Eradication attempt for an early detected invasive crayfish: the case of Pacifastacus leniusculus (Decapoda, Astacidae) in the Clitunno River (central Italy)

331 Eradication attempt for an early detected invasive crayfish: the case of Pacifastacus leniusculus (Decapoda, Astacidae) in the Clitunno River (central Italy) Antonella Carosi1, Francesca Lorenzoni1, Fatemeh Zarei1, Massimo Lorenzoni1 1 Department of Chemistry, Biology and Biotechnologies, University of Perugia, via Elce di Sotto, 06123 Perugia, Italy Corresponding author: Antonella Carosi (antonella.car[email protected]) Copyright: © Antonella Carosi et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract The signal crayfish Pacifastacus leniusculus (Dana, 1852) is one of the most ecologically impactful decapod crustaceans introduced in Europe. As a species of Union concern, early detection and rapid eradication measures are required to prevent its establishment and spread. We aimed to: i) test the effectiveness of an eradication attempt undertaken to counteract the spread and contain the demographic growth of P. leniusculus in the Clitunno River Basin (central Italy), where the species has been detected in 2020; ii) assess distribution, age structure and growth of P. leniusculus in this invaded area. The removal actions were conducted biweekly, from June 2022 to December 2024, using both traps and electrofishing. Biometric parameters were individually recorded, and the demographic features and growth of P. leniusculus population were assessed. In total, 259 removal activities were carried out and a biomass of 39.74 kg was removed. The presence of six cohorts (from 0+ to 5+), including the young-of-the-year (0+), attested to the rapid acclimatisation of P. leniusculus, which gave rise to a self-sustaining population in a short time. The greatest removal efforts have been concentrated on a small tributary, named Fosso Vecchio, where the average values of Catch Per Unit Effort (CPUE) showed a significant decreasing trend over time, suggesting the effectiveness of the removal actions. Our findings provided some evidence that early detection and eradication measures conducted on a small scale, but with high effort, represent effective management tools to limit population abundance and prevent invasive crayfish from spreading further. Key words: Biodiversity conservation, chalk streams, inland waters, invasive crayfish, removal activities Introduction Biological invasions represent one of the major causes of biodiversity loss in freshwaters (Ricciardi and MacIsaac 2011; Reid et al. 2019). Acting as powerful generalist omnivores, crayfish are included amongst the most successful invaders in these environments (Gherardi 2012; Twardochleb et al. 2013; Wacker and Harzsch 2021; Soto et al. 2023; Carvalho et al. 2025). Crayfish have strong opportunities for invading aquatic systems due to their capacity to enter the food chain at various trophic levels and to exploit the considerable energy reserves of the detrital pool (Gherardi 2007). In the invaded areas, significant management efforts are required to prevent damage from alien crayfish, since they have the potential to directly affect native species (through predation, competition for food and shelters, disease transmission, etc.) as well as indirectly Academic editor: Pedro Anastácio Received: 16 January 2025 Accepted: 12 June 2025 Published: 7 October 2025 Citation: Carosi A, Lorenzoni F, Zarei F, Lorenzoni M (2025) Eradication attempt for an early detected invasive crayfish: the case of Pacifastacus leniusculus (Decapoda, Astacidae) in the Clitunno River (central Italy). In: Anastácio P, Ribeiro F, Chainho P (Eds) Invasions in Aquatic Systems. NeoBiota 102: 331–350. https://doi. org/10.3897/neobiota.102.146951 NeoBiota 102: 331–350 (2025) DOI: 10.3897/neobiota.102.146951 Advancing research on alien species and biological invasions A peer-reviewed open-access journal NeoBiota 332 NeoBiota 102: 331–350 (2025), DOI: 10.3897/neobiota.102.146951 Antonella Carosi et al.: Alien crayfish removal activities in a Mediterranean chalk stream affect ecosystems (through food webs and habitat alterations) (Ficetola et al. 2012; Twardochleb et al. 2013). North American crayfish are of particular concern, considering their ability to assume a preponderant role in aquatic communities when introduced outside their original range (Vaeßen and Hollert 2015; Souty-Grosset et al. 2016); their