The Red Alien vs . the Blue Destructor : The Eradication of Cherax destructor by Procambarus clarkii in Latium ( Central Italy )

Cherax destructor is a crayfish endemic to south-eastern Australia and one of the last alien crayfish to be introduced in Italy. In the Laghi di Ninfa Natural Reserve (Latium region, Central Italy), the species was probably introduced in 1999, but only reported for the first time in 2008. Nearby this area, the most widespread alien crayfish is the red swamp crayfish Procambarus clarkii. In the Natural Reserve, between 2008 and 2013 and during spring and summer, crayfish sampling was carried out with baited traps to assess the distribution of C. destructor and its possible relationship with P. clarkii. Cherax destructor was first recorded in 2008; few P. clarkii were detected in the cultivation ponds where C. destructor was present in 2012 and 2013. Moreover, crayfish plague analyses evidenced a positive result in two out of the 12 sampled P. clarkii. Cherax destructor is now completely absent from the Natural Reserve, while P. clarkii has spread in the area and was probably responsible for this eradication since C. destructor is vulnerable to crayfish plague which was also detected in the area. An ecosystem restoration project in the area favoured the spread of. P. clarkii; the implications of this intervention are discussed.


Introduction
Freshwater ecosystems are widely recognized as more vulnerable to invasive alien species (IAS) than terrestrial environments due to the highly intrinsic dispersal ability of freshwater species, and the prevalent impacts of human disturbance and climate change on inland waters [1][2][3][4][5].Despite the frequent and often simultaneous invasion of freshwater ecosystems by several IAS [3], the relationship among those IAS has scarcely been investigated [2].Crayfish are one of the most introduced taxa in fresh waters with dramatic consequences on the invaded ecosystems [6]; currently, there are 11 established alien crayfish species in Europe and some coexist in the same water body [7].
Italy is one of the European countries heavily affected by the invasion of alien crayfish (reviewed in Aquiloni et al. [8]), which threaten freshwater biodiversity in general and indigenous crayfish in particular.One of the most recent alien crayfish introduced in Italy is the Australian parastacid yabby Cherax destructor Clark, 1936 [9].Cherax destructor is a south-eastern Australian crayfish that has successfully colonized many different areas across Australia [10], and it has also been established in Europe, particularly in Spain, in the provinces of Zaragoza, Aragón and Navarra [11,12].In Italy, C. destructor was probably introduced in the Laghi di Ninfa Natural Reserve (Latium region, Central Italy), at the end of the 1980s to foster an experimental aquaculture, and it was reported for the first time in 2008 by Scalici et al. [9].According to Scalici et al. [9], a large population inhabited six abandoned crayfish cultivation ponds in the Reserve, although the species seemed to be still confined in the ponds without expanding into the several surrounding fresh waters of the area.Until now, the species has only been found in Latium in Italy, but recently a few individuals were sampled in the Costanzo stream in the province of Siracusa, Sicily [13].Cherax destructor is an r-selected species, and is considered a high-risk species by Tricarico et al. [14] due to its high resistance to environmental extremes and its severe impacts on the invaded habitat.Moreover, while Scalici et al. [9] suggested that the low temperature of the surrounding waters of Laghi di Ninfa (12 • C) acts as a barrier against the natural spreading of crayfish, Veselý et al. [15] discovered the ability of C. destructor to withstand low winter temperatures by burrowing into the levees.
In the area close to Laghi di Ninfa, the most widespread alien crayfish is the red swamp crayfish Procambarus clarkii (Girard, 1852) [16], but up until 2012 the two species were never found in syntopy.In 2012, a few P. clarkii specimens and just one C. destructor individual were found in the same cultivation pond.Due to a long co-evolutionary history with the crayfish plague agent Aphanomyces astaci Schikora, 1906, North American crayfish species such as P. clarkii have evolved defence mechanisms against its growth [17].In contrast, crayfish of European and Australian origin such as C. destructor lack efficient immune responses to crayfish plague and are thus considered highly susceptible [18], although Mrugała et al. [19] found that some individuals of C. destructor can survive after exposure to the least virulent A. astaci strain used in the experiment.
The aim of this paper was twofold: (1) to assess the distribution and abundance of C. destructor in the Natural Reserve of Laghi di Ninfa, which is characterized by cold waters, and (2) to evaluate the possible relationship and the consequent coexistence of C. destructor with P. clarkii.

