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Transcription factors (TFs) have an important role in the regulation of the gene expression network. The role of TFs in the immune response of freshwater crayfish is poorly understood, but leveraging the regulatory mechanisms of immune response could augment the resistance against the invasive oomycete pathogen, Aphanomyces astaci. Previous studies indicated that the TFs CCAAT/enhancer-binding protein (C/EBP) and putative Krüppel homolog-1 protein (Kr-h1) might play a role in immune and stress response of the noble crayfish (Astacus astacus). Here, we aimed to further characterise these two gene products to gain a better understanding of their evolutionary origin, domain organisation and expression patterns across different crayfish tissues. Furthermore, we conducted an immune stimulation experiment to observe the potential changes in the gene expression of C/EBP and Kr-h1 under immune challenge in different crayfish tissues. Our results showed that both C/EBP and Kr-h1 are closely related to other C/EBPs and Kr-h1s in Malacostraca. Gene expression analysis revealed that both TFs are present in all analysed tissues, with higher expression of C/EBP in the gills and Kr-h1 in the abdominal muscle. Immune stimulation with laminarin (mimicking β-1-3-glucan in the oomycete cell wall) showed an activation of the crayfish immune system, with an overall increase in the total haemocyte count (THC) compared to untreated control and crayfish buffered saline (CBS) treatment. On the gene expression level, an up-regulation of the C/EBP gene was detected in the laminarin treated group in hepatopancreas and heart, while no changes were observed for the Kr-h1 gene. Our results indicate an early change in C/EBP expression in multiple tissues during immune stimulation and suggest its involvement in the immune response of the noble crayfish., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Ljudevit Luka Boštjančić reports financial support was provided by German Research Foundation. Odile Lecompte reports financial support was provided by French National Research Agency. Sandra Hudina reports financial support was provided by Croatian Ministry of Science and Education. Kathrin Theissinger reports financial support was provided by German Academic Exchange Service. Kathrin Theissinger reports financial support was provided by German Research Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.]., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Introduced into Europe from North America 150 years ago alongside its native crayfish hosts, the invasive pathogen Aphanomyces astaci is considered one of the main causes of European crayfish population decline. For the past two centuries, this oomycete pathogen has been extensively studied, with the more recent efforts focused on containing and monitoring its spread across the continent. However, after the recent introduction of new strains, the newly-discovered diversity of A. astaci in North America and several years of coevolution with its European host, a new assessment of the traits linked to the pathogen's virulence is much needed. To fill this gap, we investigated the presence of phenotypic patterns (i.e., in vitro growth and sporulation rates) possibly associated with the pathogen's virulence (i.e., induced mortality in crayfish) in a collection of 14 A. astaci strains isolated both in North America and in Europe. The results highlighted a high variability in virulence, growth rate and motile spore production among the different strains, while the total-sporulation rate was more similar across strains. Surprisingly, growth and sporulation rates were not significantly correlated with virulence. Furthermore, none of the analysed parameters, including virulence, was significantly different among the major A. astaci haplogroups. These results indicate that each strain is defined by a characteristic combination of pathogenic features, specifically assembled for the environment and host faced by each strain. Thus, canonical mitochondrial markers, often used to infer the pathogen's virulence, are not accurate tools to deduce the phenotype of A. astaci strains. As the diversity of A. astaci strains in Europe is bound to increase due to translocations of new carrier crayfish species from North America, there is an urgent need to deepen our understanding of A. astaci's virulence variability and its ability to adapt to new hosts and environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Biodiversity in freshwater ecosystems is declining due to an increased anthropogenic footprint. Freshwater crayfish are keystone species in freshwater ecosystems and play a crucial role in shaping the structure and function of their habitats. The Idle Crayfish Austropotamobius bihariensis is a native European species with a narrow distribution range, endemic to the Apuseni Mountains (Romania). Although its area is small, the populations are anthropogenically fragmented. In this context, the assessment of its conservation status is timely., Results: Using a reduced representation sequencing approach, we identified 4875 genomic SNPs from individuals belonging to 13 populations across the species distribution range. Subsequent population genomic analyses highlighted low heterozygosity levels, low number of private alleles and small effective population size. Our structuring analyses revealed that the genomic similarity of the populations is conserved within the river basins., Conclusion: Genomic SNPs represented excellent tools to gain insights into intraspecific genomic diversity and population structure of the Idle Crayfish. Our study highlighted that the analysed populations are at risk due to their limited genetic diversity, which makes them extremely vulnerable to environmental alterations. Thus, our results emphasize the need for conservation measures and can be used as a baseline to establish species management programs., (© 2024. The Author(s).)

