Location-Dependent DNA Methylation Signatures in a Clonal Invasive Crayfish

DNA methylation is an important epigenetic modification that has been repeatedly implied in organismal adaptation. However, many previous studies that have linked DNA methylation patterns to environmental parameters have been limited by confounding factors, such as cell-type heterogeneity and genetic variation. In this study, we analyzed DNA methylation variation in marbled crayfish, a clonal and invasive freshwater crayfish that is characterized by a largely tissue-invariant methylome and negligible genetic variation. Using a capture-based subgenome bisulfite sequencing approach that covers a small, variably methylated portion of the marbled crayfish genome, we identified specific and highly localized DNA methylation signatures for specimens from geographically and ecologically distinct wild populations. These results were replicated both biologically and technically by re-sampling at different time points and by using independent methodology. Finally, we show specific methylation signatures for laboratory animals and for laboratory animals that were reared at a lower temperature. Our results thus demonstrate the existence of context-dependent DNA methylation signatures in a clonal animal.

Time-resolved, integrated analysis of clonal genome evolution in parthenogenetic animals and in cancer

Clonal genome evolution is a key aspect for parthenogenetic species and cancer. While many studies describe precise landscapes of clonal evolution in cancer, few studies determine the underlying evolutionary parameters from molecular data, and fewer integrate theory with data. We derived theoretical results linking mutation rate, time, expansion dynamics, transition to recurrence, and survival. With this, we inferred time-resolved estimates of evolutionary parameters from mutation accumulation, mutational signatures and selection. Using this framework we traced the speciation of the rapidly emerging and invasive marbled crayfish to a time window between 1947 and 1996, which is consistent with biological records. In glioblastoma samples, we determined tumor expansion patterns, and tumor cell survival ratio at resection. Interestingly, our results suggest that the expansion pattern in the primary tumor is predictive of the progress and time to recurrence. In addition, tumor cell survival was always higher after resection and was associated with the expansion pattern and time to recurrence. We further observed selection events in a subset of tumors, with longer and purifying-only selection phases in recurrent tumors. In conclusion, our framework allowed a time-resolved, integrated analysis of key parameters in clonally evolving genomes, and provided novel insights into the evolutionary age of marbled crayfish and the progression of glioblastoma.

Phylogeographic reconstruction of the marbled crayfish origin

The marbled crayfish (Procambarus virginalis) is a triploid and parthenogenetic freshwater crayfish species that has colonized diverse habitats around the world. Previous studies suggested that the clonal marbled crayfish population descended as recently as 25 years ago from a single specimen of P. fallax, the sexually reproducing parent species. However, the genetic, phylogeographic, and mechanistic origins of the species have remained enigmatic. We have now constructed a new genome assembly for P. virginalis to support a detailed phylogeographic analysis of the diploid parent species, Procambarus fallax. Our results strongly suggest that both parental haplotypes of P. virginalis were inherited from the Everglades subpopulation of P. fallax. Comprehensive whole-genome sequencing also detected triploid specimens in the same subpopulation, which either represent evolutionarily important intermediate genotypes or independent parthenogenetic lineages arising among the sexual parent population. Our findings thus clarify the geographic origin of the marbled crayfish and identify potential mechanisms of parthenogenetic speciation.

Controlled Infection Experiment With Aphanomyces astaci Provides Additional Evidence for Latent Infections and Resistance in Freshwater Crayfish

