European Crayfish Species Research Articles

Species-specific pattern of crayfish distribution within a river network relates to habitat degradation: implications for conservation

The degradation of aquatic habitats has increasingly become one of the most important factors influencing the distribution of freshwater species worldwide. We analysed the occurrence of three crayfish species, Astacus astacus, Austropotamobius torrentium and Orconectes limosus, in relation to habitat degradation status (based on Directive 2000/60/ES), stream morphology, geographical characteristics and land cover. In total, we analysed 6,768 sites within the Czech Republic (Central Europe), of which 6,187 sites lacked crayfish; among the remainder, A. astacus was present in 507 sites, O. limosus occurred in 44 sites and A. torrentium was present in only 30 sites. The analysis revealed that A. astacus preferred streams of better water quality that were not surrounded by agricultural land or settlements. This species also preferentially occurred in smaller streams with stony bottom substrata that were located at higher altitudes. Austropotamobius torrentium occurrence was associated with the natural character of the water body and the presence of protected areas at higher altitudes. Conversely, the non-native crayfish species O. limosus was typically recorded at lower altitudes in downstream reaches surrounded by agricultural land and with deteriorated water quality. The degree of environmental (human) pressure besides differences in habitat characteristics at sites with ICS and NICS may be of general importance for conservation strategies aimed at central European native crayfish species, primarily because the habitat-driven co-occurrence pattern (and its possible changes in the future) may strongly influence interspecific relationships, such as direct competition and the spread of infectious diseases between crayfish species.

Invasive Chinese mitten crab (Eriocheir sinensis) transmits crayfish plague pathogen (Aphanomyces astaci)

The Chinese mitten crab ( Eriocheir sinensis) is a vector of the fatal crayfish plague pathogen (Aphanomyces astaci). Both species are listed among the worlds 100 worst invasive species. From its origin in China the Chinese mitten crab was introduced to Europe, presumably via ballast water from ships. A. astaci originated in North America and is assumed to have been firstly introduced to Europe through human- mediated transportation of live specimens of North American freshwater crayfish species. While the North American crayfish species are resistant to the pathogen, it causes a fatal disease to native European crayfish species. It was generally assumed that the crayfish plague could only infect freshwater crayfish species. Until recently, the Chinese mitten crab had not been recognised as a host of the pathogen. And no study has yet been carried out to investigate if they also serve as a vector. In this study, transmission experiments are carried out in combination with subsequent species-specific molecular analysis. The results establish that the Chinese mitten crab carry the crayfish plague pathogen and can transmit it to the European noble crayfish (Astacus astacus). The implications of these findings are ecologically alarming, considering the high invasive potential and the catadromous life cycle of Chinese mitten crabs, facilitating the widespread distribution of the pathogen.

A set of new and cross-amplifying microsatellite loci for conservation genetics of the endangered stone crayfish (Austropotamobius torrentium)

Like other indigenous crayfish species in Europe, the stone crayfish (Austropotamobius torrentium) is endangered due to habitat degradation, competition with invasive American crayfish and a deadly disease (crayfish plague) vectored by these invaders. To study the genetic diversity and connectivity of the remnant stone crayfish populations, eight new microsatellite loci were developed and supplemented by four cross-amplifying loci developed for related species. These twelve loci were tested for polymorphism in 130 stone crayfish from five streams in Switzerland. Variability was low with 1–7 alleles per locus, but the markers revealed strong genetic differentiation among streams. Use of these microsatellites will provide important information for the conservation and management of this endangered species.

Continental-wide distribution of crayfish species in Europe: update and maps

Recently published astacological studies substantially improved available data on distribution of crayfish in various European regions. At the same time, spread of invasive species has been recorded, additional non-indigenous species became established in various countries, and losses of populations of native species due to crayfish plague and other negative factors were observed. We overview recent advances in this knowledge, and provide updated colour maps of the distribution of all crayfish species present in Europe. These maps are originally based on the data from the Atlas of Crayfish in Europe published in 2006 as a result of the CRAYNET project, and were further updated from more recently published reports, grey literature, and especially thanks to contributions and feedback of over 70 specialists from 32 countries. Separate maps are available for all indigenous crayfish species in Europe as well as for three most widespread non-indigenous crayfish species. Additionally, two maps give locations of known findings of crayfish species introduced to Europe after 1980. These newly established alien species have so far restricted distributions; however, the frequency of recent reports suggests that findings of such species resulting from releases of aquarium pets will further increase.

