The oomycete Aphanomyces astaci is the causative agent of crayfish plague in native European freshwater crayfish. Molecular analyses showed that several distinct genotype groups of this pathogen, apparently associated with different original host taxa, are present in Europe. Tracking their distribution may contribute to understanding the introduction pathways of A. astaci. We used microsatellite markers to genotype the pathogen strains involved in 7 mass mortalities of the endangered indigenous crayfish Austropotamobius pallipes that occurred between 2009 and 2016 in the Abruzzi and Molise regions, central Italy. Three A. astaci genotype groups (A, B, and D, with the latter represented by 2 distinct multilocus genotypes) were identified, suggesting the existence of multiple infection sources even in a relatively small area. Most crayfish plague episodes were due to genotype groups associated with the North American host species Pacifastacus leniusculus and Procambarus clarkii, although these crayfish are not widespread in the study area. A. astaci genotype group A was detected not only in crayfish plague outbreaks but also in apparently healthy Astacus leptodactylus imported for human consumption from Armenia and kept alive in an aquaculture facility. Imports of chronically infected A. leptodactylus from Armenia, Turkey, and possibly Eastern Europe are an underestimated introduction pathway for A. astaci. Although we cannot exclude the presence of latently infected native populations of A. pallipes in the region, A. astaci infections in legally imported crayfish species considered vulnerable to crayfish plague may represent further reservoirs of A. astaci; this should be reflected in the policies regulating the trade of live crayfish.

The oomyceteAphanomyces astaciis the etiologic agent of crayfish plague, a disease that has seriously impacted the populations of European native crayfish species. The introduction of non-indigenous crayfish of North American origin and their wide distribution across Europe have largely contributed to spread of crayfish plague in areas populated by indigenous crayfish. TrackingA. astacigenotypes may thus be a useful tool for investigating the natural history of crayfish plague in its European range, as well as the sources and introduction pathways of the pathogen. In this study, we describe the development of real-time PCR TaqMan assays aiming to distinguish the five genotype groups ofA. astaci(A–E) previously defined by their distinct RAPD patterns. The method was evaluated using DNA extracts from pureA. astacicultures representing the known genotype groups, and fromA. astaci-positive crayfish clinical samples collected mostly during crayfish plague outbreaks that recently occurred in Central Italy and Czechia. The assays do not cross-react with each other, and those targeting genotype groups A, B, D, and E seem sufficiently specific to genotype the pathogen from infected crayfish in the areas invaded byA. astaci(particularly Europe). The unusualA. astacigenotype “SSR-Up” documented from crayfish plague outbreaks in Czechia and chronically infectedPontastacus leptodactylusin the Danube is detected by the group B real-time PCR. The assay originally developed to detect group C (one not yet documented from crayfish plague outbreaks) showed cross-reactivity withAphanomyces fennicus; theA. astacigenotype “rust1” described in the United States fromFaxonius rusticusis detected by that assay as well. Analyses of additional markers (such as sequencing of the nuclear internal transcribed spacer or mitochondrial ribosomal subunits) may complement such cases when the real-time PCR-based genotyping is not conclusive. Despite some limitations, the method is a robust tool for fast genotyping ofA. astacigenotype groups common in Europe, both during crayfish plague outbreaks and in latent infections.

Crayfish plague, caused by the oomycete Aphanomyces astaci, is a serious disease of European freshwater crayfish and has eliminated entire populations in several European countries. In September 2011, mortality was observed among the Austropotamobius pallipes population of a river basin in the Abruzzi region (central Italy), and A. astaci DNA was detected by PCR in dead crayfish. A systematic survey was carried out to evaluate the spread and the effects of the plague in the river basin. The source of the outbreak remained unknown since North American crayfish species, which frequently act as subclinical carriers of the infection, were not detected in the area. The A. pallipes population disappeared from a river stretch of ~1 km, where A. astaci infection was detected in dead crayfish. However, apparently unaffected crayfish were still present upstream of that area as well as in a tributary that joined the brook in the apparently depopulated stretch. A. astaci infection was not detected in dead individuals collected in the upstream area and tributary. A follow-up visit conducted in the following season showed the presence of A. pallipes in the river stretch hit by the plague. In this outbreak, the spread of the infection could have been limited by a low density of the crayfish population and by the geographic conformation of the river basin, which includes a dense network of small tributaries, characterized by high flow velocity and low water temperature. In this particular setting, crayfish plague outbreaks can remain undetected. This underlines the importance of active monitoring programs aimed at the prompt recognition of both episodes of mortality and the presence of non-indigenous crayfish species.

The aim of this study was to evaluate the use of gonadal alterations in the thinlip grey mullet (Liza ramada) as a biological indicator in assessing aquatic ecosystems health, with particular emphasis to river ecosystems exposed to sewage discharges. For this purpose, the reproductive status and the presence of gonadal alterations were studied in 206 mullets collected from two sites on the low course of the Tiber River, downstream of a large urban sewage treatment plant and in the estuarine area, and from an uncontaminated pond considered as reference site. Intersex and irregularly shaped gonads were observed in 20.8% of the mullets from the most polluted site, and intersex gonads in 10.3% of those from the estuarine area. No alterations were detected in the fish from the reference site, which also showed\ud \ud \ud distinct stages of gonadal development. Conversely, unclear stages of testicular and ovary development were observed in the fish from the two polluted river sites. The results of this study suggest that L. ramada may represent a sentinel species in environmental risk assessment and support the use of gonadal alterations of this species as a bioindicator for extensive monitoring of pollution in lower stretches of rivers and estuarine areas

The aim of this study was to evaluate the use of gonadal alterations in the thinlip grey mullet (Liza ramada) as a biological indicator in assessing aquatic ecosystems health, with particular emphasis to river ecosystems exposed to sewage discharges. For this purpose, the reproductive status and the presence of gonadal alterations were studied in 206 mullets collected from two sites on the low course of the Tiber River, downstream of a large urban sewage treatment plant and in the estuarine area, and from an uncontaminated pond considered as reference site. Intersex and irregularly shaped gonads were observed in 20.8% of the mullets from the most polluted site, and intersex gonads in 10.3% of those from the estuarine area. No alterations were detected in the fish from the reference site, which also showed distinct stages of gonadal development. Conversely, unclear stages of testicular and ovary development were observed in the fish from the two polluted river sites. The results of this study suggest that L. ramada may represent a sentinel species in environmental risk assessment and support the use of gonadal alterations of this species as a bioindicator for extensive monitoring of pollution in lower stretches of rivers and estuarine areas.