One of the main drivers of biodiversity loss in freshwater ecosystems are alien invasive species. In Europe, pathogen Aphanomyces astaci Schikora, 1906, is considered as one of the most problematic invasive species, as its introduction caused the severe decimation of the European freshwater crayfish stocks. The most affected are the populations of noble crayfish, keystone species native to European freshwaters. Unfortunately, even after decades of research, we do not understand the temporal dynamics of the noble crayfish immune response during Ap. astaci infection. Here, we studied the changes in the immune response of the noble crayfish during a time course challenge with a highly virulent strain of Ap. astaci. We recorded gross symptoms of the disease, changes in the total haemocyte count (THC), gene expression profiles of putative immune response regulators and pathogen load. Additionally, we conducted a preliminary histological analysis of the pathogen dissemination in host tissues. Based on the occurrence of symptoms we propose three stages in the crayfish plague disease progression: asymptomatic stage, symptomatic stage, and finally death of infected individual. Furthermore, based on the qPCR analysis we could differentiate three Ap. astaci growth phases: initial lag phase, followed by exponential growth phase and finally sporulation phase. We observed that all measured immune response parameters were significantly correlated to the observed increase in the pathogen load (qPCR). Altogether, our results point to the absence of a successful immune response in the noble crayfish to a challenge with a highly virulent strain of Ap. astaci. The noble crayfish immune system was not able to suppress the growth of the intruding pathogen. In general, our observations have to be considered in the context of the specific combination of crayfish plague pathogen virulence and disease resistance of the challenged crayfish population, which defines the temporal dynamics of their interaction., Competing Interests: Declaration of competing interest Authors express no conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Repetitive elements are a major component of DNA sequences due to their ability to propagate through the genome. Characterization of Metazoan repetitive profiles is improving; however, current pipelines fail to identify a significant proportion of divergent repeats in non-model organisms. The Decapoda order, for which repeat content analyses are largely lacking, is characterized by extremely variable genome sizes that suggest an important presence of repetitive elements. Here, we developed a new standardized pipeline to annotate repetitive elements in non-model organisms, which we applied to twenty Decapoda and six other Crustacea genomes. Using this new tool, we identified 10% more repetitive elements than standard pipelines. Repetitive elements were more abundant in Decapoda species than in other Crustacea, with a very large number of highly repeated satellite DNA families. Moreover, we demonstrated a high correlation between assembly size and transposable elements and different repeat dynamics between Dendrobranchiata and Reptantia. The patterns of repetitive elements largely reflect the phylogenetic relationships of Decapoda and the distinct evolutionary trajectories within Crustacea. In summary, our results highlight the impact of repetitive elements on genome evolution in Decapoda and the value of our novel annotation pipeline, which will provide a baseline for future comparative analyses.

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.)

Background: Austropotamobius torrentium is a freshwater crayfish species native to central and south-eastern Europe, with an intricate evolutionary history and the highest genetic diversity recorded in the northern-central Dinarides (NCD). Its populations are facing declines, both in number and size across its entire range. By extanding current knowledge on the genetic diversity of this species, we aim to assist conservation programmes. Multigene phylogenetic analyses were performed using different divergence time estimates based on mitochondrial and, for the first time, nuclear DNA markers on the largest data set analysed so far. In order to reassess taxonomic relationships within this species we applied several species delimitation methods and studied the meristic characters with the intention of finding features that would clearly separate stone crayfish belonging to different phylogroups., Results: Our results confirmed the existence of high genetic diversity within A. torrentium, maintained in divergent phylogroups which have their own evolutionary dynamics. A new phylogroup in the Kordun region belonging to NCD has also been discovered. Due to the incongruence between implemented species delimitation approaches and the lack of any morphological characters conserved within lineages, we are of the opinion that phylogroups recovered on mitochondrial and nuclear DNA are cryptic subspecies and distinct evolutionary significant units., Conclusions: Geographically and genetically isolated phylogroups represent the evolutionary legacy of A. torrentium and are highly relevant for conservation due to their evolutionary distinctiveness and restricted distribution.