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The Northern raccoon Procyon lotor is a native mesocarnivore of North America which has been introduced to many Eurasian countries, where invasive populations currently occur. Regarding Italy, the species is present in Lombardy and in the Foreste Casentinesi National Park (Tuscany and Emilia-Romagna). In this work, we conducted a review on food habits of the Northern raccoon both in the native and in the introduced ranges, and we firstly reported the evidence on the impact of this species on native crayfish in Central Italy. We retrieved 18 papers published between 1932 and 2020, 14 from the native range and four from the invasive range. We analysed the frequency of occurrence of nine food categories (mammals, birds, reptiles, amphibians, fish, insects, crustaceans, fruits, and others). After fruits, crustaceans (where available) were the main food items detected in raccoon scats (up to 60% of the total diet, in the frequency of occurrence). In September 2019, we detected a total of 37 dead adult freshwater white-clawed crayfish Austropotamobius pallipes complex in Central Italy, with evident signs of predation by the Northern raccoon. The local population of Northern raccoon is fast expanding its range, whereas the local population of A. pallipes complex is declining. This crayfish species is protected by national and international laws. Thus, a monitoring program involving all stakeholders and institutions dealing with in environmental management, aiming at removing all raccoons currently present in this area of Central Italy, should be carried out in the short term to limit the potential impacts exerted by the fast-spreading of this invasive mammal.

The white-clawed crayfish Austropotamobius pallipes complex populations are decreasing in the Foreste Casentinesi, Monte Falterona and Campigna National Park (Central Italy), due to several factors, including illegal poaching, predatory fishes, drought, and invasive alien species. Recently, the Northern raccoon Procyon lotor has been reported to be present in the area of the National Park and has started to predate on the white-clawed crayfish. The aim of the study was to update the distribution and population status of A. pallipes in the reserves, other sites of the National Park, and surrounding areas to assess the potential effects of the raccoon. Crayfish were sampled by hand or by traps in 14 sites; sampled individuals were sexed and measured. Signs of raccoon presence (e.g., footprints and predated crayfish) were also recorded. Our study confirms the impact of raccoon on native crayfish: indeed, where the invasive mammal is present (six sites), crayfish disappeared, or their populations have been dramatically reduced in number, with a size distribution skewed towards juveniles. In two sites, close to urban settlements, fresh footprints of P. lotor and predated specimens of A. pallipes were also observed. Populations of crayfish are still abundant or even increasing as compared with samplings conducted in the past where raccoon is absent (five sites). Urgent actions (e.g., control of raccoons, and monitoring and restocking of A. pallipes populations if feasible and where possible) should be taken into account to guarantee the survival of this protected species.

The microsporidiosis of the endangered white-clawed crayfish Austropotamobius pallipes complex has generally been attributed to only one species, Thelohania contejeani, the agent of porcelain disease. Species identification was mostly assessed by macroscopic examination or microscopic evaluation of muscle samples rather than by molecular or ultrastructural analyses. A survey conducted on A. pallipes complex populations in Northern Italy highlighted the presence of two different microsporidia causing similar muscular lesions, T. contejeani and an undescribed octosporoblastic species Vairimorpha austropotamobii sp. nov. Mature spores and earlier developmental stages of V. austropotamobii sp. nov. were found within striated muscle cells of the thorax, abdomen, and appendages of the crayfish. Only octosporoblastic sporogony within sporophorous vesicles (SPVs) was observed. Diplokaryotic sporonts separated into two uninucleate daughter cells, which gave rise to a rosette-shaped plasmodium, and eight uninucleate spores were produced within the persistent SPV. Ultrastructural features of stages in the octosporoblastic sequence were similar to those described for Vairimorpha necatrix, the type species. Mature spores were pyriform in shape and an average of 3.9 × 2.2 µm in size. The polar filament was coiled 11-14 times, lateral to the posterior vacuole. The small subunit ribosomal RNA gene (SSU rRNA) and the large subunit RNA polymerase II gene (RPB1) of V. austropotamobii sp. nov. were sequenced and compared with other microsporidia. The highest sequence identity of SSU rRNA (99%) and RPB1 (74%) genes was with the amphipod parasite Nosema granulosis and subsequently with V. cheracis, which infects the Australian yabby Cherax destructor. In our work we discuss about the reasons for placing this new species in the genus Vairimorpha. In addition, we provide for T. contejeani a RPB1 gene sequence, supplemental sequences of SSU rRNA gene and ultrastructural details of its sporogony in the host A. pallipes complex.

