Anne Schrimpf

Invasive crayfish and crayfish plague on the move: first detection of the plague agent Aphanomyces astaci in the Romanian Danube.

Native European crayfish, such as Astacus leptodactylus, are threatened, among other factors, by the crayfish plague agent Aphanomyces astaci, dispersed by invasive North American crayfish. Two of these invaders, Pacifastacus leniusculus and Orconectes limosus, have extended their distribution in the River Danube catchment; the latter was detected for the first time in Romania in 2008. We monitored, at monthly intervals for over 2 yr, occurrence of native A. leptodactylus and invasive O. limosus at 6 sites on the Romanian Danube and checked for the invasive species in 4 of its tributaries. Between January 2009 and March 2011, the relative abundances of O. limosus steadily increased with time, while the native A. leptodactylus dramatically decreased in abundance. O. limosus expanded downstream at a rate of ca. 15 km yr-1; in August 2011, it was already present in the upper 105 km of the Romanian Danube. An agent-specific real-time PCR analyses demonstrated the presence of A. astaci DNA in at least 32% of the analysed invasive (n = 71) and 41% of the native (n = 49) crayfish coexisting in the Danube. Furthermore, A. astaci was also detected in A. leptodactylus captured about 70 km downstream of the O. limosus invasion front (at the time of sampling). Assuming a steady rate of expansion, O. limosus may invade the sensitive Danube delta area in the mid-2060s, even without long-distance dispersal. The crayfish plague agent, however, may reach the delta substantially earlier, through dispersal downstream among populations of native crayfish.

The marbled crayfish as a paradigm for saltational speciation by autopolyploidy and parthenogenesis in animals.

ABSTRACT The parthenogenetic all-female marbled crayfish is a novel research model and potent invader of freshwater ecosystems. It is a triploid descendant of the sexually reproducing slough crayfish, Procambarus fallax, but its taxonomic status has remained unsettled. By cross-breeding experiments and parentage analysis we show here that marbled crayfish and P. fallax are reproductively separated. Both crayfish copulate readily, suggesting that the reproductive barrier is set at the cytogenetic rather than the behavioural level. Analysis of complete mitochondrial genomes of marbled crayfish from laboratory lineages and wild populations demonstrates genetic identity and indicates a single origin. Flow cytometric comparison of DNA contents of haemocytes and analysis of nuclear microsatellite loci confirm triploidy and suggest autopolyploidisation as its cause. Global DNA methylation is significantly reduced in marbled crayfish implying the involvement of molecular epigenetic mechanisms in its origination. Morphologically, both crayfish are very similar but growth and fecundity are considerably larger in marbled crayfish, making it a different animal with superior fitness. These data and the high probability of a divergent future evolution of the marbled crayfish and P. fallax clusters suggest that marbled crayfish should be considered as an independent asexual species. Our findings also establish the P. fallax–marbled crayfish pair as a novel paradigm for rare chromosomal speciation by autopolyploidy and parthenogenesis in animals and for saltational evolution in general. Summary: The triploid marbled crayfish is a rare animal example of speciation by autopolyploidisation and parthenogenesis. It seems to be a particularly suitable model to study how much genetic and epigenetic change is necessary to create a new species.

De Novo assembly and annotation of the freshwater crayfish Astacus astacus transcriptome

We generated RNA-seq data to assemble the transcriptome of the noble crayfish (Astacus astacus) from four combined tissues (abdominal muscle, hepatopancreas, ovaries, green glands). A total of 194 million read pairs with a length of 100 bp were generated. The transcriptome was assembled de novo using Trinity software, producing 158,649 non-redundant transcripts. Lowly expressed transcripts were filtered out leaving 45,415 transcripts of which 14,559 were found to contain open reading frames with predicted gene function. The Transrate software revealed that 91% of the total reads were realigned to the assembly. Furthermore, BUSCO analysis indicated that our assembly is 64% complete. A total of 13,770 transcripts were assigned at least one GO term. This first de novo transcriptome assembly is an important foundation for future genomic research on the noble crayfish and adds to the general knowledge and further characterization of transcriptomes of non-model organisms.

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

Abstract. 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.

Cherax snowden, a new species of crayfish (Crustacea, Decapoda, Parastacidae) from the Kepala Burung (Vogelkop) Peninsula in Irian Jaya (West Papua), Indonesia.

Abstract A new species, Cherax snowden sp. n., from the Oinsok River Drainage, Sawiat District in the central part of the Kepala Burung (Vogelkop) Peninsula, West Papua, Indonesia, is described, figured and compared with the closest related species, Cherax holthuisi Lukhaup & Pekny, 2006. This species is collected and exported for ornamental purposes and its commercial name in the pet trade is “orange tip” or “green orange tip”. Both species may be easily distinguished morphologically or by using sequence divergence, which is substantial, for considering Cherax snowden sp. n. to be a new species.