Two distinct evolutionary lineages of the Astacus leptodactylus species-complex (Decapoda : Astacidae) inferred by phylogenetic analyses

Narrow-clawed crayfish (Astacus leptodactylus Eschscholtz, 1823 species-complex) is one of five European freshwater crayfish species. Even though widely distributed, it hasn’t been frequently studied and its taxonomy and systematics are unresolved. The results of a recent comparative morphometric character study revealed that morphometry of Asian and European populations differ significantly. In this research, for the first time, mitochondrial molecular markers (16S rRNA and COI) were used with the aim of elucidating the phylogenetic relationship between European and Asian populations of the narrow-clawed crayfish. Analyses included crayfish from Croatia, Bulgaria, Armenia, Russia, Poland and Turkey, and three different optimality criteria were applied. Phylogenetic relationships were reconstructed using the COI dataset, as well as the concatenated one (COI + 16S rRNA). For both datasets, congruent topologies were obtained and trees were characterised by the existence of two well supported phylogroups, one that included European populations, and the other Asian. Results indicate the presence of distinct evolutionary lineages within the A. leptodactylus species-complex, and corroborate previous results obtained using morphometric analyses.

Is the distribution pattern of the stone crayfish in the Carpathians related to karstic refugia from Pleistocene glaciations?

The stone crayfish, Austropotamobius torrentium, is one of the oldest freshwater crayfish species in Europe. Most Carpathian populations are in Romania, with a distribution clustered in 2 compact metapopulations in the western part of the country. Our goal was to understand if this pattern is the result of a coincidence or a complex set of restrictive circumstances. Romania is an ideal place to analyze crayfish distributions because they have not been disturbed by species translocations or massive loss of populations. We recorded the presence/absence of crayfish and measured 15 habitat variables at 428 randomly chosen headwater sampling sites, and analyzed the crayfish distribution pattern with a boosted regression tree model. Our results show that most of the Romanian territory is ecologically suited to support stone crayfish under current conditions. The most important influences on probability of presence were water velocity, conductivity, altitude, river size, and dissolved O2. When included as a supplementary predictor in the model, the distance from karst became the strongest variable accounting for the probability of presence and explaining the current distribution of the stone crayfish. We propose the hypothesis that at least one cycle of Pleistocene glaciation shaped the current distribution pattern by causing massive extinction in the Carpathians, except in karstic underground water bodies that offered ecological refuges. After the glaciations, stone crayfish expanding from these refuges competed with colonizing noble crayfish Astacus astacus, restricting stone crayfish to insular areas.

Dose-dependent mortality of the noble crayfish (Astacus astacus) to different strains of the crayfish plague (Aphanomyces astaci)

Several reports of the European crayfish species carrying a latent infection of the crayfish plague (Aphanomyces astaci) have emerged and the discussion has focused especially on the lowered virulence of As-genotypes behind decreased mortality. The aim of this study was to compare the killing rate of different A. astaci strains in controlled infection experiments. Two separate infection experiments with three A. astaci strains (UEFT2B (As), Evira6462/06 (As) and UEF8866-2 (PsI)) were made to compare the noble crayfish populations from the Lake Viitajärvi, Tervo, (Expt I) and the Lake Mikitänjärvi, Hyrynsalmi (Expt II). In the Expt III, the Lake Koivujärvi population noble crayfish were infected with A. astaci strains UEF8866-2 (PsI) and Evira6462/06 (As) using different dosages (1, 10, 100 and 1000sporesml−1) of A. astaci zoospores. The results confirmed that PsI-genotype strain is highly virulent and kills all the crayfish within a few days. The tested two As-genotype strains caused the mortalities more slowly, and part of the challenged crayfish survived until the end of the follow-up period. Our results also confirmed the variance of virulence among A. astaci strains within the As-genotype and demonstrated that the mortality is dependent on the number of zoospores used in the infections. It also appeared, that some noble crayfish populations show increased resistance towards the crayfish plague, especially against the As-genotype of A. astaci.