2013/2014 The white-clawed crayfish A. pallipes has suffered in recent decades a strong decline throughout its entire distributional range, mainly due to the growing number of threats coming from anthropic influence, including habitat loss and degradation, overfishing, infectious diseases, and the introduction of non-indigenous crayfish species (NICS). The species is included in the red list of the IUCN (International Union for Conservation of Nature) as a species at risk of extinction. An important goal in conservation biology is to assess the genetic variability and thus the “genetic health” of populations and to identify any evolutionarily significant unit (ESU) within endangered species, before management decisions are taken. Within RARITY (http://www.life-rarity.eu), a LIFE project for the eradication of the invasive Louisiana red swamp (Procambarus clarkii) and for the preservation of the native white-clawed crayfish in Friuli Venezia Giulia (NE Italy), I was responsible for the genetic characterization of A. pallipes complex in this area, in order to define the taxonomic status, the genetic variability and the population structure and differentition. The analysis of two mitochondrial genes (COI and 16 rDNA) of about 500 individuals from 58 monitored sites showed that the FVG crayfish belong to the A. italicus species, with two different subspecies present: A.i. carsicus and A.i.meridionalis. The analysis at six polimorphic microsatellite loci revealed generally low levels of within population genetic diversity (0,0

Any initiative aimed at the management of a threatened species needs a good knowledge of its environmental requirements. Aims of this study were to individuate suitable areas for the reintroduction of crayfish species belonging to the Austropotamobius pallipes complex and to evaluate the causes of the local extinction in Tuscany (Italy) of some populations. Between May 2003 and September 2004, we collected ecological data from 19 streams of 4 catchments, 9 watercourses where crayfish were present (WI) and 10 where they were present in the recent past and absent now (WO), and we compared them. Multivariate analyses were performed using chemico-physical and biotic parameters to examine the relationships between their values and the presence of crayfish. The results did not allow us to find significant differences between the two categories of streams, suggesting their suitability for crayfish reintroduction.

A total of 99 live and 3 dead white-clawed crayfish Austropotamobius pallipes complex were collected during the summer and autumn of 2003 from one creek in Emilia Romagna region and four watercourses in Friuli Venezia Giulia region (Northern Italy) for experimental breeding and health status monitoring. Fifty-one animals from the Bidente River were maintained in tanks for breeding, and restocking, while another 30 specimens (27 living and 3 dead) were examined in the laboratory to evaluate their health status. Parasitological surveys were carried out on gills; mycological examinations on the exoskeleton (the walking legs and the abdomen); bacteriological examinations of inoculations from the haemolymph, and histopathological analysis on full transverse and sagittal sections of cephalothorax and abdomen.In the branchial-chambers, the gill filaments and the exoskeleton were observed for ectocommensal peritrich protozoans Epystilis spp. and Cothurnia sieboldii. In crayfish sampled from five streams and rivers, Branchiobdella italica, B. astaci, B. parasita and B. hexodonta were isolated. Heavily damaged gills in the presence of branchiobdellid cocoons were observed in the crayfish found dead in Orvenco creek.Hyaline and dematiaceous hyphomycetes were frequently observed and Fusarium spp. and Saprolegnia sp. were isolated from cuticular lesions.Mortalities occurred in two tanks, after 2 and 4 weeks respectively, in the adult crayfish coming from Bidente River utilized for breeding, with approximately a 23.5% cumulative mortality rate. In both cases the crayfish were weak, anorexic and lethargic. Four and eight moribund samples respectively, coming from the two breeding tanks in which the mortalities occurred, underwent examination. In the first case the mortalities were attributed to Thelohania-like organisms and in the second case the mortalities were associated with enteric bacteria Citrobacter freundii.