Genetic characterization of Western European noble crayfish populations ( Astacus astacus ) for advanced conservation management strategies

One central goal of conservation biology is to conserve the genetic diversity of species in order to protect their adaptive potential. The main objective of this study was to identify management units (MUs) for the threatened noble crayfish (Astacus astacus) in Western Europe by utilizing sequence and microsatellite analysis to determine populations in need of focused conservation programs. With the analysis of noble crayfish from 31 sampling sites from Belgium, France, The Netherlands and Germany, and further comparison of this data with a European-wide dataset, we propose four distinct MUs: the French Meuse (MU 1), the French Rhine (MU 2), the Belgian Scheldt and Meuse (MU 3) as well as populations from the French Seine (MU 4). This knowledge enables advanced A. astacus conservation management practises in these catchments by distinguishing between outbreeding and inbreeding populations and by preserving the maximum genetic diversity. When required, a high genetic diversity can be conserved by strengthen existing populations via stocking with populations that either bear the most common haplotype or population-specific private haplotypes in order to maintain recent and regional adaptions. Above all, stocking with populations that exhibit haplotypes from outside Western Europe should be avoided in these catchments. This study supports the preservation of the genetic diversity of noble crayfish in Western Europe and provides thus a proposition for advanced conservation management.

In-depth investigation of the species problem and taxonomic status of marbled crayfish, the first asexual decapod crustacean

The marbled crayfish is the only obligately parthenogenetic decapod crustacean and a novel research model and invasive animal on three continents. It is regarded either as a parthenogenetic form of slough crayfish Procambarus fallax or as a separate species named Procambarus virginalis. In order to investigate the species question of this unusual crayfish in detail we have identified the similarities and differences in morphology, life history, genetics, behaviour, ecology and biogeography between marbled crayfish and its most likely parent species P. fallax. We have investigated specimens from natural habitats, laboratory colonies and museum collections and performed a meta-analysis of our data and published data. Our COI based molecular tree with 27 Cambaridae confirms closest relationship of marbled crayfish with P. fallax. Marbled crayfish and P. fallax are similar with respect to morphological characters, coloration and body proportions, but differ considerably with respect to body size, fertility and longevity. The mitochondrial genes of both crayfish are similar, but ploidy level and haploid genome size are markedly different. Both crayfish are eurytopic and have two major annual recruitment periods, but marbled crayfish show different population structure and higher invasiveness. Marbled crayfish occur in tropical to cold temperate habitats of the old world, but P. fallax is confined to subtropical and warm-temperate habitats of the southeastern USA. Cross-breeding experiments with both crayfish revealed reproductive isolation. The application of the Evolutionary Genetic Species Concept for asexuals to all available data supports raising marbled crayfish from “forma” to species rank. A determination key is provided to discriminate Procambarus virginalis, the first asexual decapod species, from its parent species P. fallax.

Hidden sites in the distribution of the crayfish plague pathogen Aphanomyces astaci in Eastern Europe: Relicts of genetic groups from older outbreaks?

The crayfish plague agent Aphanomyces astaci is one of the worlds most threatening invasive species. Originally from North America, the pathogen is being imported alongside American crayfish species, which are used for various purposes. In this study, we investigated the marginal, currently known distribution area of the pathogen in Eastern Europe by sampling narrow-clawed crayfish (Astacus leptodactylus) and spiny-cheek crayfish (Orconectes limosus) populations. In addition, using specific real-time PCR, we tested several marine decapod species, which also occur in brackish waters of the Danube at the West coast of the Black Sea and the Dniester River basin. By sequencing the nuclear chitinase gene, mitochondrial rnnS/rnnL DNA and by genotyping using microsatellite markers, we identified the A. astaci haplogroups of highly infected specimens. The A. astaci DNA was detected in 9% of the investigated A. leptodactylus samples, both in invaded and non-invaded sectors, and in 8% of the studied O. limosus samples. None of the marine decapods tested positive for A. astaci. The results revealed that narrow-clawed crayfish from the Dniester River carried the A. astaci B-haplogroup, while A. astaci from the Danube Delta belonged to the A- and B-haplogroups. In the invaded sector of the Danube, we also identified the A-haplogroup. Microsatellite analysis revealed a genotype identical to the genotype Up. It might be that some of the detected A. astaci haplogroups are relics from older outbreaks in the late 19th century, which may have persisted as a chronic infection for several decades in crayfish populations.

Quantitative real-time PCR as a promising tool for the detection and quantification of leaf-associated fungal species – A proof-of-concept using Alatospora pulchella

Traditional methods to identify aquatic hyphomycetes rely on the morphology of released conidia, which can lead to misidentifications or underestimates of species richness due to convergent morphological evolution and the presence of non-sporulating mycelia. Molecular methods allow fungal identification irrespective of the presence of conidia or their morphology. As a proof-of-concept, we established a quantitative real-time polymerase chain reaction (qPCR) assay to accurately quantify the amount of DNA as a proxy for the biomass of an aquatic hyphomycete species (Alatospora pulchella). Our study showed discrimination even among genetically closely-related species, with a high sensitivity and a reliable quantification down to 9.9 fg DNA (3 PCR forming units; LoD) and 155.0 fg DNA (47 PCR forming units; LoQ), respectively. The assay’s specificity was validated for environmental samples that harboured diverse microbial communities and likely contained PCR-inhibiting substances. This makes qPCR a promising tool to gain deeper insights into the ecological roles of aquatic hyphomycetes and other microorganisms.