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Noble crayfish is the most widespread native freshwater crayfish species in Europe. It is threatened in its entire distribution range and listed on the International Union for Concervation Nature- and national red lists. Reliable monitoring data is a prerequisite for implementing conservation measures, and population trends are traditionally obtained from catch per unit effort (CPUE) data. Recently developed environmental DNA (eDNA) tools can potentially improve the effort. In the past decade, eDNA monitoring has emerged as a promising tool for species surveillance, and some studies have established that eDNA methods yield adequate presence-absence data for crayfish. There are also high expectations that eDNA concentrations in the water can predict biomass or relative density. However, eDNA studies for crayfish have not yet been able to establish a convincing relationship between eDNA concentrations and crayfish density. This study compared eDNA and CPUE data obtained the same day and with high sampling effort, and evaluated whether eDNA concentrations can predict relative density of crayfish. We also compared two analytical methods [Quantitative real-time PCR (qPCR) and digital droplet PCR (ddPCR)], and estimated the detection probability for eDNA monitoring compared to trapping using occupancy modeling. In all lakes investigated, we detected eDNA from noble crayfish, even in lakes with very low densities. The eDNA method is reliable for presence-absence monitoring of noble crayfish, and the probability of detecting noble crayfish from eDNA samples increased with increasing relative crayfish densities. However, the crayfish eDNA concentrations were consistently low and mostly below the limit of quantification, even in lakes with very high crayfish densities. The hypothesis that eDNA concentrations can predict relative crayfish density was consequently not supported. Our study underlines the importance of intensified sampling effort for successful detection of very low-density populations, and for substantiating presumed absence, inferred from negative results. Surprisingly, we found a higher likelihood of eDNA detection using qPCR compared to ddPCR. We conclude that eDNA monitoring cannot substitute CPUE data, but is a reliable supplement for rapid presence-absence overviews. Combined with eDNA analyses of alien crayfish species and diseases such as crayfish plague, this is a cost-efficient supplement offering a more holistic monitoring approach for aquatic environments and native crayfish conservation.

The thick–clawed crayfish Pontastacus pachypus (Rathke, 1837) is an endangered, and the least known, crayfish species in Europe. Currently, detailed information regarding the morphology, ecology and reproduction of thick–clawed crayfish is outdated. This study presents, for the first time, detailed photographs of the thick-clawed crayfish, and updated information on distinctive morphological characters and morphometric analysis. New specifications of the carapace and appendage morphological characteristics were established as: 1) the rostrum is long, sharply pointed and has three pairs of distinctive sub-apical lateral spines, 2) two well–developed pairs of post–orbital ridge on the carapace are ended by prominent spine; 3) each finger of chelae ends with a black sharp tip. Among the 18 morphometric indices, carapace width to the total length (CPW/TL), abdomen width to the total length (ABW/TL) and claw height to the claw width (CLH/CLW) clearly differentiate P. pachypus from the other representatives of Astacus genus ( A. colchicus and A. astacus ) and P. leptodactylus ( P< 0.05). Comparison of individual indices between P. pachypus and P. leptodactylus revealed that almost all indices differed significantly except head length to the total length (HEL/TL) and rostrum length to the total length (ROL/TL). This study contributes to the identification of the thick–clawed crayfish for the purpose of conservation and protection of its localities.