White-clawed crayfish is a native European species distributed naturally within water bodies of Southern and Western Europe. Due to anthropogenic influence onto their habitats and spread of crayfish plague many populations disappeared and/or were dramatically reduced in their sizes. Hence white-clawed crayfish is treated as endangered and is protected by both international and local legislation. Currently, based on molecular research, white-clawed crayfish is recognised as a complex including two distinct species: Austropotamobius pallipes and Austropotamobius italicus, with four subspecies in the latter. So far, results of white-clawed morphological traits research have not yielded unambiguous and clear separations. Therefore, the aim of this study was to use large morphological and meristical data sets originating from white- clawed crayfish caught in rivers of the Adriatic Sea drainage, and analyse them using the multivariate statistical method. In total 249 crayfish from 9 populations belonging to different subspecies were studied (analysed). Results showed that males and females differ significantly between populations in measured meristical and morphometrical traits. Meristical traits were significantly correlated to the crayfish body size, therefore not reliable for distinguishing populations. From measured morphometrical traits, the most discriminant ones for separating both males and females were those describing rostrum, carapace and claw shape. Overall, percentage of correctly classified crayfish was high (98% for males, 96% for females). Additionally, grouping and separation of studied populations obtained by canonical analyses was in accordance with their taxonomical classification (i.e. subspecies), previously achieved with a molecular approach.

From 2004 to 2006, a parasitological survey aimed at the detection of the microsporidian parasite Thelohania contejeani Henneguy was carried out on 177 wild white-clawed crayfish (Austropotamobius pallipes complex) captured in six streams and rivers of the province of Belluno in north-eastern Italy. Microscopical examination of the skeletal muscles, and histological analysis applying different histochemical stains to full transverse and sagittal sections of the cephalothorax and abdomen were carried out. Transmission electron microscopy (TEM) was also conducted on the parasites recovered during the survey. Out of 177 crayfish examined, Thelohania contejeani (Microsporidia, Thelohaniidae) was present in only one crayfish from the Vena d’oro creek. The parasite was detected in the skeletal muscles in several developmental stages, including mature spores, which represented the most common stage recovered. Sporophorous vesicles were also present. Histological examination revealed that the fibres of the skeletal, cardiac and intestinal muscles were filled with spores. Melanin infiltrations were focally present in the infected striated muscles. The gill phagocytic nephrocytes were engulfed by small masses of spores. Among the staining techniques applied, Crossman’s trichrome stain represented the most effective method of detecting T. contejeani.

Mortality of white-clawed crayfish, Austropotamobius pallipes complex, occurred in a Northeastern Italian trout hatchery during an intensive breeding trial between autumn 2004 and summer 2005. The crayfish were submitted to postmortem histological, microscopical, mycological and bacteriological examinations. Saprolegniaceae were isolated from gills, legs and abdomen of all crayfish samples, while Fusarium sp. was found in only one specimen. The ectosymbiotic peritrichous ciliate protozoan, Cothurnia sieboldii, and a clitellate annelid, Branchiobdella hexodonta, were found on gill filaments and exoskeletons of all specimens examined. Fungal hyphae were observed on gonopods, abdominal exoskeletons, eye stalks and leg joints. These infections were occasionally observed causing heavy inflammatory lesions in skeletal muscle and green gland, and necrosis in the hypoderm; in some cases the hyphae were surrounded by melanin. The cause of mortality was attributed to a Saprolegniaceae infection, induced by poor water quality conditions

The white-clawed crayfish (Austropotamobius pallipes (Lereboullet)) is a native European freshwater crayfish inhabiting Croatian freshwater habitats. It is naturally distributed in the Adriatic Sea drainage, while in Europe it is found in its southern and western parts, including British Isles. It has been frequently studied and it presents a taxon that is highly structured, so the latest suggestion is to consider A. pallipes as a species complex. The number of populations is continuously diminishing so it is treated as endangered species and it protected by Croatian and international laws. The purpose of this research was to study the phylogenetic position of Croatian populations in comparison to already studied populations of this species in the rest of Europe utilizing broad range of phylogentic methods on two mitochondrial genetic markers. Results of phylogenetic analysis are discussed in details.