detrimental effects have been linked to life history traits (early sexual maturity, high fecundity), wide tolerance to various environmental conditions, scarcity of natural predators, and competition with native crayfish (Gherardi 2006; Vaeßen and Hollert 2015). Due to the widespread diffusion of North American crayfish in Europe, many local cases of native species extinction have been observed (Kouba et al. 2014); the combination of biodiversity loss and ecosystem processes alteration often resulted in ecological and economic disruptions (Lodge et al. 2000). The signal crayfish Pacifastacus leniusculus (Dana, 1852), native to the northwestern coast of the United States, is considered one of the most widespread and ecologically impactful decapod crustaceans introduced in Europe (Rebrina et al. 2015; Chucholl and Chucholl 2021). This species is a carrier of the crayfish plague, which is lethal for the most native European crayfish (Holdich et al. 2009). Moreover, its ecological niche largely overlaps with that of native crayfish (Préau et al. 2020); other detrimental effects are related to predatory activity and environmental alterations in the invaded areas. With its feeding habits, P. leniusculus modifies the food web and alters the composition of aquatic communities, consuming macrophytes, organic detritus, and preying on macroinvertebrates, amphibians and small benthic fish (Souty-Grosset et al. 2006; Procopio 2020). Furthermore, the digging activity associated with creating shelter, increases water turbidity, reducing light penetration with a consequent drop in primary productivity. Digging activity can also damage riverbanks, facilitating their collapse (Harvey et al. 2011). The species has led to the local extinction of many native crayfish populations in Scandinavia, England, and northern Europe (Lodge et al. 2000; Green et al. 2018). Even in Italy, as in other European countries, local extinction of the native crayfish Austropotamobius pallipes complex (Lereboullet, 1858) has been reported immediately after the introduction of the signal crayfish (Ghia et al. 2017); there are few cases in which a native population of A. pallipes complex was observed in syntopy with P. leniusculus (Ghia et al. 2019). The distribution range of P. leniusculus in Italy, in the period 1981–2015, was limited to some watercourses in the northern part of the country (Capurro et al. 2007; Morpurgo et al. 2010; Bo et al. 2016; Ghia et al. 2017); the first report of the species in Central Italy dates back to 2020, when some individuals were found in the Clitunno River (Umbrian portion of the Tiber River basin) (Della Bella et al. 2021). The further spread of P. leniusculus in the Apennine watercourses raises serious concern because among all the North American crayfish, its ecological preferences largely overlap with that of A. pallipes complex (Souty-Grosset et al. 2006; Chucholl 2013), which being a cold-water species preferentially colonizes the upper river stretches (Bo et al. 2016). According to the European Invasive Alien Species Regulation (EU 1143/2014), due to the detrimental multi-level impacts posed by the species on native biodiversity and ecosystem services, P. leniusculus is deemed as species of Union Concern. Therefore, population monitoring and quick removal actions shortly after its detection are required, to prevent a further spread of 333 NeoBiota 102: 331–350 (2025), DOI: 10.3897/neobiota.102.146951 Antonella Carosi et al.: Alien crayfish removal activities in a Mediterranean chalk stream the species. Based on these regulatory requirements, within the LIFE project (IMAGINE IPE/IT/000015) the Umbria Region prepared, an eradication plan that was started in 2022. The purpose of the eradication attempt was to contain the dispersal and the population growth of P. leniusculus in the Clitunno River Basin. The LIFE Programme is the EU’s funding instrument for supporting Environment, Nature Conservation and Climate Action projects throughout the European Union (EU). Two crucial traits expressing species fitness, population persistence and habitat adaptation are: population growth and demographic characteristics (Hoffmann et al. 2017). Thus, a deeper knowledge of these aspects plays a major role in crayfish invasions management (Guan and Wiles 1999), and it is essential for predicting potential range expansion of invasive species (Chuang and Peterson 2016). Within this context, the present study