Crayfish plague agent detected in populations of the invasive North American crayfish Orconectes immunis (Hagen, 1870) in the Rhine River, Germany

Crayfish plague, caused by the parasitic oomycete Aphanomyces astaci, has driven indigenous European crayfish species to regional extinction in many parts of Europe and is among the leading threats to the remaining populations. A. astaci is known to be carried by longestablished invasive crayfish species of North American origin, which are also the main vectors of the plague pathogen. In this study, we examined whether a new invasive crayfish of North American origin, the calico crayfish (Orconectes immunis), also carries A. astaci. Orconectes immunis is a recent invader of the Upper Rhine plain, where it seems to displace its invasive predecessor Orconectes limosus, which is a known carrier of the agent of the crayfish plague. Using real-time PCR, we identified the calico crayfish as the fourth invasive crayfish species to be a carrier of the crayfish plague pathogen in Europe and we confirmed the infection with A. astaci in O. limosus. These findings support the concern that all North American crayfish species in European waters are carriers of the crayfish plague pathogen. Such knowledge should prove useful for conservation efforts, management, legislation, and public education about the spread of crayfish plague and non-indigenous crayfish species.

Temporal dynamics of spore release of the crayfish plague pathogen from its natural host, American spiny-cheek crayfish (Orconectes limosus), evaluated by transmission experiments

The crayfish plague pathogen, Aphanomyces astaci, is one of the most serious threats to indigenous European crayfish species. The North American invasive spiny-cheek crayfish, Orconectes limosus, is an important source of this pathogen in central and western Europe. We evaluated potential changes in A. astaci spore release rate from infected individuals of this species by experiments investigating the pathogen transmission to susceptible noble crayfish, Astacus astacus. We filtered defined volumes of water regularly to quantify spore concentration, and sampled crayfish tissues at the end of the experiment. The filters and tissues were then tested for the presence of A. astaci DNA by species-specific quantitative PCR. Additionally, we tested the efficiency of horizontal transmission to apparently uninfected O. limosus. The experiments confirmed that A. astaci can be transmitted to susceptible crayfish during intermoult periods, and that the pathogen was more frequently detected in noble crayfish recipients than in American ones. The pathogen spore concentrations substantially varied in time, and significantly increased during moulting of infected hosts. Our study strengthens the evidence that although the likelihood of crayfish plague transmission by water transfer from localities with infected American crayfish might increase when these are moulting or dying, no time-periods can be proclaimed safe.

Absence of the crayfish plague pathogen (Aphanomyces astaci) facilitates coexistence of European and American crayfish in central Europe

Summary1. Most European crayfish species are strongly threatened, mainly as a result of the introduced pathogen, Aphanomyces astaci, transmitted by invasive North American crayfish. Long‐term coexistence of American and European crayfish species is therefore regarded as almost impossible, even though some coexisting populations have been observed.2. In this study, crayfish were collected from presently coexisting populations of the introduced spiny‐cheek crayfish (Orconectes limosus) and the native noble crayfish (Astacus astacus) from nine standing waters in central Europe. Our aim was to resolve whether the coexistence resulted from reduced virulence in local strains of A. astaci, increased immunity in the native crayfish or an absence of the pathogen in these populations. We used highly sensitive A. astaci‐specific real‐time PCR to evaluate the crayfish latent carrier status, combined with transmission experiments to further validate the molecular results.3. From the total of 523 crayfish tested (490 spiny‐cheek crayfish, 33 noble crayfish), none positive for A. astaci was detected. Transmission experiments confirmed these results: No abnormal mortality or behavioural changes were seen in noble crayfish kept together with American crayfish from the coexisting populations. If we assume a very low prevalence of A. astaci of 10% in a carrier population, there is a 98% probability of disease being absent in five of the nine coexisting populations tested. Hence, a consistent absence, or an extremely low prevalence, of A. astaci seems to allow the coexistence of European and American crayfish in these central European populations.4. The results are important for native crayfish conservation and management and demonstrate that disease transmission risk may vary substantially between the different populations of spiny‐cheek crayfish in central Europe.

