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The European noble crayfish Astacus astacus is threatened by crayfish plague caused by the oomycete Aphanomyces astaci, which is spread by the invasive North American crayfish (e.g. signal crayfish Pacifastacus leniusculus). Surveillance of crayfish plague status in Norway has traditionally relied on the monitoring survival of cage‐held noble crayfish, a method of ethical concern. Additionally, trapping is used in crayfish population surveillance. Here, we test whether environmental DNA (eDNA) monitoring could provide a suitable alternative to the cage method, and a supplement to trapping. We took advantage of an emerging crayfish plague outbreak in a Norwegian watercourse following illegal introduction of disease‐carrying signal crayfish, and initiated simultaneous eDNA monitoring and cage‐based surveillance, supplemented with trapping. A total of 304 water samples were filtered from several sampling stations over a 4‐year period. eDNA data (species‐specific quantitative real‐time PCR [qPCR]) for the presence of A. astaci, noble and signal crayfish within the water samples were compared to cage mortality and trapping. This is the first study comparing eDNA monitoring and cage surveillance during a natural crayfish plague outbreak. We show that eDNA monitoring corresponds well with the biological status measured in terms of crayfish mortality and trapping results. eDNA analysis also reveals the presence of A. astaci in the water up to 2.5 weeks in advance of the cage method. Estimates of A. astaci and noble crayfish eDNA concentrations increased markedly during mortality and vanished quickly thereafter. eDNA provides a snapshot of the presence, absence or disappearance of crayfish regardless of season, and constitutes a valuable supplement to the trapping method that relies on season and legislation. Synthesis and applications. Simultaneous eDNA monitoring of Aphanomyces astaci (crayfish plague) and relevant native and invasive freshwater crayfish species is well‐suited for early warning of invasion or infection, risk assessments, habitat evaluation and surveillance regarding pathogen and invasive/native crayfish status. This non‐invasive, animal welfare friendly method excludes the need for cage‐held susceptible crayfish in disease monitoring. Furthermore, eDNA monitoring is less likely to spread A. astaci than traditional methods. This study resulted in the implementation of eDNA monitoring for Norwegian crayfish plague and crayfish surveillance programmes, and we believe other countries could improve management strategies for freshwater crayfish using a similar approach. Sammendrag Europeisk edelkreps (Astacus astacus) trues av krepsepest som forårsakes av eggsporesoppen Aphanomyces astaci. Smitten spres av fremmed nordamerikansk ferskvannskreps (f.eks. signalkreps; Pacifastacus leniusculus). Overvåking av krepsepest i Norge har tradisjonelt basert seg på burforsøk, en etisk problematisk metode hvor dødelighet hos edelkreps i bur overvåkes ved relevante lokaliteter. Overvåking av edelkrepsbestander blir gjort ved bruk av teiner. Vi har testet om overvåking basert på innsamling av miljø‐DNA (eDNA) kan være et egnet alternativ til burforsøk, og et supplement til teinefangst. Etter en ulovlig introduksjon av smittebærende signalkreps i en innsjø med edelkreps, utnyttet vi vissheten om et kommende krepsepestutbrudd til å initiere eDNA‐overvåking og burforsøk samtidig, supplert med teinefangst. Tilsammen ble 304 vannprøver filtrert fra ulike prøvetakingsstasjoner over en fire‐års periode. eDNA data (arts‐spesifikk qPCR) for tilstedeværelse av A. astaci, edelkreps og signalkreps i vannprøver ble sammenlignet med dødelighet i burforsøk og teinefangst. Dette er den første studien som sammenligner eDNA‐overvåkning og burforsøk under et naturlig krepsepestutbrudd. Vi viser at eDNA‐overvåking korresponderer godt med biologisk status målt i form av dødelighet hos burkreps og resultater fra teinefangst. eDNA‐analyser avslører også tilstedeværelsen av A. astaci smittestoff i vannet opptil 2,5 uker før edelkreps dør i burforsøk. Mengdeestimater av eDNA fra A. astaci og edelkreps i vannet økte markant under dødelighet, og forsvant deretter raskt. Uansett årstid gir eDNA et øyeblikksbilde av tilstedeværelse, fravær eller bortfall av edelkreps, og utgjør derfor også et verdifullt supplement til teinefiske, som avhenger av sesong og nasjonal lovgivning. Syntese og