aimed to i) test the effectiveness of the eradication actions, in terms of limiting P. leniusculus spread and population abundance in the Clitunno River Basin; and ii) assess distribution, age structure and growth of P. leniusculus population in the invaded area. Material and methods Study area The Clitunno River (Tiber River Basin, central Italy) is a chalk stream that flows for 18 km, in an agricultural context, with an almost constant slope (0.18%), and stable thermal and hydrological regime (water temperature range: 11.8–20 °C; average flow rate: 1.5 m3/s) (Lorenzoni et al. 2010, 2023). The stream originates from a rheo-limnocrenic spring system, called “Fonti del Clitunno”. Despite the high cultural, naturalistic, and landscape value of the Clitunno River, which has inspired artists and poets since Roman times (Pascual et al. 2024), its riverbed is characterised by the presence of significant silt deposits that have built up over the years because of urban and industrial discharges (Lorenzoni et al. 2010). The Clitunno River has been classified as having “moderate ecological status”, in line with the assessments carried out by the regional Environmental Protection Agency (ARPA Umbria) in the years 2016–2017 (Della Bella et al. 2021). The hydrographic network of the Clitunno River Basin is very complex and articulated, due to the numerous human-made hydraulic system modifications carried out over the centuries (Lorenzoni et al. 2023). The Clitunno River hosts an abundant macrophyte community (Cingolani et al. 2008), while amongst the most widespread fish species, several are currently facing the threat of extinction according to the IUCN Red List (IUCN 2024). These include Anguilla anguilla (Linnaeus, 1758) (CR), Sarmarutilus rubilio (Bonaparte, 1837) (VU), and Barbus tyberinus (EN). Additionally, according to the IUCN Red List of Italian Vertebrates (Rondinini et al. 2022), the cyclostome Lampetra planeri (Bloch, 1784) is listed as vulnerable. The native crayfish A. pallipes complex is locally extinct, while the invasive Procambarus clarkii (Girard, 1852) is widely distributed throughout the Basin, including the area considered in this study. Here the species seems to find suitable conditions for reproduction only in the downstream warmer river stretches (Lorenzoni, personal observation). 334 NeoBiota 102: 331–350 (2025), DOI: 10.3897/neobiota.102.146951 Antonella Carosi et al.: Alien crayfish removal activities in a Mediterranean chalk stream Experimental design Fig. 1 schematically shows the selected area for P. leniusculus eradication activities, located in the upper part of the Clitunno River Basin. The eradication programme, conducted using both trapping and electrofishing, was based on the results of preliminary monitoring carried out in 2021 (Lorenzoni et al. 2023). The entire area has been divided into 13 stretches. Nine sites (from -1 to -9) were situated upstream from the first detection point of P. leniusculus (site 0); three sites, numbered from 1 to 3 and located downstream of site 0, were selected as “control sites”, to check the possible range expansion, given that, in these areas, the species was not detected during the preliminary monitoring. The most abundant P. leniusculus population, occurring in the site identified as the “invasion core”, was chosen to conduct the demographic and growth analyses. Table 1 shows the main environmental characteristics for each river stretch, including canopy cover (i.e. the percentage of vegetation coverage of the riverbed), prevalent substrate (i.e. the dominant particle size of the sediments), and shaded area (i.e. the percentage of riparian vegetation shading). In the present research we tested the hypothesis that the eradication actions were effective in limiting P. leniusculus spread and population abundance in the investigated area. Figure 1. Location of the sites selected for P. leniusculus removal activities. a. Tiber River watershed; b. Topino River sub-watershed; c. Hydrographic network of the upper Clitunno River Basin and location of the eradicated stretches. An asterisk indicates the first detection site. The shaded area includes the species occurrence stretches. The dashed arrow indicates the spread direction of the signal crayfish. 