Materials and Methods
The Laghi di Ninfa Natural Reserve (106 ha) is located in Latina Province (Central Italy), and within this reserve there is a Special Area of Conservation (SAC IT6040002 "Ninfa").Also an ecosystem restoration project has transformed a farm in the wetland area of Pantanello (12 ha), an important site for aquatic birds composed of interconnected ponds, marshes, wet grasslands, and streams (Figure 1).destructor was probably introduced in the Laghi di Ninfa Natural Reserve (Latium region, Central Italy), at the end of the 1980s to foster an experimental aquaculture, and it was reported for the first time in 2008 by Scalici et al. [9].According to Scalici et al. [9], a large population inhabited six abandoned crayfish cultivation ponds in the Reserve, although the species seemed to be still confined in the ponds without expanding into the several surrounding fresh waters of the area.Until now, the species has only been found in Latium in Italy, but recently a few individuals were sampled in the Costanzo stream in the province of Siracusa, Sicily [13].Cherax destructor is an r-selected species, and is considered a high-risk species by Tricarico et al. [14] due to its high resistance to environmental extremes and its severe impacts on the invaded habitat.Moreover, while Scalici et al. [9] suggested that the low temperature of the surrounding waters of Laghi di Ninfa (12 °C) acts as a barrier against the natural spreading of crayfish, Veselý et al. [15] discovered the ability of C. destructor to withstand low winter temperatures by burrowing into the levees.
In the area close to Laghi di Ninfa, the most widespread alien crayfish is the red swamp crayfish Procambarus clarkii (Girard, 1852) [16], but up until 2012 the two species were never found in syntopy.In 2012, a few P. clarkii specimens and just one C. destructor individual were found in the same cultivation pond.Due to a long co-evolutionary history with the crayfish plague agent Aphanomyces astaci Schikora, 1906, North American crayfish species such as P. clarkii have evolved defence mechanisms against its growth [17].In contrast, crayfish of European and Australian origin such as C. destructor lack efficient immune responses to crayfish plague and are thus considered highly susceptible [18], although Mrugała et al. [19] found that some individuals of C. destructor can survive after exposure to the least virulent A. astaci strain used in the experiment.
The aim of this paper was twofold: (1) to assess the distribution and abundance of C. destructor in the Natural Reserve of Laghi di Ninfa, which is characterized by cold waters, and (2) to evaluate the possible relationship and the consequent coexistence of C. destructor with P. clarkii.

Materials and Methods
The Laghi di Ninfa Natural Reserve (106 ha) is located in Latina Province (Central Italy), and within this reserve there is a Special Area of Conservation (SAC IT6040002 "Ninfa").Also an ecosystem restoration project has transformed a farm in the wetland area of Pantanello (12 ha), an important site for aquatic birds composed of interconnected ponds, marshes, wet grasslands, and streams (Figure 1).Pantanello is also connected on the south side to the Pontina plain, where P. clarkii is present with an average density of 0.60 ind/m 2 [20].No individuals of the indigenous Austropotamobius pallipes complex have been found in the area since 1986, and it is probably extinct due to pollution and overexploitation.
In the Reserve, close to the entrance, there are six abandoned cultivation ponds where C. destructor was found [9] (50 × 8 m, with a muddy bottom and a water depth of 60 to 30 cm).The pond water is provided by a stream (that is connected to the Pantanello area).
Crayfish samplings were carried out between 2008 and 2013 with baited traps during the spring and summer for a total of 16,392 h of sampling.In the cultivation ponds, 10-30 traps were used (minimum six per pond, according to water presence and level).In the outside cultivation ponds, 18-40 traps were used.Population density was estimated using the Catch per Unit Effort (CPUE, the total number of trapped crayfish/(total used traps*hours of sampling)).The collected crayfish were sexed and measured (carapace length).Nine and three individuals of P. clarkii found in 2012 and 2013, respectively, and one individual of C. destructor found in 2012 were preserved under ethanol 90 • and sent to the laboratory of Instituto Zooprofilattico Sperimentale delle Venezie for the detection of A. astaci.The analyses were conducted by end-point PCR following Oidtmann et al. [21], with sequencing of the amplified products.Animals were sampled and processed following the standard procedure recommended by the Italian Health Authority and the Research Organization for Animal Health and Food Safety.