Branchiobdellidan samples obtained from host crustaceans from 60 localities in the Balkan region (Slovenia, Croatia, Montenegro, Bosnia and Herzegovina, North Macedonia, Serbia) were studied based on collections of the University of Ljubljana, Biotechnical Faculty, Department of Biology (Slovenia) and new sampling. The crayfish worms were assigned to seven species: Branchiobdella balcanica, B. pentadonta, B. italica, B. parasita, B. hexadonta, B. astaci and Xironogiton victoriensis. Species-specific microlocations of the different branchiobdellidan species and their cocoons on the crayfish body are described. The wider type locality of the endemic subspecies Branchiobdella balcanica sketi was re-surveyed, and fresh samples were obtained. Based on morphological analysis of these samples, we formally reject the subspecies status of B. balcanica sketi. Članek obravnava vzorce branhiobdelidov ali račjih pijavčic z gostiteljskih rakov deseteronožcev, nabrane na 60 lokalitetah na območju zahodnega Balkana (Slovenija, Hrvaška, Bosna in Hercegovina, Črna gora, Makedonija, Srbija in Bolgarija). Vzorci so bili pridobljeni iz zoološke zbirke Oddelka za biologijo Biotehniške fakultete Univerze v Ljubljani ter z nedavnim terenskim delom. Ugotovljenih je bilo sedem vrst branhiobdelidov: Branchiobdella balcanica, B. pentadonta, B. italica, B. parasita, B. hexadonta, B. astaci in tujerodna Xironogiton victoriensis. Opisana so vrstno specifična mesta pričvrščanja odraslih branhiobdelidov in njihovih kokonov na telesu gostiteljev. Podrobno je bilo pregledano širše območje tipske lokalitete endemičnega taksona Branchiobdella balcanica sketi. Morfološka analiza novih vzorcev je razkrila, da razlikovalni znaki niso zanesljivi, zaradi česar je predlagana formalna ukinitev statusa samostojne podvrste za B. balcanica sketi.

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.

Background For over a century, scientists have studied host-pathogen interactions between the crayfish plague disease agent Aphanomyces astaci and freshwater crayfish. It has been hypothesised that North American crayfish hosts are disease-resistant due to the long-lasting coevolution with the pathogen. Similarly, the increasing number of latent infections reported in the historically sensitive European crayfish hosts seems to indicate that similar coevolutionary processes are occurring between European crayfish and A. astaci . Our current understanding of these host-pathogen interactions is largely focused on the innate immunity processes in the crayfish haemolymph and cuticle, but the molecular basis of the observed disease-resistance and susceptibility remain unclear. To understand how coevolution is shaping the host’s molecular response to the pathogen, susceptible native European noble crayfish and invasive disease-resistant marbled crayfish were challenged with two A. astaci strains of different origin: a haplogroup A strain (introduced to Europe at least 50 years ago, low virulence) and a haplogroup B strain (signal crayfish in lake Tahoe, USA, high virulence). Here, we compare the gene expression profiles of the hepatopancreas, an integrated organ of crayfish immunity and metabolism. Results We characterised several novel innate immune-related gene groups in both crayfish species. Across all challenge groups, we detected 412 differentially expressed genes (DEGs) in the noble crayfish, and 257 DEGs in the marbled crayfish. In the noble crayfish, a clear immune response was detected to the haplogroup B strain, but not to the haplogroup A strain. In contrast, in the marbled crayfish we detected an immune response to the haplogroup A strain, but not to the haplogroup B strain. Conclusions We highlight the hepatopancreas as an important hub for the synthesis of immune molecules in the response to A. astaci . A clear distinction between the innate immune response in the marbled crayfish and the noble crayfish is the capability of the marbled crayfish to mobilise a higher variety of innate immune response effectors. With this study we outline that the type and strength of the host immune response to the pathogen is strongly influenced by the coevolutionary history of the crayfish with specific A. astaci strains .