Crayfish plague pathogen detected in the Danube Delta – a potential threat to freshwater biodiversity in southeastern Europe

The crayfish plague, caused by the oomycete Aphanomyces astaci, is probably the most significant reason for declines in European freshwater crayfish species. One of its hosts, the North American spiny-cheek crayfish Orconectes limosus, extends its range in the river Danube and recently reached the territory of Romania. We used highly sensitive A. astaci-specific real-time PCR to test if the native narrowclawed crayfish Astacus leptodactylus in the highly protected Danube Delta about 970 km downstream of the current invasion front of American crayfish is a carrier of the crayfish plague. Thirteen out of 40 analysed native A. leptodactylus tested positive for the crayfish plague pathogen, infected individuals were found at both sampled localities within the Danube Delta. Therefore A. astaci has a much wider range in this river than assumed. The pathogen seems to persist in local populations, as neither crayfish mass mortalities nor alien crayfish species have been reported from the region. Aphanomyces astaci may have reached the Delta by long-range passive dispersal of infected hosts or pathogen spores, or by gradually infecting populations of native crayfish in upstream regions of the Danube in a stepping-stone manner. Alternatively, the crayfish plague may have persisted in the Danube Delta as chronic infection from an old plague wave in the 19th century. In any case, the presence of this pathogen in the lower Danube may become a threat to conservation of European crayfish and to freshwater biodiversity in many regions of southeastern Europe, at present considered “crayfish plague-free”.

Horizontal transmission of Thelohania contejeani in the endangered white-clawed (Austropotamobius pallipes) and the invasive signal crayfish (Pacifastacus leniusculus)

The microsporidian parasite Thelohania contejeani causes porcelain disease and has been implicated in mass mortalities in populations of the endangered European crayfish Austropotamobius pallipes. However, the route of parasite transmission is not known. This paper investigates the horizontal transmission of T. contejeani between A. pallipes hosts as well as its transmissibility to the invasive signal crayfish (Pacifastacus leniusculus). Field collected juvenile A. pallipes and P. leniusculus were assigned to 1 of 3 experimental treatments; fed heavily infected A. pallipes tissue, exposed to water from tanks housing heavily parasitized A. pallipes, and a control group to provide an estimate of the baseline infection levels in the field. After 26 weeks, abdominal muscle samples were screened by PCR for T. contejeani. Infection was significantly higher in the treatment groups (83% in the cannibalism treatment, 42% in the water exposure treatment) than in the control group (4%), providing evidence for horizontal transmission of the parasite between A. pallipes hosts. Cannibalism and scavenging are common amongst crayfish, providing transmission opportunities in the field. The study also provides the first direct evidence for transmission of the parasite from an indigenous European crayfish species to the invasive signal crayfish, with 50% of P. leniusculus in each treatment, and 8% of control animals infected. We discuss the possibility that high density populations of the invasive signal crayfish may serve either as reservoirs or sinks for the parasite.

Socio-economic drivers and non-indigenous freshwater crayfish species in Europe

Non-indigenous freshwater crayfish species (NICS) outnumber the indigenous ones in many European countries, representing a major threat to biodiversity via the spread of crayfish ‘plague’ and aggressive invasion andantagonismperformance.Althoughthebiologicalbasisofthissituation is well studied, the possible role of social, economic and demographic factors on this situation has been ignored. In an attempt to explore such relationships, we suggest that human population density and economic growth (measured as gross domestic product (GDP) per capita) are positivelyrelatedtotheincreasednumberofNICSintheEUarea.Moreover, this pattern is evident in countries with higher overall footprint (i.e. the human demand of biologically productive land and sea in global hectares required to provide resources and services on waste assimilation) compared to biocapacity. Within the above context, actions are urgently needed to restore/balance existing and projected metabolic rifts (i.e. rupturesinthenormalmetabolicprocessesinnaturalsystems)created by the presence of NICS.