Results
An individual of P. clarkii was found occasionally near the Reserve during 2006, while an abundant population of this species was detected in 2008, outside the Reserve, in the Pontina plain (CPUE = 11).At the same time, in 2008, an abundant population of C. destructor was found in the cultivation ponds in Laghi di Ninfa (CPUE = 10.3;Figures 1 and 2).The subsequent samplings highlighted the complete reduction of this species in the study area (to the best of our knowledge no C. destructor specimens have been found in the neighboring ditches and areas from 2013 until now) and the exponential increase of the P. clarkii population in Laghi di Ninfa (r 2 = 0.84; Figures 1 and 2).
Pantanello is also connected on the south side to the Pontina plain, where P. clarkii is present with an average density of 0.60 ind/m 2 [20].No individuals of the indigenous Austropotamobius pallipes complex have been found in the area since 1986, and it is probably extinct due to pollution and overexploitation.
In the Reserve, close to the entrance, there are six abandoned cultivation ponds where C. destructor was found [9] (50 × 8 m, with a muddy bottom and a water depth of 60 to 30 cm).The pond water is provided by a stream (that is connected to the Pantanello area).
Crayfish samplings were carried out between 2008 and 2013 with baited traps during the spring and summer for a total of 16,392 h of sampling.In the cultivation ponds, 10-30 traps were used (minimum six per pond, according to water presence and level).In the outside cultivation ponds, 18-40 traps were used.Population density was estimated using the Catch per Unit Effort (CPUE, the total number of trapped crayfish/(total used traps*hours of sampling)).The collected crayfish were sexed and measured (carapace length).Nine and three individuals of P. clarkii found in 2012 and 2013, respectively, and one individual of C. destructor found in 2012 were preserved under ethanol 90° and sent to the laboratory of Instituto Zooprofilattico Sperimentale delle Venezie for the detection of A. astaci.The analyses were conducted by end-point PCR following Oidtmann et al. [21], with sequencing of the amplified products.Animals were sampled and processed following the standard procedure recommended by the Italian Health Authority and the Research Organization for Animal Health and Food Safety.

Results
An individual of P. clarkii was found occasionally near the Reserve during 2006, while an abundant population of this species was detected in 2008, outside the Reserve, in the Pontina plain (CPUE = 11).At the same time, in 2008, an abundant population of C. destructor was found in the cultivation ponds in Laghi di Ninfa (CPUE = 10.3;Figures 1 and 2).The subsequent samplings highlighted the complete reduction of this species in the study area (to the best of our knowledge no C. destructor specimens have been found in the neighboring ditches and areas from 2013 until now) and the exponential increase of the P. clarkii population in Laghi di Ninfa (r 2 = 0.84; Figures 1 and 2).A few individuals of P. clarkii were also detected in the cultivation ponds where C. destructor was present in 2012 (n = 1) and in 2013 (n = 3).In 2012, the only specimen of P. clarkii found in the ponds was infested by the alien temnocephalan crayfish ectoparasite Temnosewellia minor Haswell, 1888 (Figure 3), an ectoparasite always carried by C. destructor and described by morphological and molecular investigations in Chiesa et al. [22].The crayfish plague analyses evidenced a positive result in two out of the 12 sampled P. clarkii, the first found in the output canal of the cultivation ponds, the second found in Stagni della Flora (Pantanello area).

Cherax ponds Procambarus ponds Procambarus out of ponds
A few individuals of P. clarkii were also detected in the cultivation ponds where C. destructor was present in 2012 (n = 1) and in 2013 (n = 3).In 2012, the only specimen of P. clarkii found in the ponds was infested by the alien temnocephalan crayfish ectoparasite Temnosewellia minor Haswell, 1888 (Figure 3), an ectoparasite always carried by C. destructor and described by morphological and molecular investigations in Chiesa et al. [22].The crayfish plague analyses evidenced a positive result in two out of the 12 sampled P. clarkii, the first found in the output canal of the cultivation ponds, the second found in Stagni della Flora (Pantanello area).