For several hundred years freshwater crayfish (Crustacea-Decapoda-Astacidea) have played an important ecological, cultural and culinary role in Scandinavia. However, many native populations of noble crayfish Astacus astacus have faced major declines during the last century, largely resulting from human assisted expansion of non-indigenous signal crayfish Pacifastacus leniusculus that carry and transmit the crayfish plague pathogen. In Denmark, also the non-indigenous narrow-clawed crayfish Astacus leptodactylus has expanded due to anthropogenic activities. Knowledge about crayfish distribution and early detection of non-indigenous and invasive species are crucial elements in successful conservation of indigenous crayfish. The use of environmental DNA (eDNA) extracted from water samples is a promising new tool for early and non-invasive detection of species in aquatic environments. In the present study, we have developed and tested quantitative PCR (qPCR) assays for species-specific detection and quantification of the three above mentioned crayfish species on the basis of mitochondrial cytochrome oxidase 1 (mtDNA-CO1), including separate assays for two clades of A. leptodactylus. The limit of detection (LOD) was experimentally established as 5 copies/PCR with two different approaches, and the limit of quantification (LOQ) were determined to 5 and 10 copies/PCR, respectively, depending on chosen approach. The assays detected crayfish in natural freshwater ecosystems with known populations of all three species, and show promising potentials for future monitoring of A. astacus, P. leniusculus and A. leptodactylus. However, the assays need further validation with data 1) comparing traditional and eDNA based estimates of abundance, and 2) representing a broader geographical range for the involved crayfish species.

The noble crayfish, Astacus astacus, is an indigenous European freshwater species. Its populations show significant declines caused by anthropogenic pressure on its habitats, climate change and the spread of invasive species. Diminishing populations’ trends and loss of genetic diversity highlight the need for effective conservation that will ensure their long-term survival. We combined population genetics and species distribution modelling (SDM) to reveal the impact of climate change and invasive species on the noble crayfish, and to guide future conservation programs of current populations. Our study showed that Croatian populations of A. astacus harbour an important part of species genetic diversity and represent significant genetic reservoir at the European level. The SDM results predicted substantial reductions of suitable habitats for A. astacus by the 2070; only 13% of its current potential distribution is projected to remain stable under pessimistic Representative Concentration Pathway (RCP 8.5) emission scenario. Moreover, most of the populations with high genetic diversity are located in the areas predicted to become unsuitable, and consequently have a high probability of being lost in the future. Further, SDM results also indicated considerable decrease of future habitat suitability for invasive crayfish species in Croatia, suggesting that climate change poses a major threat to already endangered A. astacus . The obtained results help in the identification of populations and areas with the highest conservation value which should be given the highest priority for protection. In order to preserve present diversity in areas that are predicted as suitable, we propose assisted migration and repopulation approaches, for enhancing populations’ size and saving maximum genetic variability. The result of our research emphasizes once again the benefits of multidisciplinary approach in the modern biodiversity conservation.

The oomycete Aphanomyces astaci, the causative agent of crayfish plague, is listed as one of the 100 worst invasive species in the world, destroying the native crayfish populations throughout Eurasia. The aim of this study was to examine the potential of selected mitochondrial (mt) genes to track the diversity of the crayfish plague pathogen A. astaci. Two sets of primers were developed to amplify the mtDNA of ribosomal rnnS and rnnL subunits. We confirmed two main lineages, with four different haplogroups and five haplotypes among 27 studied A. astaci strains. The haplogroups detected were (1) the A-haplogroup with the a-haplotype strains originating from Orconectes sp., Pacifastacus leniusculus and Astacus astacus; (2) the B-haplogroup with the b-haplotype strains originating from the P. leniusculus; (3) the D-haplogroup with the d1 and d2-haplotypes strains originating from Procambarus clarkii; and (4) the E-haplogroup with the e-haplotype strains originating from the Orconectes limosus. The described markers are stable and reliable and the results are easily repeatable in different laboratories. The present method has high applicability as it allows the detection and characterization of the A. astaci haplotype in acute disease outbreaks in the wild, directly from the infected crayfish tissue samples.

