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The marbled crayfish, Procambarus fallax f. virginalis, is a taxon widely available in the aquarium pet trade, which has been introduced to open waters in several European countries and in Madagascar. Recent studies confirmed this parthenogenetically reproducing crayfish as a high-risk invasive species, and vector of the crayfish plague pathogen, Aphanomyces astaci. It has been first discovered in Slovakia in 2010, but the status of the local population was not studied since then. Due to enlarged sampling area around the first report and one locality, where we presupposed the crayfish occurrence, we identified new marbled crayfish populations. Here, we report presence of three newly established marbled crayfish populations in Slovakia. Two populations are located critically close to the Váh River, a major tributary of the Danube River; one of them being directly connected to the Váh River via a side channel during occasional floods. The third established marbled crayfish population was found at the mouth of a thermal stream flowing into the Nitra River, a tributary of the Váh River. In this stream, crayfish coexist with other exotic fish and gastropod species of aquarium origin. We presume that the reported localities may serve as a source for further expansion of the marbled crayfish in the mid-part of the Danube catchment. Floods, active dispersal (including overland), passive dispersal by zoochory or anthropogenic translocations are among the major drivers facilitating the marbled crayfish colonization. We have not detected the crayfish plague pathogen in any of the studied populations. However, if spreading further, the marbled crayfish will encounter established populations of crayfish plague carriers in the Danube River, in which case they may acquire the pathogen by horizontal transmission and contribute to spread of this disease to indigenous European crayfish species.
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