect them from direct UV radia- Animals 2025,15, 285 7 of 20 tion, which can accelerate DNA degradation. Sampling was conducted during daylight hours but was structured to avoid the central parts of the day (12:00 to 15:00), as these coincide with the peak solar radiation and higher temperatures. A total of 34 samples of 2 litres each were collected from 17 sampling sites (Figure S2). Sampling points were clustered into 3 study areas: positive control area (Iznájar, Pantalán, and De la Hoz), upper river area (Viar, El Gergal, Lora del Río, Cantillana, Brenes, Alcalá del Río, and La Rinconada) and lower river area (North Sevilla, Puerto, Tomares, South Sevilla, Gelves, Coria del Río and La Señuela). Niskin-type oceanographic bottles were used to collect water samples to a depth of at least one metre to avoid the shallowest layer (without touching the bottom), which is the hottest and most affected by solar radiation. A HannaR HI98494 (Hanna Instruments, Woonsocket, RI, USA) worked to measure these parameters in the water column at 1–2 m avoiding contact with the sediment, including pH, oxidation-reduction potential (mV), dissolved oxygen percentage (%), dissolved oxygen concentration (mg/L), electrical conductivity ( µ S/cm), total dissolved solids (ppm), turbidity (NTU), and temperature ( ◦ C), during sampling (Table S2). Sampling was performed using sterile equipment and disposable nitrile gloves. All the materials were sterilised between sampling points using 10% v/vcommercial bleach (final [Cl − ] 0.3% v/v). Any residual bleach was then rinsed off with distilled water and the Niskin bottle was then rinsed again with reservoir water at the next sampling point. Benzalkonium chloride (BAC) was added to the samples to preserve the DNA using a pipette with sterile and filtered tips to a final concentration of 0.01% w/vBAC in the sample [ 58 , 59 ]. Two samples were collected at each sampling site (R1 and R2) and sampling was carried out attempting to separate the localities by a minimum of 2 km whenever possible. After sampling, the water samples were stored in sterile 2 L graduated bottles (REF. 407008.O) (Deltalab, Barcelona, Spain) at 4 ◦C until further processing. 2.2.2. Filtration and DNA Extraction Water samples were filtered within 24 h of collection in a decontaminated laboratory using a filtration ramp with three filtration units (Labbox Labware SL, Barcelona, Spain). Two litres per sample were filtered. Sample (2 L in total): Sampling replicate 1 (1 L) − Subsamples R1_1 (0.5 L) + R1_2 (0.5 L)/Sampling replicate 2 (1 L) − Subsamples R2_1 (0.5 L) + R2_2 (0.5 L). We filtered 2 L of each point, with 1 L per sample (0.5 × 4), using a dual-filter strategy to avoid clogging. The filters used were a cellulose nitrate membrane filter (CN) with a 1.2 µ m pore size (SARTORIUS Cellulose Nitrate Filter 11403—50----ACN) and, below, a second CN filter with a 0.20 µ m pore size (PRAT DUMAS France MCNE247-100). Filtration equipment was sterilised with 10% v/vcommercial bleach solution for 10 min and rinsed twice with sterile distilled water between samples. Filters were folded using sterile tweezers and placed in bead tubes provided by the PowerWater ® Kit (Qiagen, Hilden, Germany) and stored at − 20 ◦ C until DNA extraction. Filtration negative controls were run once a day between the samples to check for contamination (n = 6). Environmental DNA was extracted using DNeasy PowerWater ® Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions in a dedicated clean room for the processing of eDNA samples. Three extraction negative controls were added. 