bruksområder. Parallell eDNA‐overvåkning av Aphanomyces astaci (krepsepest agens) og relevante stedegne og fremmede arter av ferskvannskreps er velegnet for tidlig varsling av invasjon og smitte, risikovurderinger, evaluering av habitatstatus, og overvåking av status for smittestoff og fremmed/stedegen ferskvannskreps. Metoden er dyrevelferdsvennlig, og utelukker behovet for burforsøk med levende kreps i sykdomsovervåking. Videre gir eDNA‐overvåkning mindre sannsynlighet for å spre Aphanomyces astaci smitte enn tradisjonelle metoder. Denne studien har bidratt til å implementere eDNA‐overvåking i norsk overvåkning av krepsepest, edelkreps og signalkreps, og vi tror at andre land også kan forbedre sine forvaltningsstrategier for ferskvannskreps ved hjelp av en lignende tilnærming. Simultaneous eDNA monitoring of Aphanomyces astaci (crayfish plague) and relevant native and invasive freshwater crayfish species is well‐suited for early warning of invasion or infection, risk assessments, habitat evaluation and surveillance regarding pathogen and invasive/native crayfish status. This non‐invasive, animal welfare friendly method excludes the need for cage‐held susceptible crayfish in disease monitoring. Furthermore, eDNA monitoring is less likely to spread A. astaci than traditional methods. This study resulted in the implementation of eDNA monitoring for Norwegian crayfish plague and crayfish surveillance programmes, and we believe other countries could improve management strategies for freshwater crayfish using a similar approach.

Microorganisms are an important part of productivity, water quality, and biogeochemical cycles in an aquaculture ecosystems and play a key role in determining the growth and fitness of aquaculture animals. Coculture ecosystems are widely applied with great significance in agricultural production worldwide. The crayfish-rice coculture ecosystem (CRCE) and crayfish-waterweed coculture ecosystem (CWCE) are two high-profile artificial ecosystems for crayfish culture. However, the bacterial communities of the environmental water, sediment, and intestine in the CRCE and CWCE remain elusive. In this study, we investigated the diversity, composition, and function of bacterial communities in water, sediment, and intestine samples from the CRCE to CWCE. The physicochemical factors of water [such as ORP (oxidation-reduction potential), TC (total carbon), TOC (total oxygen carbon), and NO3--N] and sediment [such as TC, TOC, TN (total nitrogen), and TP (total phosphate)] were significantly different in the CRCE and CWCE. The abundances of Proteobacteria, Actinobacteria, Verrucomicrobia, Cyanobacteria, Chlorobi, Chloroflexi, and Firmicutes were significantly different in the water bacterial communities of the CRCE and CWCE. The abundance of Vibrio in the crayfish intestine was higher in the CRCE than in the CWCE. The most abundant phyla in the CRCE and CWCE sediment were Proteobacteria and Bacteroidetes. The abundances of genes involved in transporters and ABC transporters were different in water of CRCE and CWCE. The abundances of genes involved in oxidative phosphorylation were significantly higher in the crayfish intestine of the CRCE than in that of the CWCE. Furthermore, the functional genes associated with carbon metabolism were significantly more abundant in the sediment of the CRCE than in that of the CWCE. Spearman correlation analysis and redundancy analysis (RDA) showed that the bacterial communities of the water and sediment in the CRCE and CWCE were correlated with environmental factors (pH, total carbon (TC), total oxygen carbon (TOC), total nitrogen (TN), and total phosphorus (TP)). Our findings showed that the composition, diversity and function of the bacterial communities were distinct in the environmental water, sediment, and intestine of the CRCE and CWCE crayfish coculture ecosystems due to their different ecological patterns. These results can help guide healthy farming practices and deepen the understanding of bacterial communities in crayfish-plant coculture ecosystems from the perspective of bacterial ecology.Key points• The composition of bacterial communities in the environmental water, sediment, and intestine of the CRCE and CWCE were distinct.̉• The abundances of genes involved in transporters and ABC transporters were different in the water of the CRCE and CWCE.• The bacterial communities of the water and sediment in the CRCE and CWCE were correlated with some environmental factors.

