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Background: The noble crayfish ( Astacus astacus ) is a native European species in decline, with a contracting range and diminishing populations and abundance. Previous studies revealed this species significant genetic diversity in the south-eastern Europe, with populations from the western and the southern part of the Balkan Peninsula being the most divergent. However, sampling of populations from the western part of the Balkans was limited and insufficient for investigating genetic diversity and population divergence for the purpose of conservation planning and management. Thus, the major aim of this study was to fill in this knowledge gap by studying mitochondrial and microsatellite DNA diversity, using 413 noble crayfish from 18 populations from waterbodies in the western part of the Balkan Peninsula., Methods: Phylogenetic analysis of studied populations and their mitochondrial diversity were studied using COI and 16S sequences and population genetic structure was described using 15 microsatellite loci., Results: Phylogeographic analysis revealed new divergent mitochondrial haplotypes for the populations in the westernmost part of the Balkan Peninsula in the tributaries of the Sava and Drava rivers. Microsatellite data indicated that these populations harbour an important component of genetic diversity within A. astacus . The results suggest that the western part of the Balkans played an important role as microrefugia during the Pleistocene climate fluctuations, allowing the long term persistence of A. astacus populations in this region. These results will also be important to supporting conservation decision making and planning., Competing Interests: The authors declare there are no competing interests., (©2021 Gross et al.)

Pontastacus leptodactylus is a native European crayfish species found in both freshwater and brackish environments. It has commercial importance for fisheries and aquaculture industries. Up till now, most studies concerning P. leptodactylus have focused onto gaining knowledge about its phylogeny and population genetics. However, little is known about the chromosomal evolution and genome organization of this species. Therefore, we performed clustering analysis of a low coverage genomic dataset to identify and characterize repetitive DNA in the P. leptodactylus genome. In addition, the karyogram of P. leptodactylus (2 n = 180) is presented here for the first time consisting of 75 metacentric, 14 submetacentric, and a submetacentric/metacentric heteromorphic chromosome pair. We determined the genome size to be at ~18.7 gigabase pairs. Repetitive DNA represents about 54.85% of the genome. Satellite DNA repeats are the most abundant type of repetitive DNA, making up to ~28% of the total amount of repetitive elements, followed by the Ty3/ Gypsy retroelements (~15%). Our study established a surprisingly high diversity of satellite repeats in P. leptodactylus . The genome of P. leptodactylus is by far the most satellite-rich genome discovered to date with 258 satellite families described. Of the five mapped satellite DNA families on chromosomes, PlSAT3-411 co-localizes with the AT-rich DAPI positive probable (peri)centromeric heterochromatin on all chromosomes, while PlSAT14-79 co-localizes with the AT-rich DAPI positive (peri)centromeric heterochromatin on one chromosome and is also located subterminally and intercalary on some chromosomes. PlSAT1-21 is located intercalary in the vicinity of the (peri)centromeric heterochromatin on some chromosomes, while PlSAT6-70 and PlSAT7-134 are located intercalary on some P. leptodactylus chromosomes. The FISH results reveal amplification of interstitial telomeric repeats (ITRs) in P. leptodactylus . The prevalence of repetitive elements, especially the satellite DNA repeats, may have provided a driving force for the evolution of the P. leptodactylus genome., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Boštjančić, Bonassin, Anušić, Lovrenčić, Besendorfer, Maguire, Grandjean, Austin, Greve, Hamadou and Mlinarec.)