335 NeoBiota 102: 331–350 (2025), DOI: 10.3897/neobiota.102.146951 Antonella Carosi et al.: Alien crayfish removal activities in a Mediterranean chalk stream Table 1. List of sampling sites along geographical coordinates, and main environmental features. Canopy cover, Prevalent substrate and Shaded area refer to: vegetation coverage of the riverbed, dominant particle size of the sediments, and riparian vegetation shading, respectively. River stretches code and locality Geographic coordinates Average depth (m) Average width (m) Canopy cover (%) Prevalent substrate Shaded area (%) Anthropisation level Type of disturbance CL0 Piatto Bridge first detection site 33 T 4747402.16 m N, 315656.63 m E 0.9 6 75 Silt 30 Very high Canalisation, Diffuse organic pollution CL1 Borgo Trevi 33 T 4749177.40 m N, 314823.23 m E 0.8 12 90 Silt 90 High Canalisation, Diffuse organic pollution, Presence of obstacles FS2 Pietrarossa 33 T 4750596.97 m N, 313932.25 m E 0.4 1 20 Silt 50 Very high Canalisation, Point-source organic pollution IR3 Borgo Trevi 33 T 4750793.29 m N, 314195.95 m E 0.5 2 10 Silt 10 Very high Canalisation , Diffuse organic pollution CL(-1) Pigge 33 T 4746511.93 m N, 316183.45 m E 0.7 12 70 Silt - Fine Gravel 10 Moderate Canalisation FI(-2) Pigge 33 T 4746497.39 m N, 316178.65 m E 0.7 2.5 30 Silt 20 Moderate Canalisation, Diffuse organic pollution FP(-3) Treponti 33 T 4745034.65 m N, 316789.89 m E 1 2.5 20 Silt 50 High Canalisation, Diffuse organic pollution FM(-4) Treponti 33 T 4745058.25 m N, 316802.64 m E 0.4 4 20 Silt - Fine Gravel 80 High Canalisation, Diffuse organic pollution CL(-5) Treponti 33 T 4745128.83 m N, 316837.82 m E 1 10 100 Silt - Sand 30 Moderate Canalisation, Diffuse organic pollution FV(-6) Treponti 33 T 4745008.15 m N, 316869.76 m E 0.5 3 80 Silt 80 High Canalisation, Diffuse organic pollution CL(-7) Fonti 33 T 4744708.80 m N, 317445.71 m E 1 8 90 Silt - Sand 70 Moderate Canalisation FV(-8) Fonti 33 T 4744664.53 m N, 317446.05 m E 0.3 2.5 20 Silt 100 Very high Canalisation, Diffuse organic pollution, Presence of obstacles FN(-9) Treponti 33 T 4744929.48 m N, 316845.18 m E 0.5 2.5 70 Silt 50 Very high Canalisation, Point-source organic pollution Removal activities Baited traps and artificial refuge traps The removal actions were conducted biweekly, from June 2022 to December 2024, using two types of traps: baited and artificial refuge traps (ART). Baited traps consisted of double entry cylindrical (50 × Ø 30 cm) plastic cages, with a mesh size of 0.5 cm. They were baited with dry food for cats or fish. In total, up to 22 baited traps were used, with an average value of 15. The traps were placed, approximately 25 m apart from each other, in a semi-submerged position along the banks, to avoid causing disturbance to non-target species that could be attracted by the bait, following indications reported in the signal crayfish National Management Plan (Tricarico et al. 2021). Following Green et al. (2018), to collect more individuals representative of small size ranges, the use of baited traps was considered more effective in combination with ART. ARTs were made of seven PVC tubes, of which two of 10-, 20-, 32diameter, respectively, and one of 50-mm diameter, and all 400 mm long, joined by a clamp, and closed by a fine mesh net at one end. Up to 10 ARTs were employed in total, with an average value of seven; they were placed close to baited traps, in the areas most frequented by juveniles. The trend in catches over time was assessed by Catch per Unit Effort (CPUE), calculated as a daily average of catches per trap (baited and ART). As P. leniusculus is a species of Union concern, all individuals were frozen immediately after capture and all specimens were brought to the laboratory for sex determination and biometric measurements. 336 NeoBiota 102: 331–350 (2025), DOI: 10.3897/neobiota.102.146951 Antonella Carosi et al.: Alien crayfish removal activities in a Mediterranean chalk stream Electrofishing Limited to wadable stretches, in all the 13 river stretches, the removal activities included electrofishing, using a 4.5 kW electro shocker. The interventions were carried out on a seasonal basis, excluding winter, applying a time of 45/60 minutes at each sampling site. The average length of the river stretches was 40 m (range: 15–120 m); they were sampled by at least three operators, proceeding from downstream to upstream. In some parts of the Clitunno River, where the water was too deep for electrofishing, the catches were made along the riverbanks. Environmental characterisation To investigate the environmental conditions that could most influence the