For 150 years the crayfish plague disease agent Aphanomyces astaci has been the cause of mass mortalities among native European crayfish populations. However, recently several studies have highlighted the great variability of A. astaci virulence and crayfish resistance toward the disease. The main aim of this study was to compare the response of two crayfish species, the European native noble crayfish (Astacus astacus) and the invasive alien marbled crayfish (Procambarus virginalis), to an A. astaci challenge with a highly virulent strain from haplogroup B and a lowly virulent strain from haplogroup A. In a controlled infection experiment we showed a high resistance of marbled crayfish against an A. astaci infection, with zoospores from the highly virulent haplogroup B strain being able to infect the crayfish, but unable to cause signs of disease. Furthermore, we demonstrated a reduced virulence in the A. astaci strain belonging to haplogroup A, as shown by the light symptoms and the lack of mortality in the generally susceptible noble crayfish. Interestingly, in both marbled crayfish and noble crayfish challenged with this strain, we observed a significant decrease of the detected amount of pathogen’s DNA during the experiment, suggesting that this A. astaci haplogroup A strain has a decreased ability of penetrating into the cuticle of the crayfish. Our results provide additional evidence of how drastically strains belonging to A. astaci haplogroup B and haplogroup A differ in their virulence. This study confirmed the adaptation of one specific A. astaci haplogroup A strain to their novel European hosts, supposedly due to reduced virulence. This feature might be the consequence of A. astaci’s reduced ability to penetrate into the crayfish. Finally, we experimentally showed that marbled crayfish are remarkably resistant against the crayfish plague disease and could potentially be latently infected, acting as carriers of highly virulent A. astaci strains.

High degree of non-genetic phenotypic variation in the vascular system of crayfish: a discussion of possible causes and implications

In this study, the hemolymph vascular system (HVS) in two cambarid crayfishes, i.e. the Marbled Crayfish, Procambarus virginalis Lyko, 2017 and the Spiny Cheek Crayfish, Faxonius limosus (Rafinesque, 1817), is investigated in regard of areas of non-genetic phenotypic variation. Despite their genetic identity, specimens of P. virginalis show variability in certain features of the HVS. Thus, we describe varying branching patterns, sporadic anastomoses, and different symmetry states in the vascular system of the marbled crayfish. We visualize our findings by application of classical and modern morphological methods, e.g. injection of casting resin, micro-computed tomography and scanning electron microscopy. By comparing our findings for P. virginalis to the vasculature in sexually reproducing crayfishes, i.e. F. limosus and Astacus astacus, we discuss phenotypic variation of the HVS in arthropods in general. We conclude that constant features of the HVS are hereditary, whereas varying states identified by study of the clonal P. virginalis must be caused by non-genetic factors and, that congruent variations in sexually reproducing F. limosus and A. astacus are likely also non-genetic phenotypic variations. Both common causal factors for non-genetic phenotypic variation, i.e., phenotypic plasticity and stochastic developmental variation are discussed along our findings regarding the vascular systems. Further aspects, such as the significance of non-genetic phenotypic variation for phylogenetic interpretations are discussed.

Evaluating Invasive Marbled Crayfish as a Potential Livestock for Sustainable Aquaculture

The marbled crayfish (Procambarus virginalis) is a recently discovered freshwater crayfish species, which reproduces by apomictic parthenogenesis, resulting in a monoclonal, and all-female population. The animals were widely distributed through the aquarium trade and have established numerous stable wild populations through anthropogenic releases. They are highly prevalent in Madagascar, where they have become a popular source of nutritional protein. As freshwater crayfish aquaculture in open systems is a thriving, but ecologically damaging global industry, alternatives are urgently needed. Although marbled crayfish are often branded by their invasive mode of reproduction, their overall invasiveness is not higher than for other cultured crayfish species. Furthermore, their resiliency and high adaptability provide a strong rationale for evaluating them for closed, and environmentally safe aquaculture approaches. Here we describe a novel population of marbled crayfish in a former German coal mining area that is characterized by acid and polluted water. Even under these adverse conditions, animals grew to sizes, and weights that are comparable to commercially farmed freshwater crayfish. Tailored feed development and laboratory testing demonstrated highly efficient feed conversion, suggesting a considerable capacity for sustainable production in closed systems. We further show that marbled crayfish meat can be readily introduced into European meals. Finally, chemical analysis of marbled crayfish exoskeletons revealed comparably high amounts of chitin, which is a valuable source for the synthesis of chitosan and bioplastics. Our results thus suggest that production of marbled crayfish in closed systems may represent a sustainable alternative for crayfish aquaculture.