Crayfish are regarded as useful indicators of environmental quality and freshwater biodiversity. However, reliable methods for monitoring their populations are needed so that this potential can be fully utilised. We report and discuss methodological aspects of the white-clawed crayfish (Austropotamobius pallipes complex) survey conducted in Piedmont, Italy, with the use of mark-recapture. The results suggest that the method can serve as a convenient tool for estimating the size of crayfish populations and inferring their temporal trends. The two populations investigated appeared closed except for wintertime and July. Consequently, the Robust Design, which is regarded as the most reliable mark-recapture approach, can be easily applied. The minimum effective sampling plan for monitoring purposes should comprise one primary period per year, conducted in the summer–autumn season, and consisting of three capture sessions. If gaining insight into the ecology of the investigated species is the prime objective and sufficient resources are available, the optimal plan should include two primary periods (in spring and the summer–autumn season) of five capture sessions each. Capture sessions need to be separated by roughly 2-week intervals in order to avoid the strong, but short-term, negative effect of capturing crayfish on their recapture chances. As the model without heterogeneity in capture probabilities ensures better estimate precision we recommend that data collected for both sexes are analysed separately. Taking into consideration higher male catchabilities and sex ratio being invariably 1:1, it also seems beneficial to estimate only male numbers and double them to achieve total population sizes.

A massive and apparently total mortality of white-clawed crayfish (Austropotamobius pallipes complex) occurred in Rossenna (September 2004), Lerna (February 2005) and Selve (June 2005) streams in Modena province, Northern Italy. In all three streams the deceased crayfish were in different states of preservation and only recently dead or moribund crayfish were collected and submitted to mycological, parasitological, bacteriological, virological, and histopathological analyses. Extended Biotic Index (EBI) was evaluated for each water course and chemical-physical and bacteriological water analyses were carried out., Saprophytes, ectosymbionts and ectocommensal organisms (sessile peritrich ciliate protozoa and branchiobdellida) were frequently observed in crayfish. No virus was found. Fusarium sp. was the most frequently isolated mycete and it occurred in pure culture from the specimens coming from creek Selve. Furthermore Mucor sp. and Penicillium sp. were detected on the crayfish from the river Rossenna, while Trichoderma sp., Alternaria sp., Gliocladium sp. and Mucor sp. from Lerna specimens. The bacteriological investigation recorded Hafnia alvei from both Lerna and Rio Selve samples, while Aeromonas hydrophila only from Lerna. During the histological investigation,, all crayfish showed severe fungal hyphae infiltrations from the exoskeleton through the superficial skeletal to the muscle hypodermis evidencing inflammatory reactions and melanization. All water courses showed environmental pollution. A prolonged exposure to toxic substances in the water could have exhausted the defense mechanisms so that the crayfish were more susceptible to the action of opportunistic pathogenic agents and subsequently succumbed.