presence and activity of the signal crayfish, at the same time as electrofishing, the following physico-chemical parameters were measured: water temperature (°C), pH (units), electric conductivity (μS cm-1) and dissolved oxygen (mg l-1). The measurements were conducted simultaneously with the electrofishing removal activities, using a multiparametric probe (Hanna Instruments, Padova, Italy). Laboratory activities For each individual identified as P. leniusculus based on morphological characters (i.e. white oval patch at the joint of the claw fingers and two pairs of post-orbital ridges), sex was determined, the total weight was measured using a digital scale (accuracy ± 0.1 g) and the following biometric parameters were measured using a calliper (accuracy ± 1 mm): cephalothorax length (CL) from the tip of the rostrum to the posterior edge of the carapace and the total length (TL) from the tip of the rostrum to the telson. Cheliped damage (absent or regenerating) or the presence of other macroscopic lesions were also recorded. Demographic features and growth of P. leniusculus population For the FV(-6) population, the total length-weight relationship (LWR) was estimated by the least-squares method (Ricker 1975), based on the logarithmic equation: log10 W (g) = a + b log10 CL (cm), where a is the intercept on the Y-axis and b is the regression coefficient. The standard error was calculated for the slope (b) of LWR. Isometric growth was tested through a t-test, using the equation: ts = b-3/Sb where Sb is the standard error of the slope (b), for α = 0.05 (Sokal and Rohlf 1987). The LWR was fitted both to the total sample and separately for males and females. Age classes were estimated by Bhattacharya’s method based on the CL frequency distribution data (Bhattacharya 1967), using the software FiSAT II (FAOICLARM Stock Assessment Tools v. 1.2.2). Theoretical growth was estimated by the von Bertalanffy growth curve model (VBGE) (von Bertalanffy 1938): where CLt is the carapace length of the crayfish at time t, L∞ the theoretical maximum carapace length (cm), k the rate of approach to L∞ and t0 the theoretical age (in years) at which 337 NeoBiota 102: 331–350 (2025), DOI: 10.3897/neobiota.102.146951 Antonella Carosi et al.: Alien crayfish removal activities in a Mediterranean chalk stream CLt = 0. Furthermore, the index of growth performance (Φ’) was calculated by the equation of Pauly and Munro (1984): Φ’ = log10 k + 2 log10 L∞ where k and L∞ are the growth parameters of the von Bertalanffy model. Statistical analysis To test the differences between mean CPUE amongst sites, amongst years and the interaction between these variables (site x year), a two-way factorial ANOVA was performed. A one-way ANOVA was performed to compare physico-chemical parameters amongst sites. For LWR, differences between sexes were assessed by ANCOVA. To compare the length of P. leniusculus caught by different removal methods, a Kruskal-Wallis ANOVA was performed. The normality of length data distribution was tested by Shapiro–Wilk test. All statistical tests were conducted using Dell STATISTICA 13 software for Windows. Results Removal activities and P. leniusculus distribution Baited traps and artificial refuge traps A total of 180 removal activities were conducted. The overall effort applied through setting traps was equal to 2057 trap days in 2022, 5583 trap days in 2023, and 8708 trap days in 2024. Overall, 1075 Pacifastacus leniusculus individuals were removed for a total biomass of 37.9 kg. This species was only caught upstream from the first detection site (Fig. 1), where the largest sample (91.63% of total individuals) has been removed from Fosso Vecchio, a small tributary of the Clitunno upper stretch. In FV(-6) we observed a progressive decreasing trend over time of the mean annual CPUE values (± SE), which dropped from 0.284 ± 0.071 ind/trap day recorded in 2022 to 0.065 ± 0.005 in 2024 (Fig. 2). A drastic reduction of CPUE was also observed in the same period for FM(-4) and CL(-5), where the mean annual values (± SE) decreased from 0.070 ± 0.055 to 0.024 ± 0.008 and from 0.018 ± 0.061 to 0.001 ± 0.014 ind/trap day, respectively. At all other sites, no P. leniusculus individuals were captured in 2024 by traps. There are differences between the mean values amongst sites (two-way factorial ANOVA: F = 32.19; P = 0.004), and a significant interaction between these variables (two-way factorial ANOVA: F = 2.51; P = 0.001). During the eradication interventions, a progressive significant decreasing trend in the average daily CPUE values was observed through time for traps in the FV(-6) (Fig. 3). The computed equation was y = 2.83 - 0.025 · x (F = 6.56; P = 0.022; r2 = 0.46); the regression slope significantly differed from 0 (t = 2.79; df = 9; P = 0.022). For baited traps, the analysis of the monthly trend in CPUE values allowed us to identify two peaks, occurring in April and August (Fig. 4), with mean values (± SE) recorded at 0.27 ± 0.05 and 0.21 ± 0.02, respectively. The lowest values were observed in January (mean ± SE = 0.02 ± 0.02) and February (mean ± SE = 0.02 ± 0.01). The differences amongst the mean values were statistically significant (one-way ANOVA: F = 2.69; P = 0.003). For ARTs, the monthly mean values were always lower than those of baited traps; the lowest values were observed in December (0.004 ± 0.003) and January (zero catches). However, in this case, the differences amongst the mean values were not statistically significant (one-way ANOVA: F = 1.74; P = 0.08). 338 NeoBiota 102: 331–350 (2025), DOI: 10.3897/neobiota.102.146951 Antonella Carosi et al.: Alien crayfish removal activities in a Mediterranean chalk stream Electrofishing A total of 79 electrofishing removals were carried out, during which overall 28 specimens were collected. The mean total length of individuals (± SE) was 10.0 ± 2.59 cm (range 4.3–13.5 cm); the mean weight (± SE) was 38.66 ± 23.77 g (range 2.3– 72.0 g). This method proved its effectiveness in CL0 and FV(-6), with captures recorded exclusively in 2022 and 2023. In 2024, two individuals were also captured from FM(-4) (Fig. 5). Figure 3. Trend over time in mean seasonally CPUE values for P. leniusculus during eradication interventions conducted with baited traps and artificial refuge traps in the Fosso Vecchio River. The solid line represents linear regression (equation: y = 2.83–0.0251*x; P = 0.0218; r2 = 0.46). The dotted lines represent confidence limits (95%). Figure 2. Baited traps and artificial refuge traps: mean annual CPUE values for P. leniusculus are broken down by year and removal sites falling within the invaded area. Vertical bars denote standard errors. 339 NeoBiota 102: 331–350 (2025), DOI: 10.3897/neobiota.102.146951 Antonella Carosi et al.: Alien crayfish removal activities in a Mediterranean chalk stream Selectivity of removal methods The mean length (± SE) of individuals caught with ARTs (7.36 ± 0.27 cm) was significantly lower from those of both baited traps (10.04 ± 0.04 cm) and electrofishing (9.60 ± 0.58 cm) at the Kruskal-Wallis ANOVA (χ2 = 38.44; P < 0.001). Sex ratio did not differ significantly amongst different removal methods at the chisquare test (χ2 = 1.10; P = 0.578). Environmental characterisation Water temperature ranged from 11.20 °C, recorded in FM(-4), to 19.36 °C, recorded in FN(-9) (Fig. 6a). The differences between the mean values calculated for the 13 sites were statistically significant (one-way ANOVA: F = 5.27; P < 0.001). Figure 4. Monthly mean CPUE values for P. leniusculus during eradication interventions conducted with baited traps and artificial refuge traps in the Fosso Vecchio River. Vertical bars denote confidence limits. Figure 5. Electrofishing: mean annual CPUE values for P. leniusculus are broken down by year and removal sites falling within the invaded area. Vertical bars denote standard errors. 346 NeoBiota 102: 331–350 (2025), DOI: 10.3897/neobiota.102.146951 Antonella Carosi et al.: Alien crayfish removal activities in a Mediterranean chalk stream Use of AI No use of AI was reported. Funding This study was supported and funded by LIFE IMAGINE – LIFE19 IPE/IT/000015 within the framework of the European Commission Life Projects. Author contributions Conceptualization: ML, AC. Data curation: FZ. Formal analysis: AC. Funding acquisition: ML. Investigation: FZ, FL, ML. Methodology: ML. Supervision: ML. Writing - original draft: AC. Writing - review and editing: FL, ML. Author ORCIDs Antonella Carosi https://orcid.org/0000-0003-0513-7287 Massimo Lorenzoni https://orcid.org/0000-0002-8548-9487 Data availability All of the data that support the findings of this study are available in the main text. References Águas M, Banha F, Marques M, Anastácio PM (2014) Can recently-hatched crayfish cling to moving ducks and be transported during flight? 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