Phoma glomerata, a potential new threat to Italian inland waters

Non-indigenous crayfish species, carrier of the oomycete Aphanomyces astaci, are the most important threats to European crayfish species. However, other ectoparasite species have been reported to be the pathogen of aquatic organisms. As little is known about other potential pathogens carried by the red swamp crayfish Procambarus clarkii, a total of 86 individuals of this species was collected in Lake Trasimeno from June 2007 to 2008. Phoma glomerata, never recorded in Italy, colonised more than 65% of the captured crayfish. No significant differences in prevalence were recorded between sexes, among life stages and between soft- and hard-shelled crayfish. This Coelomycetes is the causal agent of many diseases in plants, animals and humans. These results may contribute to a better understanding of the implications of epibiontic microfungi on the invasive crayfish P. clarkii, for both ecological and sanitary purposes.

Comparative analyses of Astacus leptodactylus morphological characteristics from Croatia and Armenia

Narrow-clawed crayfish, Astacus leptodactylus is a native European freshwater crayfish species, also distributed in Croatian freshwater systems belonging to the Black Sea drainage. Its taxonomical status is still in the process of change and discussion, and the data on morphological, molecular, ecological and zoogeographical characteristics of this species are scarce. Therefore, comparative analyses of morphological characteristics were applied with the aim to contribute to the knowledge on the morphometrical and meristical characteristics of A. leptodactylus. Recent research proved that measurements of a large number of morphometrical characteristics, in combination with multivariate statistical analysis, could provide a good instrument for identification and differentiation between populations. In this research altogether 143 animals were analysed (121 from two Croatian populations and 22 from Armenia). 22 morphometrical characteristics and 4 meristical characteristics, per crayfish, were measured. It was found that males and females differ between populations in measured meristical and morphometrical characteristics. None of recorded meristical characteristics proved itself to be reliable characteristic for distinguishing populations. From measured morphometrical characteristics the most discriminant characteristics for separating males from different populations were those describing carapace shape and for females those that describe shape of the claws.

Identification of two GH18 chitinase family genes and their use as targets for detection of the crayfish-plague oomycete Aphanomyces astaci

BackgroundThe oomycete Aphanomyces astaci is regarded as the causative agent of crayfish plague and represents an evident hazard for European crayfish species. Native crayfish populations infected with this pathogen suffer up to 100% mortality. The existence of multiple transmission paths necessitates the development of a reliable, robust and efficient test to detect the pathogen. Currently, A. astaci is diagnosed by a PCR-based assay that suffers from cross-reactivity to other species. We developed an alternative closed-tube assay for A. astaci, which achieves robustness through simultaneous amplification of multiple functionally constrained genes.ResultsTwo novel constitutively expressed members of the glycosyl hydrolase (GH18) gene family of chitinases were isolated from the A. astaci strain Gb04. The primary amino acid sequence of these chitinase genes, termed CHI2 and CHI3, is composed of an N-terminal signal peptide directing the post-translational transport of the protein into the extracellular space, the catalytic GH18 domain, a proline-, serine-, and threonine-rich domain and a C-terminal cysteine-rich putative chitin-binding site. The A. astaci mycelium grown in a pepton-glucose medium showed significant temporal changes in steady-state CHI2 and CHI3 mRNA amounts indicating functional constraint. Their different temporal occurrence with maxima at 48 and 24 hours of incubation for CHI2 and CHI3, respectively, is in accordance with the multifunctionality of GH18 family members. To identify A. astaci-specific primer target sites in these novel genes, we determined the partial sequence homologs in the related oomycetes A. frigidophilus, A. invadans, A. helicoides, A. laevis, A. repetans, Achlya racemosa, Leptolegnia caudata, and Saprolegnia parasitica, as well as in the relevant fungi Fusarium solani and Trichosporon cutaneum. An A. astaci-specific primer pair targeting the novel genes CHI2 and CHI3 as well as CHI1 - a third GH18 family member - was multiplexed with primers targeting the 5.8S rRNA used as an endogenous control. A species was typed unambiguously as A. astaci if two peaks were concomitantly detected by melting curve analysis (MCA). For sensitive detection of the pathogen, but also for quantification of agent levels in susceptible crayfish and carrier crayfish, a TaqMan-probe based real-time PCR (qPCR) assay was developed. It targets the same chitinase genes and allows quantification down to 25 target sequences.ConclusionThe simultaneous qualitative detection of multiple sequences by qPCR/MCA represents a promising approach to detect species with elevated levels of genetic variation and/or limited available sequence information. The homogenous closed-tube format, reduced detection time, higher specificity, and the considerably reduced chance of false negative detection achieved by targeting multiple genes (CHI1, CHI2, CHI3, and the endogenous control) at least two of which are subject to high functional constraint, are the major advantages of this multiplex assay compared to other diagnostic methods.Sensitive quantification achieved with TaqMan qPCR facilitates to monitor infection status and pathogen distribution in different tissues and can help prevent disease transmission.