The European native, noble crayfish ( Astacus astacus) has suffered from a serious and long term population decline due to habitat destruction, water pollution and the impact of the invasive North American crayfish that are carriers of the crayfish plague ( Aphanomyces astaci ). The latter being the major factor currently confining noble crayfish to uninvaded (parts of) waterbodies. However, recently wild populations of apparently healthy noble crayfish carrying the crayfish plague have been found. As crayfish are known for their inter- and intraspecific agonistic behaviour which may be key for their competitive success, this raised the interesting question what would happen if the crayfish plague would not be a dominant factor anymore in the interaction between native and invasive species. Since the outcome of those encounters is still unclear, this study explores whether the noble crayfish can stand its ground towards invasive species in such agonistic interactions. Furthermore, the ability of the noble crayfish and invasive crayfish to acquire shelter through agonistic interaction is also assessed. Through pairwise staged interactions, agonistic behaviour and shelter competition between the native A . astacus and the invasive Faxonius limosus and Procambarus acutus were examined. The results showed that A . astacus triumphs over F . limosus and P . acutus in agonistic encounters and in competition for shelter. In turn, P . acutus dominates F . limosus in staged encounters and shelter. In possible future situations were crayfish plague does no longer eradicate noble crayfish populations, our results show that the native noble crayfish might still have a promising future when confronted with invasive species.

The narrow-clawed crayfish ( Pontastacus leptodactylus ) is an alien species in Finland with only a few populations reported from the southeastern region during the last century. We discovered a productive population in the lake Jängynjärvi, which is upstream from the previously reported wild narrow-clawed crayfish population in that region. Preliminary studies indicated that this population is not infected with Aphanomyces astaci . We collected narrow-clawed crayfish samples from the lake Jängynjärvi population for both infection challenge and genetic studies, in order to investigate possible A. astaci resistance among this Finnish population and to evaluate their phylogenetic position that would enable us to speculate different scenarios of distribution pathways or origin of the population. The infection studies indicated that the narrow-clawed crayfish in this population were more resistant against A. astaci infection (B haplogroup A. astaci ) compared to the noble crayfish ( Astacus astacus ) from the lake Rytky in North Savo, while all crayfish of both species in the B haplogroup A. astaci challenged groups died within 58 days post-infection. Results of the phylogenetic reconstruction indicate that the lake Jängynjärvi narrow-clawed crayfish are closely related to narrow-clawed crayfish from the lake Bolshoye near Krasnoye, located on the White Sea island of Solovestky and also populations from Tyumen region, both in Russia. This could confirm previous speculations about introduction of the narrow-clawed crayfish from Russia into Finland or could indicate previous hydrological connection. L'écrevisse à pattes grêles ( Pontastacus leptodactylus ) est une espèce exotique en Finlande, avec seulement quelques populations signalées dans la région sud-est au cours du siècle dernier. Nous avons découvert une population productive dans le lac Jängynjärvi, qui se trouve en amont de la population sauvage d'écrevisses à pinces étroites signalée précédemment dans cette région. Des études préliminaires ont indiqué que cette population n'est pas infectée par Aphanomyces astaci . Nous avons prélevé des échantillons d'écrevisses à pinces étroites dans la population du lac Jängynjärvi, à la fois pour des études sur le test de l'infection et des études génétiques, afin d'étudier la résistance possible à A. astaci dans cette population finlandaise et d'évaluer leur position phylogénétique, ce qui nous permettrait de spéculer sur différents scénarios de voies de diffusion ou d'origine de la population. Les études d'infection ont montré que les écrevisses à pinces étroites de cette population étaient plus résistantes à l'infection par A. astaci (haplogroupe B A. astaci ) que les écrevisses nobles ( Astacus astacus ) du lac Rytky dans le nord du Savo, tandis que toutes les écrevisses des deux espèces de l'haplogroupe B A. astaci ayant été infectées sont mortes dans les 58 jours suivant l'infection. Les résultats de la reconstitution phylogénétique indiquent que les écrevisses à pinces étroites du lac Jängynjärvi sont étroitement apparentées aux écrevisses à pinces étroites du lac Bolshoye près de Krasnoïe, situé sur l'île de Solovestky dans la mer Blanche, ainsi qu'aux populations de la région de Tioumen, toutes deux en Russie. Cela pourrait confirmer les hypothèses antérieures sur l'introduction en Finlande d'écrevisses à pinces étroites en provenance de Russie ou pourrait indiquer un lien hydrologique antérieur.