We explore the prospect of applying mineral element and stable isotope data in origin tracing Procambarus clarkii to establish an origin tracing system. Microwave digestion–atomic absorption spectrometry and stable isotope ratio mass spectrometry determined the contents of 14 mineral elements (Na, Mg, Al, K, Ca, Mn, Zn, Cu, Fe, Sr, Ba, As, Se and Cd) and the abundances of C and N stable isotopes in the muscle tissue of P. clarkii from Guangdong, Hunan and Hubei regions. The one-way ANOVA and Duncan multiple comparison results revealed Na, Sr, Ba, Cu, Mn, Fe, Al, Se, δ[sup.13] C and δ[sup.15] N varied significantly between the three regions (p < 0.05). A systematic clustering analysis revealed the stable isotopes combined with the mineral elements easily distinguished samples into the three different regions. Multivariate statistical analysis allowed us to establish a discriminant model for distinguishing P. clarkii from the three geographical regions. When stable isotopes were combined with mineral elements, the accuracy of the linear discriminant analysis of the samples from Guangdong, Hunan and Hubei were 95%, 95% and 100%, respectively. The initial overall discriminant accuracy was 96.7%, and the cross-validation discriminant accuracy was 93.3%. Principal component analysis identified three main components which were based on eleven major factors, including Cu, Ba, Cd, Mn, δ[sup.13] C, δ[sup.15] N, Al and Mg, resulting in a cumulative variance contribution rate of 78.77%. We established a three-dimensional coordinate system using the three principal components to create scatter diagrams with the samples from the three regions in the coordinate system. The results revealed the samples clearly differentiated into the three regions. Therefore, mineral elements combined with stable isotopes can distinguish the regional origin of P. clarkii.

Analysis of Croatian freshwater crayfish populations, aiming to gather new distributional data and complement previous surveys (2005–2011), was performed during 2014–2016, within the frame of Natura 2000 Integration Project. The research included different waterbodies across the whole of Croatia, harbouring both indigenous and non-indigenous crayfish species (ICS and NICS, respectively). Field work was conducted in 117 grid squares with dimensions 10 × 10 km, and up to four waterbodies were surveyed per grid square, making a total of 450 studied sites. Out of those, crayfish were not recorded in 368 sites. In the sites with crayfish presence ( i.e. , 82), the most frequently observed ICS was Astacus astacus (registered in 33 sites) followed by Austropotamobius pallipes (16 sites), Austropotamobius torrentium (12 sites), and Astacus leptodactylu s (5 sites). Concerning NICS, the majority of records were for Orconectes limosus (13 sites), followed by Pacifastacus leniusculus (2 sites), whereas Procambarus fallax f. virginalis was registered in only one locality. Comparisons of obtained data with those from previous surveys showed that NICS are progressively spreading and displacing ICS, as recorded for A. leptodactylus that was almost completely displaced by O. limosus in waterbodies of the east Croatia. Existing ICS populations are under growing anthropogenic preassure, frequently fragmented and isolated. Moreover, this survey showed that in the last decade 55% of A. astacus and 67% of A. pallipes populations disappeared mainly as a consequence of anthropogenic influence onto their habitats. Further monitoring and conservation actions for ICS should be urgently applied to mitigate negative impacts of both NICS and anthropogenic influence. L'analyse des populations croates d'écrevisses d'eau douce, visant à recueillir de nouvelles données de répartition et à compléter les relevés précédents (2005–2011), a été réalisée au cours de la période 2014–2016, dans le cadre du projet d'intégration Natura 2000. La recherche a porté sur différents cours d'eau dans toute la Croatie, abritant des écrevisses indigènes et non indigènes (ICS et NICS, respectivement). Le travail sur le terrain a été effectué dans un quadrillage de 117 quadrats de dimensions 10 × 10 km, et jusqu' à quatre plans d'eau ont été étudiés par quadrat, ce qui fait un total de 450 sites étudiés. Parmi ceux-ci, les écrevisses n'ont pas été recensées dans 368 sites. Dans les sites où il y avait des écrevisses (c. -à-d. 82), l’ICS le plus fréquemment observé était Astacus astacus (enregistré dans 33 sites), suivi de Austropotamobius pallipes (16 sites), Austropotamobius torrentium (12 sites) et Astacus leptodactylus (5 sites). En ce qui concerne le NICS, la majorité des signalements concernaient Orconectes limosus (13 sites), suivi de Pacifastacus leniusculus (2 sites), tandis que Procambarus fallax f. virginalis n' a été enregistrée que dans une seule localité. Les comparaisons des données obtenues avec celles des enquêtes précédentes ont montré que les NICS se répandent et déplacent progressivement les ICS, comme cela a été enregistré pour A. leptodactylus qui a été presque complètement déplacé par O. limosus dans les masses d'eau de l'est de la Croatie. Les populations actuelles d’ICS sont de plus en plus souvent exposées à une pression anthropique croissante, fragmentées et isolées. En outre, cette enquête a montré qu'au cours de la dernière décennie, 55% des populations d' A. astacus et 67% des populations d' A. pallipes ont disparu principalement en raison de l'influence anthropique sur leurs habitats. D'autres mesures de surveillance et de conservation des ICS devraient être appliquées d'urgence pour atténuer les impacts négatifs des NICS et de l'influence anthropique.