2.2.3. The qPCR and ddPCR Analyses Primer3 software implemented in Geneious Prime ® 2023.1.1 [ 60 ] was used for primers and probe design (see Supplementary Materials for more details). Two specific primers and a probe (FAM-labelled) were developed for a fragment of the 12S rRNA mitochondrial gene of the genus Silurus: Sil1-F (5 ′ -TTTTCCCCGCCTATATACCGCC-3 ′ ) Sil1-R Animals 2025,15, 285 8 of 20 (5 ′ -CTTCGGGCACTTACTTTCAAGG-3 ′ ) and Sil1-probe (**FAM-AACGTCAGGTCGAGGT GTAGCGTACG-MGB). After a strict validation of the primers and probe (SI.1), the qPCR reactions were conducted in a dedicated pre-PCR laboratory where bench surfaces were sterilised with UV radiation. Each PCR contained 6 µ L of template DNA, 1.2 µ L of each primer (10 µ M), 0.8 µ L of probe (5 µ M), 10 µ L of TaqMan ® Environmental Master Mix 2.0 (Thermo Fisher Scientific, Waltham, MA, USA), and DNase/RNase-free water to a final volume of 20 µ L. Each PCR plate contained a standard curve consisting of a 1:10 dilution series to generate a standard curve of known amounts of DNA. Three technical (PCR) replicates were performed for each biological sample, resulting in a total of twelve replicates per sampling point. In addition, 3 no template controls (NTCs) per plate were included, as well as filtration and extraction controls. The filtration, extraction and technical controls returned negative results. Samples were run on an Applied Biosystems 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific) under the following thermal cycling conditions: 2 min at 50 ◦ C, 10 min at 95 ◦ C, 50 cycles of 15 s at 95 ◦ C, and 60 s at 60 ◦ C. Sequence Detection Software SDS 2.4 (Thermo Fisher Scientific) was used to analyse the results. To include locations/sites for the rest of the analyses, two of the three technical replicates, besides one of the two subsamples (e.g., R1_1 (0.5 L) + R1_2 (0.5 L)) in at least one of the sites replicates (R1 or R2), should reveal catfish eDNA (as advised following a precautionary framework recommended by Goldberg et al. [61]). Further optimisation and validation of the ddPCR assay were performed (SI.1). In this case, and due to logistical restrictions, subsamples (e.g., R1_1 (0.5 L) + R1_2 (0.5 L)) were pooled, and for each location/site, two replicates (Replicate 1 and Replicate 2) were analysed. Each ddPCR reaction mix (20 µ L) comprised 10 µ L of sample, 900 nM of each primer, and 250 nM of TaqMan probe, completed with Supermix for Probes (No dUTP) (1863023) (Bio-Rad, Hercules, CA, USA) at a concentration of 1 × . This mixture was then combined with Bio-Rad’s droplet generation oil and divided into 15,000–20,000 droplets using the QX-100 droplet generator (Bio-Rad). Individual sample droplets were placed separately into each well of a 96-well PCR reaction plate. PCR was performed in the sealed 96-well plate using the Bio-Rad T-100 thermocycler (Bio-Rad). The final PCR conditions were 10 min at 95 ◦ C, 40 cycles of denaturation for 30 s at 94 ◦ C, and extension for 90 s at 57 ◦ C with a temperature ramp of 2 ◦ C/second, followed by 10 min at 98 ◦ C, and a hold at 4 ◦ C until plate reading. After PCR amplification, the plate was transferred to the Bio-Rad QX-200 droplet reader (Bio-Rad). Bio-Rad’s QuantaSoft software, version 1.7.4.0917, was used to quantify copies of the target DNA. To include locations/sites for the rest of the analyses, at least one of the site replicates (R1 or R2) should reveal catfish eDNA. 2.3. Mapping and Statistical Analyses For the map graphics, we used QGIS v. 3.32.3 Lima (https://qgis.org/en/site/, accessed on 20 December 2024) with hydrography data from the Spatial Reference Data of Andalusia, Institute of Statistics and Cartography of the Government of Andalusia. Water physicochemical parameters were assessed for collinearity using a correlation matrix in R software v. 4.3.3. [ 62 ]. As a result, the following variables were dropped from the final regression analysis: pH, dissolved oxygen percentage (%), electrical conductivity ( µ S/cm), and total dissolved solids (ppm). Pearson correlation analyses [ 63 ] were conducted between log-transformed total qPCR and ddPCR eDNA copy numbers by site. Principal component analysis (PCA) [ 64 , 65 ] was performed on physicochemical variables and qPCR and ddPCR catfish eDNA values using PAST V.4.17 [ 66 ]. A PCA was also performed using the total catfish eDNA values obtained by qPCR and ddPCR per site, the hydroacoustic catfish detections (aggregate counts) in river sections between 1 and Animals 2025,15, 285 9 of 20 10 km upstream of the eDNA sampling sites (the mid-point between replicates R1 and R2 was taken as the reference point), and finally the distances from each eDNA sampling site to the river mouth (km). Stepwise linear regression analyses were also performed to identify associations between log-transformed response variables (eDNA ddPCR and qPCR copy numbers), and potentially explanatory variables in river and reservoir habitats wherever possible. Moreover, and despite temporal and spatial heterogeneity between the two detection methodologies used in this work, Fisher’s exact test (presence/absence) [ 67 ] and Spearman’s rank correlation were used to assess possible relationships between the abundance of catfish detected by hydroacoustics (estimated as counts upstream of eDNA sampling points as in the previous analyses), molecular techniques (qPCR and ddPCR catfish eDNA values), and distances from the river mouth (km). 3. Results 3.1. Hydroacoustic Survey Results and Spatial Distribution of S.glanis Both vertical and horizontal surveys confirmed the presence of catfish in the lower Guadalquivir as well as in the El Gergal and Alcalá del Río Reservoirs (Figure 1). A total of 200 individuals were positively identified as adult catfish measuring 1 m or more. In the El Gergal Reservoir, a total of 11 individuals were detected, with the highest number in the dam area (9 individuals); in the Alcalá del Río Reservoir, 54 catfish detections were evenly distributed across the study area and, overall, were greater than in the El Gergal Reservoir. The highest number of catfish was detected in the river, particularly in the stretch between the Alcalá del Río dam and North Sevilla, with 78 detections decreasing progressively downstream, 54 detections in the rest of the Sevilla area, and 3 in the Coria del Río area, the last location where catfish was detected. Most of the detected individuals fell within the size range of 1.0 to 1.2 m (Figure S3). 3.2. Environmental DNA-Specific Detections, Quantifications and Spatial Distribution of S.glanis The mean and standard deviation values for each physicochemical parameters from the sampling locations were estimated (Table S2): pH ( µ = 8.2 ± 0.45), oxidation-reduction potential (ORP) ( µ = 33.2 ± 9.55 mV), dissolved oxygen percentage ( µ = 71.8 ± 42.80%), dissolved oxygen concentration ( µ = 5.5 ± 3.23 mg/L), electrical conductivity ( µ = 1769.2 ± 1118.84 µ S/cm), total dissolved solids ( µ = 885.2 ± 561.06 ppm), turbidity ( µ = 0.9 ± 0.59 NTU), and temperature ( µ = 28.6 ± 1.92 ◦ C) showing high heterogeneity among sampling sites for some of the variables and evident correlation between some of them (Figure S4). In the PCA analysis, PC1 and PC2 accounted for 74.8% of the total variability based on the eigenvalues obtained (Figure S4). Stepwise linear regression analyses with logarithmic transformation were performed among the variables, showing that in rivers, ddPCR copies were indeed positively associated with dissolved oxygen (mg/L) (p-value < 0.01) and negatively associated with turbidity (NTU) (p-value < 0.01). In reservoirs, copies detected by qPCR were also weakly negatively associated with turbidity (NTU) (p-value < 0.05) (Table S3). Overall, both molecular assays (qPCR and ddPCR) demonstrated specific detection of catfish. There was no detection in any of the negative technical controls using either methodology. All filter and extraction controls showed no amplification, indicating the absence of contamination at all stages of the study. The qPCR data reported positive catfish detection in 13 sites (76.5%), while we obtained 11 positive sites with ddPCR (64.7%) from the 17 sample sites (Figure S5). 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