Discussion and Conclusions
The results of our study show that C. destructor is now completely absent from the Laghi di Ninfa Natural Reserve, while P. clarkii is widespread in the area and was probably responsible for this eradication.In 2011, only 11 specimens of C. destructor were found in the cultivation ponds while P. clarkii was absent from the same ponds.In 2012, remains of dead C. destructor individuals (and only one alive) were found in the cultivation ponds together with a few P. clarkii, while in 2013 no specimens of C. destructor were found.Thus, the decline occurred before they came in syntopy in 2012, supporting the hypothesis of the crayfish plague.Indeed, Cherax destructor is vulnerable to the crayfish plague that is present in the area, as evinced by the positive outcomes on sampled P. clarkii.This case clearly demonstrated how an IAS can lead to the eradication of another IAS, not due to direct competition or predation but due to transmission of pathogens.In the Laghi di Ninfa area, all the water bodies are interconnected, and this allowed not only the spread of P. clarkii that reached the cultivation ponds where C. destructor was present but also the spread of the crayfish plague.Similarly, an episode of mortality was reported in farmed red claw crayfish, Cherax quadricarinatus (von Martens, 1868), reared in Sicily (Italy), due to A. astaci probably being carried by P. clarkii reared in the same facility [23].As the native river crab Potamon fluviatile Herbst, 1785, protected under the regional law 18/88, is present in the area (and was even found in the same trap with P. clarkii), the presence of alien crayfish carrying plague elicits great concern for the conservation of this species.Other crustaceans known to be susceptible to infection by A. astaci are the Chinese mitten crab (Eriocheir sinensis) (Benisch, 1940) [24], the crab Potamon potamios (Olivier, 1804) [25], and the Asian