We report results of a study that made reciprocal comparisons of environmental DNA (eDNA) assays for two major invasive crayfishes between their disparate invasive ranges in North America. Specifically, we tested for range expansions of the signal crayfish Pacifastacus leniusculus (Dana, 1852) into the Laurentian Great Lakes region known to be invaded by the rusty crayfish Orconectes rusticus (Girard, 1852), as well as for the invasion of O. rusticus into large lakes of California and Nevada, US known to be invaded by P. leniusculus. We compared eDNA detections to historic localities for O. rusticus within the Great Lakes, and to recent sampling for presence/absence and relative abundance of P. leniusculus in California and Nevada via overnight sets of baited traps. We successfully detected O. rusticus eDNA at six sites from the Great Lakes and P. leniusculus from six of seven lakes where it was known to occur in California and Nevada, but did not detect any range expansions by either species across the North American continent. eDNA appears suitable to detect benthic arthropods from exceptionally large lakes, and will likely be useful in applications for monitoring of new biological invasions into these and other freshwater and marine habitats.

Background The marbled crayfish ( Procambarus virginalis ) is a monoclonal, parthenogenetically reproducing freshwater crayfish species that has formed multiple stable populations worldwide. Madagascar hosts a particularly large and rapidly expanding colony of marbled crayfish in a unique environment characterized by a very high degree of ecological diversity. Results Here we provide a detailed characterization of five marbled crayfish populations in Madagascar and their habitats. Our data show that the animals can tolerate a wide range of ecological parameters, consistent with their invasive potential. While we detected marbled crayfish in sympatry with endemic crayfish species, we found no evidence for the transmission of the crayfish plague pathogen, a potentially devastating oomycete. Furthermore, our results also suggest that marbled crayfish are active predators of the freshwater snails that function as intermediate hosts for human schistosomiasis. Finally, we document fishing, farming and market sales of marbled crayfish in Madagascar. Conclusions Our results provide a paradigm for the complex network of factors that promotes the invasive spread of marbled crayfish. The commercial value of the animals is likely to result in further anthropogenic distribution.

Glyphosate is an herbicide that inhibits the growth of weed plants, while chlorpyrifos is an insecticide commonly applied to control the pests’ population. This study aimed to investigate the combined effects of chlorpyrifos and glyphosate on biochemical, immunological parameters, and oxidative stress biomarkers in freshwater crayfish Pontastacus leptodactylus for 21 days. The experimental design of this study was factorial (3 × 3), including 0.0, 0.4, and 0.8 mg L−1 glyphosate and 0.0, 2.5, and 5 µg L−1 chlorpyrifos. The exposure to chlorpyrifos, glyphosate alone and a mixture of them significantly decreased acetylcholinesterase, alkaline phosphatase, phenoloxidase activities, and total protein levels. The lactate dehydrogenase, glutamic-pyruvic-transaminase, and catalase activities, the contents of glucose, and malondialdehyde levels were increased in the crayfish. No significant changes were detected in glutamic-oxaloacetic-transaminase (SGOT) activity, triglyceride, and total antioxidant (TAO) levels in the crayfish treated with 0.4 mg L−1 glyphosate and the control group. Co-exposure of crayfish to chlorpyrifos and glyphosate increased SGOT activity and TAO levels. Although chlorpyrifos combined with glyphosate decreased the γ-Glutamyltransferase (GGT) activity, the GGT activity was significantly increased in the P. leptodactylus exposed during 21 days to 5 µg L−1 chlorpyrifos alone and 0.8 mg L−1 glyphosate alone. In comparison with the reference group, no significant changes were evidenced in the cholesterol levels in the P. leptodactylus treated with 2.5 µg L−1 chlorpyrifos, but its levels were significantly increased in the other treatment groups. In conclusion, the mix of glyphosate and chlorpyrifos exhibited synergic effects on the different toxicological biomarkers in the narrow-clawed crayfish. Co-exposure to pesticides may result in disruption of homeostasis in the crayfish by altering the biochemical and immunological parameters.