First Record of the Alien Species Procambarus virginalis Lyko, 2017 in Fresh Waters of Sardinia and Insight into Its Genetic Variability

In the fresh waters of Sardinia (Italy), the non-indigenous crayfish species Procambarus clarkii has been reported from 2005, but, starting from 2019, there have been several reports of a new non-indigenous crayfish in southern and central areas of this Mediterranean island, and its morphology suggests that this species may be the marbled crayfish Procambarus virginalis. Forty-seven individuals of this putative species were analyzed, using the mitochondrial gene Cytochrome c Oxidase subunit I as molecular marker to identify this crayfish and investigate the level of genetic variability within the recently established population. Phylogenetic and phylogeographic analyses were carried out on a dataset including sequences from the Sardinian individuals and from all congenerics available in GenBank. Results showed that the new Sardinian crayfish belong to the species P. virginalis. All the sequences belonging to P. virginalis from European countries are identical, with only few exceptions found among Sardinian individuals. In conclusion, this paper highlights the occurrence of a new further alien species in the Sardinian fresh waters, which are already characterized by the high presence of non-indigenous species.

Comparative transcriptome analysis of noble crayfish and marbled crayfish immune response to Aphanomyces astaci challenges

Introduction of invasive North American crayfish species and their pathogen Aphanomyces astaci has significantly contributed to the decline of European freshwater crayfish populations. In this study, noble crayfish, a susceptible native European species, and marbled crayfish, an invasive disease-resistant species, were challenged with haplogroup A (low virulence) and haplogroup B (high virulence) strain of A. astaci. Hepatopancreatic tissue was isolated 3 and 21 days post-challenge. Our results revealed strong up-regulation in expression levels of the prophenoloxidase cascade immune-related genes in the haplogroup B challenged noble crayfish 3 days post-challenge. In the marbled crayfish, we observed an up-regulation of immune system relevant genes (DSCAM, AP, ALFs, CTLs and hemocyanin) 3 days post-challenge. This response highlights the marbled crayfish capability of building the immune tolerance. Furthermore, we successfully characterised several novel immune related gene groups in both crayfish species, contributing to our current understanding of crayfish immune related genes landscape.

Survival, Growth, and Reproduction: Comparison of Marbled Crayfish with Four Prominent Crayfish Invaders

Biological invasions are increasingly recognized ecological and economic threats to biodiversity and are projected to increase in the future. Introduced freshwater crayfish in particular are protruding invaders, exerting tremendous impacts on native biodiversity and ecosystem functioning, as exemplified by the North American spiny-cheek, signal and red swamp crayfish as well as the Australian common yabby. The marbled crayfish is among the most outstanding freshwater crayfish invaders due to its parthenogenetic reproduction combined with early maturation and high fecundity. As their introduced ranges expand, their sympatric populations become more frequent. The question of which species and under what circumstances will dominate in their introduced communities is of great interest to biodiversity conservation as it can offer valuable insights for understanding and prioritization of management efforts. In order to examine which of the aforementioned species may be more successful as an invader, we conducted a set of independent trials evaluating survival, growth, claw injury, and reproduction using single-species stocks (intraspecific interactions) and mixed stocks (interspecific interactions) of marbled crayfish vs. other crayfish invaders since the onset of exogenous feeding. In both single and mixed stocks, red swamp crayfish and yabby grew faster than marbled crayfish, while marbled crayfish were superior to both spiny-cheek and signal crayfish in terms of growth. With the exception of signal crayfish, the faster-growing species consistently reached a higher survival rate. The faster-growing species tended to negatively impair smaller counterparts by greater claw injury, delayed maturation, and reduced fecundity. Only marbled crayfish laid eggs as early as 14 weeks in this study, which is earlier than previously reported in the literature. Thus, the success of marbled crayfish among invasive crayfish is significantly driven by relatively fast growth as well as an early and frequent reproduction. These results shed light on how interactions between invasive populations can unfold when their expansion ranges overlap in the wild, thereby contributing to the knowledge base on the complex population dynamics between existing and emerging invasive species.