Lenka Filipová

Prevalence of the Crayfish Plague Pathogen Aphanomyces astaci in Invasive American Crayfishes in the Czech Republic

In Central Europe invasive North American crayfishes are carriers of the oomycete Aphanomyces astaci, which causes crayfish plague. This lethal disease currently represents one of the major threats to native European crayfishes. We used molecular methods-species--specific amplification and sequencing of the pathogen DNA--to investigate the prevalence of individuals latently infected with A. astaci in 28 populations of two invasive American crayfish species (6 of the signal crayfish [Pacifastacus leniusculus] and 22 of the spiny-cheek crayfish [Orconectes limosus]) in the Czech Republic. The pathogen occurred in 17 investigated populations. We recorded a high variation in positive reactions, ranging from 0% to 100%, in populations of O. limosus. In P. leniusculus, however, only one individual out of 124 tested positive for the pathogen. There was a clear relationship between the water body type and pathogen prevalence in O. limosus. Infection ratios in isolated standing waters were usually low, whereas in running waters, pathogen prevalence often exceeded 50%. Other evaluated characteristics of potential plague pathogen carriers (size, sex, and the presence of melanized spots in the cuticle) seemed to be unrelated to infection. Our data suggest that in contrast to other European countries, O. limosus seems to be the primary reservoir of crayfish plague in the Czech Republic. Although all populations of alien American crayfishes may be potential sources of infections and should be managed as such, knowledge on the prevalence of the plague pathogen at various localities may allow managers to focus conservation efforts on the most directly endangered populations of native crayfishes.

Prevalence of the Crayfish Plague Pathogen Aphanomyces astaci in Populations of the Signal Crayfish Pacifastacus leniusculus in France: Evaluating the Threat to Native Crayfish

Aphanomyces astaci, the crayfish plague pathogen, first appeared in Europe in the mid-19th century and is still responsible for mass mortalities of native European crayfish. The spread of this parasite across the continent is especially facilitated by invasive North American crayfish species that serve as its reservoir. In France, multiple cases of native crayfish mortalities have been suggested to be connected with the presence of the signal crayfish Pacifastacus leniusculus, which is highly abundant in the country. It shares similar habitats as the native white-clawed crayfish Austropotamobius pallipes and, when infected, the signal crayfish might therefore easily transmit the pathogen to the native species. We investigated the prevalence of A. astaci in French signal crayfish populations to evaluate the danger they represent to local populations of native crayfish. Over 500 individuals of Pacifastacus leniusculus from 45 French populations were analysed, plus several additional individuals of other non-indigenous crayfish species Orconectes limosus, O. immunis and Procambarus clarkii. Altogether, 20% of analysed signal crayfish tested positive for Aphanomyces astaci, and the pathogen was detected in more than half of the studied populations. Local prevalence varied significantly, ranging from 0% up to 80%, but wide confidence intervals suggest that the number of populations infected by A. astaci may be even higher than our results show. Analysis of several individuals of other introduced species revealed infections among two of these, O. immunis and P. clarkii. Our results confirm that the widespread signal crayfish serves as a key reservoir of Aphanomyces astaci in France and therefore represents a serious danger to native crayfish species, especially the white-clawed crayfish. The prevalence in other non-indigenous crayfish should also be investigated as they likely contribute to pathogen transmission in the country.

Re-examination of the prevalence of Aphanomyces astaci in North American crayfish populations in Central Europe by TaqMan MGB real-time PCR

We applied quantitative TaqMan minor groove binder real-time polymerase chain reaction (PCR) on DNA isolates from soft abdominal cuticle of 460 North American crayfish Orconectes limosus and Pacifastacus leniusculus, previously tested for Aphanomyces astaci presence by conventional semi-nested PCR. Both approaches target the internal transcribed spacers of the pathogen nuclear ribosomal DNA, but apply different specific sequence motifs and technologies. The real-time PCR approach seems to provide higher sensitivity; the number of crayfish that tested positive increased from 23 to 32%, and 10 additional crayfish populations were indicated as hosting the disease agent. However, the vast majority of newly recorded positives contained very low agent levels, from 5 to 50 PCR-forming units. An isolate producing a false positive result by the semi-nested PCR (apparently undescribed Aphanomyces related to A. astaci) remained negative using the real-time PCR. The present study shows that previous results based on the semi-nested PCR were not substantially influenced by false positives but might have suffered from some false negatives at low agent levels. Combining alternative methods may therefore provide more reliable conclusions on the pathogens presence. Further, we found positive correlation between the prevalence of infection carriers in American crayfish populations and the average amounts of A. astaci DNA detected in infected local crayfish individuals.

