Hydrobiologia DOI 10.1007/s10750-016-2841-4 PRIMARY RESEARCH PAPER First detection of the crayfish plague pathogen Aphanomyces astaci in South America: a high potential risk to native crayfish Douglas Fernando Peiro´ . Mauricio P. Almera˜o . Carine Delaunay . Japo Jussila . Jenny Makkonen . Didier Bouchon . Paula B. Araujo . Catherine Souty-Grosset Received: 30 June 2015 / Revised: 22 May 2016 / Accepted: 24 May 2016 Ó Springer International Publishing Switzerland 2016 Abstract The crayfish plague pathogen, Aphano- Furthermore, our results indicate preliminary evidence myces astaci, is a fungal-like organism (Oomycetes), of possible A. astaci infection, agent level A2, in two specialized in parasitizing freshwater crayfish species. sympatric native species, namely Parastacus defossus Crayfish plague is a disease that has caused losses of and Parastacus pilimanus. In this study, we provide indigenous crayfish populations, especially in Europe. the first overview concerning the presence of the The pathogen chronically infects North American crayfish plague pathogen, A. astaci, in South America. endemic crayfish, such as Procambarus clarkii, which is considered an invasive species in several continents, Keywords Procambarus clarkii Á Parastacus Á including South America. Using molecular tools, Astacidea Á PCR Á qPCR Á RT-PCR Á Alien species Á quantitative PCR, and conventional PCR, we detected Invasive species Á Conservation Á Molecular tools this pathogen in feral P. clarkii populations estab- lished in southeastern Brazil. This is an alarming result Introduction because in South America, especially in Brazil, there is considerable endemic crayfish species diversity, Crayfish plague is a disease caused by the pathogen especially in the genus Parastacus. Possible contacts Aphanomyces astaci Schikora (Oomycetes, Saproleg- between P. clarkii and the endemic crayfish could be niaceae) (Souty-Grosset et al., 2006). One of the many seen as a major threat to the native crayfish, mainly carriers of A. astaci is Procambarus clarkii (Girard, because of the possibility of A. astaci transmission. 1852) (Astacidea, Cambaridae), originally from North America, and popularly known as the red swamp crayfish (Barbaresi et al., 2007;Ha¨nfling et al., 2011). Handling editor: John Havel ´ & D. F. Peiro´ Á C. Delaunay Á D. Bouchon Á D. F. Peiro ( ) Á M. P. Almera˜o Á P. B. Araujo C. Souty-Grosset Laborato´rio de Carcinologia, Departamento de Zoologia, Laboratoire Ecologie & Biologie des Interactions - UMR Universidade Federal do Rio Grande do Sul, Av. Bento CNRS 7267, Universite´ de Poitiers, Equipe Ecologie Gonc¸alves 9500, Bloco IV, pre´dio 43435, Porto Alegre, Evolution Symbiose. 5, rue Albert Turpain, TSA 51106, RS 91501-970, Brazil 86073 Poitiers Cedex 9, France e-mail: [email protected] D. F. Peiro´ J. Jussila Á J. Makkonen Laborato´rio de Biologia Aqua´tica-Crusta´ceos, Department of Environmental and Biological Sciences, Departamento de Cieˆncias Biolo´gicas e da Sau´de, University of Eastern Finland, Kuopio Campus, Universidade de Araraquara, Rua Carlos Gomes 1338, P.O. Box 1627, 70211 Kuopio, Suomi-Finland Araraquara, SP 14801-340, Brazil 123 Hydrobiologia This crayfish may become infected without develop- freshwater crayfish, such as in Porto Alegre City in ing the gross symptoms of the disease or increased south Brazil (Loureiro et al., 2015). The distribution of mortality (Unestam, 1969; Aquiloni et al., 2011), and, P. clarkii in Brazil follows predictions from dispersal thus, is capable of acting as a chronic carrier and models for this species (Palaoro et al., 2013), and permanent reservoir of the pathogen. In contrast, some indicates a possible threat to native Brazilian freshwater other crayfish of North American origin may show crayfish in the future. increased mortality due to A. astaci infection (Tho¨rn- A total of 13 crayfish species, belonging to three qvist & So¨derha¨ll, 1993; Aydin et al., 2014; Jussila genera (Parastacus, Virilastacus and Samastacus), are et al., 2014a, b, 2015b; Edsman et al., 2015). native to South America. They are distributed in High levels of mortality are usually observed within southern Brazil, Uruguay, northeast and southern a few months of when A. astaci first infects a crayfish Argentina, and central-southern Chile (Buckup, 2003; population of a susceptible species (Alderman, 1996; Rudolph, 2013; Almera˜oetal.,2014). Brazil hosts Souty-Grosset et al., 2006; Makkonen et al., South American endemic parastacid crayfish species, 2012, 2014; Jussila et al., 2015a). In Europe, mass which may be infected by A. astaci via the introduced mortalities caused by crayfish plague epidemics had P. clarkii (Loureiro et al., 2015). The contact among P. already started before the introduction of the North clarkii (infected with A. astaci) with the native crayfish American crayfish, as the pathogen A. astaci was first may result in epidemics, thus, negative consequences to introduced into the continent with an unknown host. diversity, similar to that experienced in Europe This host, based on current understanding, is not found (Holdich et al., 2009; Tuffs & Oidtmann, 2011). The in Europe (Grandjean et al., 2014; Kozubı´kova´- threat is heightened by the highly invasive nature of P. Balcarova´ et al., 2014). A second wave of epidemics clarkii (Barbaresi & Gherardi, 2001; Gherardi et al., was caused by the massive introduction of North 2002; Gherardi, 2006), and also by the possibility of American crayfish species from the 1960s onwards, human translocations (Magalha˜es et al., 2005; Silva & inducing further damage and establishing widespread Bueno, 2005). P. clarkii also seems to be capable of permanent host populations for A. astaci (Unestam, adapting to cooler climatic conditions (as in southern 1972; Alderman, 1996;So¨derha¨ll & Cerenius, 1999; South America), as has been observed in northern Souty-Grosset et al., 2006; Kozubı´kova´-Balcarova´ Germany (Chucholl, 2011;Vesely´ et al., 2015;Jo¨rn et al., 2014). Currently, one of the main transmission Panteleit, personal communication), The Netherlands, routes of A. astaci is the colonization of new habitats and southern England (Kouba et al., 2014). by the North American crayfish host species (Holdich Our aim was to investigate the presence of the et al., 2009; OIE, 2012). crayfish plague pathogen A. astaci in Brazilian feral In Brazil, the first reported occurrence of P. clarkii populations of P. clarkii using molecular tools [TaqMan was in the State of Sa˜o Paulo (Huner, 1986), and further MGB quantitative PCR (qPCR) and conventional PCR]. reports have been made of several feral P. clarkii In addition, we investigated the presence of the A. astaci populations in recent years (Magalha˜es et al., 2005; in selected native crayfish species, namely Parastacus Silva & Bueno, 2005; Loureiro et al., 2015). Commer- brasiliensis (Von Martens, 1869), Parastacus pilimanus cial and illegal trading of crayfish as ornamental species (Von Martens, 1869), and Parastacus defossus Faxon, has been suggested as the pathway of introducing P. 1898, which have not yet been recorded in sympatry clarkii into Brazil (Loureiro et al., 2015). Procambarus with P. clarkii populations, to study possible latent A. clarkii maintained as pets, but released later alive to the astaci infections in native Brazilian crayfish. environment, may have adapted quickly and success- fully, establishing populations in some ponds and lakes (Loureiro et al., 2015). This has happened in spite of the Material and methods fact that the trade or possession of P. clarkii has been prohibited by federal laws since 2008 by the Brazilian Crayfish sampling Institute of Environment and Renewable Natural Resources (IBAMA, 2008). Illegal pet trade of P. Procambarus clarkii were collected (from April 2012 clarkii has been reported not only in the State of Sa˜o to February 2013) from different populations estab- Paulo, but also in other regions inhabited by native lished in the State of Sa˜o Paulo, Brazil, and a sample 123 Hydrobiologia was obtained from a pet shop (PS), in Porto Alegre Filipova´ et al. (2013). Individual crayfish were City, State of Rio Grande do Sul, Brazil (Fig. 1; dissected aseptically to remove presumably infected Table 1). The specimens from the wild were captured tissues (Oidtmann et al., 2006): abdominal ventral using hand-nets and traps, and were individually cuticle: 0.5 cm2, both uropods, and two pereopod euthanized by freezing, and then stored in 100% coxa. All dissections were conducted at the Labo- ethanol. The same sampling procedures were applied rato´rio de Carcinologia, Universidade Federal de Rio to P. brasiliensis, P. pilimanus, and P. defossus, which Grande do Sul, Porto Alegre City, Brazil. All material were collected in the State of Rio Grande do Sul, was preserved in 70% ethanol, and then transported to Brazil. A total of 160 individuals from 15 different France for the following procedures at the Laboratoire populations or origins were collected. Ecologie & Biologie des Interactions, Universite´ de Poitiers, Poitiers City, France. Crayfish dissection and DNA extraction The dissected tissues were aseptically cut by scissors and dried on a heating block (75°C) for about The following procedures are based on and modified 5 min. Then, 360 ll of ATL buffer (DNeasy tissue kit, from Oidtmann et al. (2006), Vra˚lstad et al. (2009) and Qiagen), and sterilized steel beads (one portion: Fig. 1 Brazil and southern states of Brazil. A Locations in the AV, 9 CEU, and 10* PS. Parastacus brasiliensis populations: 11 State of Sa˜o Paulo. B Locations in State of Rio Grande do Sul. MP, 12 MS, and 13 ZS. Parastacus pilimanus population: 14* *Population or origins identified Aphanomyces astaci-positive EA. Parastacus defossus population: 15* EA. Please find the (by qPCR or ITS-PCR). Procambarus clarkii populations or acronyms list of the populations/origins in Table 1 origins: 1* EM, 2 VRE, 3 APA, 4 CO, 5 SRO, 6* SD, 7 JAR, 8* 123 Hydrobiologia Table 1 Location list of Procambarus clarkii, Parastacus brasiliensis, Parastacus pilimanus, and Parastacus defossus collected in Brazil Population Location, city, state Geographical #of #of ITS- Level of acronyms location analyzed Aphanomyces PCR infection: no.