Isolation and characterization of 11 novel microsatellite DNA markers in the noble crayfish, Astacus astacus

Description: Noble crayfish (Astacus astacus L.) is the most common and the most highly appreciated freshwater crayfish species in Europe. Once abundant, it is now listed as vulnerable by the IUCN Red List of Threatened Species, and there is increased interest in the aquaculture of this species for stocking and consumption purposes. Allozyme studies revealed little variation in A. astacus and stressed the need for the development of more informative microsatellite markers. We report here the isolation and characterization of 11 polymorphic microsatellite markers that, together with eight loci from our previous study, allow us to assess and monitor genetic variation in natural and farmed populations of A. astacus.

Invasive non-indigenous crayfish species in Europe: Recommendations on managing them

The expansion of non-indigenous crayfish in Europe and the damage caused by them on indigenous species and habitats is a cause for increasing concern. Yet there is a great deal of variation in the measures put in place to control introductions of crayfish in different countries in Europe and indeed within countries. Recommendations from a conference on the future of native crayfish in Europe were that there is a need for much tighter regulation on the sale of non-indigenous crayfish within Europe, including a need to prevent the introduction of more species via the aquarium trade. There is a clear need to identify the threats to remaining populations of indigenous crayfish regionally and nationally and to provide well-enforced biosecurity measures to help protect these populations. It was recognised that this can only be done with concerted efforts on education and information sharing, because without public cooperation, the on-going trend of decline in indigenous crayfish will continue.

Current techniques, approaches and knowledge in diagnosis of crayfish plague and other crayfish diseases

Crayfish diseases are one of the main causes of crayfish decline in Europe and specifically, crayfish plague (Edgerton et al., 2004). Knowledge of its causative agent, Aphanomyces astaci (Oomycetes), is still one of the crucial key aspects in crayfish management, and designing and implementing action plans for restoring the European indigenous crayfish species (ICS). In the Regional European Crayfish Workshop dealing with the Future of Native Crayfish in Europe held in Pisek, Czech Republic, a number of researchers joined a round table focused on crayfish diseases, and discussed the main problems and needs in crayfish disease studies. The data discussed only refer to the countries represented in this group discussion (Czech Republic, Estonia, Finland, France, Italy, Spain and Sweden) and the main topics are discussed next.

The old menace is back: Recent crayfish plague outbreaks in the Czech Republic

The crayfish plague pathogen, Aphanomyces astaci (Saprolegniales, Oomycetes), is lethal for native European crayfish species and eliminated a number of their populations during the second half of the 19th and throughout the 20th centuries. The disease is spread by non-native North American crayfish species, known to be carriers of the pathogen, which are currently thriving in many European countries. This represents one of the major threats to the recovery of populations of native crayfish (the noble crayfish Astacus astacus and the stone crayfish Austropotamobius torrentium) in the Czech Republic. While only two suspected plague-caused crayfish mass mortalities were reported in the second half of the 20th century, five mass mortalities involving both native species were found to be caused by A. astaci between 2004 and 2007, and several additional ones, though unconfirmed, are likely. We discuss possible reasons for this apparent increase in the number of crayfish plague outbreaks, and we report details of the four most recent mass mortalities in which the presence of A. astaci has been confirmed by molecular methods (PCR with species-specific primers and sequencing of the pathogen DNA) and microscopic analysis, and of additional suspicious cases of crayfish disappearance. As these plague outbreaks have been found only accidentally, it is likely that their actual frequency is higher. The Czech Republic is probably not exceptional in this respect, and plague monitoring within the whole Central Europe deserves more attention.