Vengono descritti 3 gravi episodi di mortalità in Austropotamobius pallipes complex verificatisi in corsi d’acqua dell’Appennino Modenese: nel torrente Rossenna (09/04), Lerna (02/05) e Rio Selve (06/05). Campioni di gamberi appena morti o moribondi, sono stati raccolti e sottoposti ad esame micologico, parassitologico, virologico, batteriologico ed istopatologico. Nei torrenti sono stati valutati l’indice biotico esteso ed i parametri chimico-fisici dell’acqua. Sono stati riscontrati organismi saprofiti ectosimbionti ed ectocommensali. Nessun virus è stato riscontrato. All’esame micologico Fusaruim sp. è stato isolato dai campioni di tutti e tre i torrenti e, in coltura pura, da quelli del Rio Selve. Nei soggetti provenienti dal Rossenna, erano presenti Mucor sp., Penicillium sp. e Dematiaceae; Trichoderma sp., Alternaria sp., Gliocladium sp., Mucor sp., Geotrichum sp. dal Lerna. All’esame batteriologico Hafnia alvei si isolava nei campioni provenienti sia da Rio Selve che dal Lerna ed in quest’ultimo anche Aeromonas hydrophila. All’esame istologico di tutti i campioni si riscontravano ife fungine infiltranti l’esoscheletro che penetravano la cuticola invadendo l’epidermide ed il muscolo sottostante. Tutti i torrenti mostravano segni di inquinamento. Una prolungata esposizione dei gamberi a sostanze tossiche potrebbe avere determinato una riduzione dei meccanismi di difesa dei crostacei rendendoli vulnerabili all’azione di agenti patogeni opportunisti, con conseguente massiva mortalità.

To fill the existing gaps in the knowledge of the crayfish species distribution in Trentino region (North-East Italy), we surveyed fourteen lakes, six ponds and sixty-nine creeks and streams in 2010-2012. We recorded four populations of the invasive alien species Orconectes limosus in four lakes and twenty of the native Austropotamobius pallipes complex from four lakes, three ponds and thirteen creeks; three of these populations went extinct during the three years of our survey. Crayfish populations were monitored in spring, summer and early autumn, to assess the distribution, density and seasonal dynamics of the two species. Statistical analysis of morphometric data showed differences in body size and growth rates among sampling sites, possibly related to local environmental factors, with higher growth rates in males of both species; A. pallipes grew faster in creeks than in lakes. The extinction of twenty-two native populations of A. pallipes complex in the last century was likely due to habitat modifications, in particular to the loss of riparian habitat, in few cases to overfishing, and, more recently, to the spread of alien species and the related transmission of their parasite Aphanomyces astaci. Some populations of A. pallipes complex were infested with the parasite Thelohania sp., and only one population with ectosymbiotic Branchiobdellida. In Trentino, small creeks with well developed riparian vegetation and good hydromorphological conditions, flowing through mountain slopes, can represent potential refuge and recruitment areas for of A. pallipes complex, without significant management intervention, although a sustainable management of piedmont water bodies would allow preserving or increasing the number and density of the relict populations.

The Austropotamobius pallipes complex is formed by the two species A. pallipes and A. italicus , recently defined only on genetic basis, without obvious morphological features for their differentiation. Dealing with this problem, we have conducted a breeding experiment between these two presumed species under controlled conditions, in order to investigate the possible existence of reproductive barriers. Using 80 breeders (56 females and 24 males) collected from populations where their species identity was genetically well established, we performed four series of trials: F ita × M pal and F pal × M ita interspecific mating; F ita × M ita and F pal × M pal intraspecific mating as a control. We have monitored only the main phases of the reproduction in order to avoid excessive interference for the crayfish. In November 2009 the 91.7% of females having intraspecific mating, as well as the 100% of females having interspecific mating, produced eggs. In June 2010 the eggs from the 36.4% of females having intraspecific mating, and those from the 42.3% of females having interspecific mating did hatch. We emphasize the hatching of hybrids and their vitality.

Information was gathered on the distribution of white-clawed crayfish Austropotamobius pallipes complex in the watercourses of the Alessandria province (NW Italy), on the biological and ecological preferences of the species, and on the features of the biotopes in which it is found. A total of 409 sites on 361 watercourses connected to the main sub-basins of the Po River were analysed, with data gathered during the field research phase, conducted for three consecutive summers from 2002 through 2004. Thirteen percent of the sites investigated were found to currently house crayfish populations. The persistence in time of superficial water and natural morphology of the watercourse were found to be the chief requirements for the presence of crayfish. Data on land use, human density and other factors which potentially limit the survival of crayfish populations were also analysed.