The pathogenic oomycete Aphanomyces astaci, transmitted mainly by invasive North American crayfish, causes the crayfish plague, a disease mostly lethal for native European crayfish. Due to its decimating effects on native crayfish populations in the last century, A. astaci has been listed among the 100 worst invasive species. Importantly, detecting the pathogen in endangered native crayfish populations before a disease outbreak would provide a starting point in the development of effective control measures. However, current A. astaci-detection protocols either rely on degradation-prone eDNA isolated from large volumes of water or, if focused on individual animals, include killing the crayfish. We developed a non-destructive method that detects A. astaci DNA in the microbial biofilm associated with the cuticle of individual crayfish, without the need for destructive sampling. Efficiency of the new method was confirmed by PCR and qPCR and the obtained results were congruent with the traditional destructive sampling method. Additionally, we demonstrated the applicability of the method for A. astaci monitoring in natural populations. We propose that the new method should be used in future monitoring of A. astaci presence in endangered European native crayfish individuals as an alternative to eDNA-based monitoring., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In this study, the evolutionary history of the white-clawed crayfish (WCC) was evaluated using large-scale datasets comprising >1350 specimens from the entire distribution range. Using species delimitation methods on mitochondrial DNA (mtDNA) sequences, we propose four primary species hypotheses for WCC. Sequences for several nuclear regions were screened but none showed significant variation within WCC. This result favours a single secondary species hypothesis and indicates the existence of a mito-nuclear discordance in WCC. Therefore, mtDNA groups were considered only as genetic units that carry information about ancient divergences within WCC and not as taxonomic units. The reconstruction of ancestral ranges and divergence time estimates were used to link the current genetic structure with paleogeographic processes. These results showed that the emergence of mtDNA groups in WCC could be related to the Messinian Salinity Crisis, the climate cooling during the Pliocene and Pleistocene, and (paleo)shifting of the Adriatic Sea coastline in the Padanovenezian Plain. The most recent common ancestor of the mtDNA groups most likely originated from Dalmatia (eastern Adriatic coast) as indicated by the reconstruction of ancestral ranges. This ecoregion, along with the Gulf of Venice Drainages, harbours a high genetic diversity and should be emphasised as an area of the highest conservation priority., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The Oomycete Aphanomyces astaci is an obligate crayfish parasite that co-evolved with American crayfish species, and they therefore generally live in a balanced relationship. On the contrary, European native crayfish are highly susceptible to A. astaci, and infestation with it causes development of the lethal disease termed crayfish plague. Until now, 5 A. astaci strains have been described from the freshwater crayfish present in Europe. In this study we aimed to investigate the occurrence of the pathogen A. astaci in Croatian native and non-native crayfish populations, as well as to genotype established strains using microsatellite markers and obtain information on the pathogen's epidemiology. Our results showed that the pathogen is widespread in both native and non-native crayfish populations. Agent level, when positive, in non-native crayfish was generally low; in native species it was higher. Genotyping from microsatellites proved the presence of the B (Ps) strain in non-native species (Pacifastacus leniusculus), while the A (As) strain was detected from viable native species (Astacus astacus and Austropotamobius torrentium) that are distributed in areas lacking non-native crayfish. The genotype from A. torrentium differed from a typical A (As) by 1 allele. Strain B (Ps) was identified in native Astacus leptodactylus from the population that co-occurs with P. leniuscuslus. Interestingly, in 1 A. leptodactylus population both A (As) and B (Ps) strains were present.

The aim of this research was to investigate influence of different environmental stressors, such as temperature increase, air exposure and food deprivation on DNA integrity of a bioindicator species, freshwater crayfish Astacus leptodactylus. DNA damage was measured in crayfish haemocytes using Comet assay and micronucleus test. Crayfish haemolymph was subsequentially sampled during their 7 days of exposure to increased temperatures (25 and 30 degrees C) and during 24 h of air exposure. Both groups were also monitored through the following 7 days of recovery period. Food deprived crayfish were monitored over a period of 2 weeks. Alterations of measured physiological and immunological haemolymph parameters (THC, lactate, glucose and protein concentration) indicated stress response in exposed crayfish. However, only the stress induced by increased temperature significantly increased DNA damage in freshwater crayfish while food deprivation or air exposure did not cause a significant genotoxic effect., (Copyright © 2010 Elsevier Inc. All rights reserved.)