Cryptic diversity within the invasive virile crayfish Orconectes virilis (Hagen, 1870) species complex: new lineages recorded in both native and introduced ranges

The virile crayfish (Orconectes virilis) represents a cryptic species complex with several lineages known in the USA, and a wide introduced range. In Europe, O. virilis is an emerging invader, established during the last decade in at least two areas—one in the United Kingdom and another in the Netherlands. We assessed the position of both known European populations within the species complex by sequencing part of the mitochondrial gene for cytochrome c oxidase subunit I. Tested UK and Dutch individuals did not belong to any mitochondrial lineage recorded in North America so far but formed a separate clade, the original distribution area of which is unknown. Additionally sequenced virile crayfish from Iowa (USA) also represented a new clade, suggesting that undiscovered lineage variation within O. virilis remains high. This exemplifies that genetic analyses of invading populations may provide new insights into diversity of a taxon in its original range.

Microsatellite markers for direct genotyping of the crayfish plague pathogen Aphanomyces astaci (Oomycetes) from infected host tissues.

Aphanomyces astaci is an invasive pathogenic oomycete responsible for the crayfish plague, a disease that has devastated European freshwater crayfish. So far, five genotype groups of this pathogen have been identified by applying random amplified polymorphic DNA analysis on axenic cultures. To allow genotyping of A. astaci in host tissue samples, we have developed co-dominant microsatellite markers for this pathogen, tested them on pure cultures of all genotype groups, and subsequently evaluated their use on tissues of (1) natural A. astaci carriers, i.e., North American crayfish species, and (2) A. astaci-infected indigenous European species from crayfish plague outbreaks. Out of over 200 potential loci containing simple sequence repeat (SSR) motifs identified by 454 pyrosequencing of SSR-enriched library, we tested 25 loci with highest number of repeats, and finally selected nine that allow unambiguous separation of all known RAPD-defined genotype groups of A. astaci from axenic cultures. Using these markers, we were able to characterize A. astaci strains from DNA isolates from infected crayfish tissues when crayfish had a moderate to high agent level according to quantitative PCR analyses. The results support the hypothesis that different North American crayfish hosts carry different genotype groups of the pathogen, and confirm that multiple genotype groups, including the one originally introduced to Europe in the 19th century, cause crayfish plague outbreaks in Central Europe. So far undocumented A. astaci genotype seems to have caused one of the analysed outbreaks from the Czech Republic. The newly developed culture-independent approach allowing direct genotyping of this pathogen in both axenic cultures and mixed genome samples opens new possibilities in studies of crayfish plague pathogen distribution, diversity and epidemiology.

Cross-species amplification of microsatellite markers in the invasive spiny-cheek crayfish (Orconectes limosus): assessment and application.

The North American spiny-cheek crayfish,Orconectes limosus (Cambaridae), endangered in its native range, is a widespread invasive species in European waters and conservationally important carrier of crayfish plague. However, its population structure is poorly known, and no informative genetic markers for the species are available. We tested cross-species transfer of microsatellite loci to spiny-cheek crayfish from 5 other crayfish species. Variability of 10 successfully amplifying loci derived from 4 species was then tested in 60 individuals ofO. limosus originating from 3 natural populations: the river Danube at Bogyiszló in Hungary, a pond in Starý Klíčov, and the brook Černovický, both in the Czech Republic. The allele number within the populations ranged from 4 to 10 alleles per locus, while heterozygosity levels varied from 0.650 to 0.900 forHo and from 0.660 to 0.890 forHe. No linkage disequilibrium and no null alleles were detected. The selected markers are useful for assessing population structure, intraspecific variation, and paternity studies in spiny-cheek crayfish.

First results on the genetic diversity of the invasive signal crayfish Pacifastacus leniusculus (Dana, 1852) in Europe using novel microsatellite loci

The introduction of non-native crayfish in aquatic ecosystems is very common due to human activities (e.g. aquaculture, recreational and commercial fisheries). The signal crayfish, Pacifastacus leniusculus (Dana, 1852), is one of the most widespread invasive species in Europe. Although several important ecological and economic impacts of this species have been reported, its European population genetic characterisation has never been undertaken using nuclear markers. Thus, the aim of this study was to develop and characterise new microsatellite markers for signal crayfish that can be useful in future studies in its invaded range, since only five are available so far. In total, 93 individuals from four geographically distinct European populations (Portugal, Great Britain, Finland and Sweden) were scored for the new markers and for those previously described, with the Bayesian analysis revealing a clear distinction among populations. These markers are suitable for future studies of the population genetic structure of this important invasive species, by increasing information about the possible pathways of introduction and dispersal, and by giving insights about the most important vectors of introduction.