Discussion and Conclusions
The results of our study show that C. destructor is now completely absent from the Laghi di Ninfa Natural Reserve, while P. clarkii is widespread in the area and was probably responsible for this eradication.In 2011, only 11 specimens of C. destructor were found in the cultivation ponds while P. clarkii was absent from the same ponds.In 2012, remains of dead C. destructor individuals (and only one alive) were found in the cultivation ponds together with a few P. clarkii, while in 2013 no specimens of C. destructor were found.Thus, the decline occurred before they came in syntopy in 2012, supporting the hypothesis of the crayfish plague.Indeed, Cherax destructor is vulnerable to the crayfish plague that is present in the area, as evinced by the positive outcomes on sampled P. clarkii.This case clearly demonstrated how an IAS can lead to the eradication of another IAS, not due to direct competition or predation but due to transmission of pathogens.In the Laghi di Ninfa area, all the water bodies are interconnected, and this allowed not only the spread of P. clarkii that reached the cultivation ponds where C. destructor was present but also the spread of the crayfish plague.Similarly, an episode of mortality was reported in farmed red claw crayfish, Cherax quadricarinatus (von Martens, 1868), reared in Sicily (Italy), due to A. astaci probably being carried by P. clarkii reared in the same facility [23].As the native river crab Potamon fluviatile Herbst, 1785, protected under the regional law 18/88, is present in the area (and was even found in the same trap with P. clarkii), the presence of alien crayfish carrying plague elicits great concern for the conservation of this species.Other crustaceans known to be susceptible to infection by A. astaci are the Chinese mitten crab (Eriocheir sinensis) (Benisch, 1940) [24], the crab Potamon potamios (Olivier, 1804) [25], and the Asian shrimp Macrobrachium dayanum (Henderson, 1893) and Neocaridina davidi (Bouvier, 1904) [26], thus, we cannot discard the hypothesis that P. fluviatile could be affected.
The connectivity of the Natural Reserve has been increased by the ecosystem restoration project in Pantanello, which was planned to create an area of conservation interest for several species without considering its potential for favouring the dispersal of P. clarkii.However, the risk of invasion should be addressed at the earliest possible stage of the planning of protected areas or any other intervention, starting from the earliest design or management plan for any new protected area [27].The landscape configuration of the geographic context in which a protected area is established, and the natural corridors connecting the protected area with surrounding areas, affect not only the interconnectivity which is vital for sustaining biodiversity but also the permeability of the protected area, and are crucial in determining the future patterns of invasions [28].A recent review on biological invasions in conservation planning evidenced that alien species were considered to be of concern for conservation in only 46% of the analysed cases (70) while mitigation measures were proposed in only 13% of the cases.Most of the studies (73%) ignored alien species in conservation planning even if their negative impacts were recognized [29].Thus, there is an urgent need to tackle this issue as protected areas are planned to maintain and increase the local biodiversity.Similarly, ecosystem restoration projects, often undertaken by or within protected areas, should consider the risk of causing or facilitating further IAS invasions, especially in aquatic habitats, and should adopt risk assessment protocols and a precautionary approach when data relating to biosecurity are lacking.However, limiting aquatic connectivity for IAS can disproportionately reduce potential connectivity restoration for desirable species [30].It is necessary to carry out a cost-benefit analysis (including the potential cost of IAS management) to assess the feasibility and effectiveness of a restoration intervention.Up to now, the spread of P. clarkii, favoured by the ecosystem restoration in the area of Pantanello has led to contrasting results.In addition to the eradication of C. destructor, an increase in the number of birds, especially aquatic birds, has been reported (from 709 in 2007 to 1159 in 2013, A. Monaco, pers.comm.)Aquatic birds mainly include the small grebe Tachybaptus ruficollis Pallas, 1764, the cormorant Phalacrocorax carbo (Linnaeus, 1758), the cattle egret Bubulcus ibis Linnaeus, 1758, the grey heron Ardea cinerea Linnaeus, 1758, the teal Anas crecca Linnaeus, 1758, the mallard Anas platyrhynchos Linnaeus, 1758, the common moorhen Gallinula chloropus (Linnaeus, 1758) and the coot Fulica atra Linnaeus, 1758), and this could be due to the abundant population of P. clarkii, as already observed in other areas highly invaded by this species (e.g., Massaciuccoli Lake, Tuscany).Conversely, P. clarkii may cause negative impacts on the local biodiversity, especially within the SAC area, where the fish Sarmarutilus rubilio (Bonaparte, 1837), endemic to Central Italy and classified as Near Threatened, is present.Further monitoring activities are thus necessary to assess the long-term effects of the restoration activities, particularly focussing on the spread of P. clarkii in the Laghi di Ninfa Natural Reserve.
Author Contributions: All authors conceived, outlined and conducted the field study.T.P. conducted crayfish plague analysis.G.M. and E.T. were responsible for data analysis, interpretation.and wrote the article.All authors read and accepted the final version of the manuscript.

Funding:
The research was part of the PASAL project (Progetto Atlante Specie Alloctone del Lazio-project no.13/a "Studi e censimenti nelle riserve del Lazio"-IV Accordo Integrativo dell'Accordo di Programma Quadro "Aree sensibili: parchi e riserve").PASAL was carried on by the Regional Parks Agency (ARP)-Regione Lazio and NEMO RTI, under the scientific supervision of the ISPRA (Institute for Environmental Protection and Research).

Figure 1 .Figure 1 .
Figure 1.The study area.Orange dots indicate the localities within the Laghi di Ninfa Natural Reserve where Procambarus clarkii was found between 2008 and 2013, the blue dot indicates the onlyFigure 1.The study area.Orange dots indicate the localities within the Laghi di Ninfa Natural Reserve where Procambarus clarkii was found between 2008 and 2013, the blue dot indicates the only population of Cherax destructor present in the area up until 2012.Numbers indicate the different ponds of the wetland area of Pantanello.The circle indicates the area of cultivation ponds.

Figure 2 .
Figure 2. Trend of Catch per Unit Effort (CPUE) of Cherax destructor and Procambarus clarkii from 2008 to 2013.

Figure 2 .
Figure 2. Trend of Catch per Unit Effort (CPUE) of Cherax destructor and Procambarus clarkii from 2008 to 2013.