As crayfish invasions continue to threaten native freshwater biota, a detailed understanding of crayfish distribution and population structure becomes imperative. Nonetheless, most current survey methods provide inadequate demographic data. The quantitative ‘Triple Drawdown’ (TDD) dewatering method has highlighted the importance of such data, yet practical constraints prevent its large‐scale application. Here, we introduce the ‘Pritchard Trap’, a novel passive sampling method that reliably generates quantitative crayfish population data while requiring substantially lower sampling effort than TDDs. This quadrat‐style sampler was extensively tested in headwater streams of North Yorkshire, England, along an invasion gradient for signal crayfish (Pacifastacus leniusculus) from well‐established sites to mixed populations of signal crayfish and native white‐clawed crayfish (Austropotamobius pallipes). The Pritchard Trap was trialled over several time intervals to determine the minimum required trap deployment time. TDDs at the same sites allowed for a robust evaluation of Pritchard Trap sampling accuracy in representing crayfish densities and population structure. The Pritchard Trap successfully sampled both invasive and native crayfish (8–42 mm carapace length). A minimum passive deployment time of 4 days was required. At low crayfish densities (0.5 individuals m−2), increased trapping effort was necessary to achieve accurate population density and size class distribution estimates. The Pritchard Trap required substantially less sampling effort (working hours) and resources than the TDD, whilst also posing less risk to non‐target species. The Pritchard Trap, for the first time, affords logistically simple, truly quantitative investigations of crayfish population demographics for headwater systems. It could be integrated into crayfish research and management, for example to explore density‐dependent ecological impacts of invasive crayfish and their management responses or to monitor populations and recruitment in native crayfish conservation initiatives. The ‘Pritchard Trap’ (PT) is a novel passive survey method for crayfish, that was tested in rocky headwaters in Northern England. PTs are filled with naturally occurring substrate and deployed for a minimum of four days. The PTs reliably generated quantitative crayfish population data, including density and size structure, along an invasion gradient from well‐established invasive signal crayfish populations to mixed populations of signal crayfish and native white‐clawed crayfish.

This study explores the geospatial relationship between the invasive crayfish species Faxonius limosus and the native Austropotamobius bihariensis and A. torrentium crayfish populations in Eastern Europe, identifying the environmental factors which influence the invasion. We used species distribution modelling based on several climatic, geophysical and water quality variables and crayfish distributional data to predict sectors suitable for each species within the river network. Thus, we identified the sectors potentially connecting invasive and native population clusters and quantified the degree of proximity between competing species. These sectors were then extensively surveyed with trapping and hand searching, doubled by eDNA methods, in order to assess whether any crayfish or the crayfish plague pathogen Aphanomyces astaci are present. The predictive models exhibited excellent performance and successfully distinguished between the analysed crayfish species. The expansion of F. limosus in streams was found to be limited by flash-flood potential, resulting in a range that is constrained to lowland rivers. Field surveys found neither crayfish nor pathogen presence in the connective sectors. Another interesting finding derived from the screening efforts, which are among the most extensive carried out across native, apparently healthy crayfish populations, was the existence of a latent infection with an A. astaci strain identified as A-haplogroup. Our results provide realistic insights for the long-term conservation of native Austropotamobius species, which appear to be naturally protected from F. limosus expansion. Conservation efforts can thus focus on other relevant aspects, such as ark-sites establishment for preventing the spread of more dangerous invasive crayfish species and of virulent crayfish plague pathogen strains, even in locations without direct contact between crayfish hosts.