Taxonomic re-examination of Cambaroides (Decapoda: Cambaridae) with a re-description of C. schrenckii from Sakhalin Island Russia and phylogenetic discussion of the Asian cambarids based on morphological characteristics

Within the freshwater crayfish genus Cambaroides, there has been taxonomic uncertainly as to the population from Sakhalin Island, Russia, represent Cambaroides schrenckii (Kessler, 1874) or whether it warrants full recognition as C. sachalinensis Birstein and Winogradov, 1934. Detailed morphological examination of specimens from Sakhalin, combined with molecular analysis of the mitochondrial 16S rRNA and COI genes, has shown that they can be regarded as C. schrenckii. We also provide detailed information on the geographic distribution, life history, and habitat of this little known species. Morphology of the epistome, merus of the third maxilliped, and the male second pleopod has phylogenetic importance for freshwater crayfishes in the Northern Hemispere (Astacidae and Cambaridae in Asia and North America). These morphological features are described and illustrated in detail for four species of Cambaroides (Asian Cambaridae), and compared with all representative genera in Astacidae and American Cambaridae. Members of Cambaroides share the same features, and the male second pleopod of Cambaroides has a unique character within Astacidea. This indicates the Asian Cambaridae are monophyletic, and separate from the American cambarid clade, which is consistent with previous molecular data.

Uniformity under in vitro conditions: Changes in the phenotype of cancer cell lines derived from different medulloblastoma subgroups.

Medulloblastoma comprises four main subgroups (WNT, SHH, Group 3 and Group 4) originally defined by transcriptional profiling. In primary medulloblastoma tissues, these groups are thought to be distinguishable using the immunohistochemical detection of β-catenin, filamin A, GAB1 and YAP1 protein markers. To investigate the utility of these markers for in vitro studies using medulloblastoma cell lines, immunoblotting and indirect immunofluorescence were employed for the detection of β-catenin, filamin A, GAB1 and YAP1 in both DAOY and D283 Med reference cell lines and the panel of six medulloblastoma cell lines derived in our laboratory from the primary tumor tissues of known molecular subgroups. Immunohistochemical detection of these markers was performed on formalin-fixed paraffin-embedded tissue of the matching primary tumors. The results revealed substantial divergences between the primary tumor tissues and matching cell lines in the immunoreactivity pattern of medulloblastoma-subgroup-specific protein markers. Regardless of the molecular subgroup of the primary tumor, all six patient-derived medulloblastoma cell lines exhibited a uniform phenotype: immunofluorescence showed the nuclear localization of YAP1, accompanied by strong cytoplasmic positivity for β-catenin and filamin A, as well as weak positivity for GAB1. The same immunoreactivity pattern was also found in both DAOY and D283 Med reference medulloblastoma cell lines. Therefore, we can conclude that various medulloblastoma cell lines tend to exhibit the same characteristics of protein marker expression under standard in vitro conditions. Such a finding emphasizes the importance of the analyses of primary tumors in clinically oriented medulloblastoma research and the urgent need to develop in vitro models of improved clinical relevance, such as 3D cultures and organotypic slice cultures.

High genetic variation of invasive signal crayfish in Europe reflects multiple introductions and secondary translocations

The signal crayfish Pacifastacus leniusculus, native to northwestern North America, has become the most widespread invasive crayfish species in Europe. It has been introduced repeatedly since 1959, and altogether >60,000 individuals were imported. Secondary introductions across the continent followed, resulting in its current presence in ≥26 European countries. Recent studies indicate that multiple highly divergent lineages of signal crayfish exist in its North American range, and previous investigators have suggested that >1 signal crayfish subspecies are present in Europe. We investigated its genetic variation by sequencing a fragment of the mitochondrial gene for cytochrome c oxidase subunit I (COI) to clarify the diversity of this invasive species in its European range. We analyzed 348 signal crayfish from 68 European populations and compared the resulting patterns with reference sequences from North America. All European individuals studied fell within 1 clade of P. leniusculus, but we observed substantial variation at the analyzed marker. Altogether, we recorded 27 COI haplotypes (4 very widespread) in Europe, but no clear distributional pattern of these haplotypes corresponds to numerous secondary introductions across Europe. Maximum pairwise divergence at COI among haplotypes detected in Europe was up to 4%. Such extent of genetic variation should be considered when developing and validating species-specific DNA-based probes for environmental detection of this invasive species because mitochondrial genes (and COI in particular) are often the markers of choice for this purpose.