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The crayfish plague caused by the pathogen Aphanomyces astaci has decimated the European and Asian populations of freshwater crayfish and represents an important threat to the other highly susceptible crayfish species in the world, such as the Australian, Madagascar, and South American species. The development and application of molecular methods addressed to the identification of A. astaci has increased exponentially during the last decades in contrast to a slow trend of the pathogen biology and host interaction. There is still a need for a better comprehension of the A. astaci-crayfish interactions, specifically the resistance and tolerance immune mechanism. These types of studies required a robust basic knowledge on the developmental biology of the pathogen in order to reproduce life stages and to perform infection experiments. A great piece of work in this area was carried out during the 1960 s to 80 s in University of Uppsala. Thus, the purpose of this work was to update previous protocols as well as to generate new guidelines to reproduce key developmental biology stages of A. astaci, to eventually identify crayfish populations with higher resistance and tolerance to this pathogen. This work also refers to other methodologies and guidelines for the diagnosis of crayfish plague, the pathogen isolation, and the in vitro production of zoospores.

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European crayfish species are a clear example of the drastic decline that freshwater species are experiencing. In particular, the native species of the Iberian Peninsula, the white clawed-crayfish (WCC) Austropotamobius pallipes, is listed as "endangered" by the IUCN and included in Annex II of the EU Habitat Directive and requires especially attention. Currently, implemented conservation management strategies require a better understanding of the genetic diversity and phylogeographic patterns, as well as of its evolutionary history. For this purpose, we have generated the largest datasets of two informative ribosomal mitochondrial DNA regions, i.e., cytochrome oxidase subunit I and 16S, from selected populations of the WCC covering its geographical distribution. These datasets allowed us to analyze in detail the (i) genetic diversity and structure of WCC populations, and (ii) divergence times for Iberian populations by testing three evolutionary scenarios with different mtDNA substitution rates (low, intermediate, and high rates). The results indicate high levels of haplotype diversity and a complex geographical structure for WCC in the Iberian Peninsula. The diversity found includes new unique haplotypes from the Iberian Peninsula and reveals that most of the WCC genetic variability is concentrated in the northern and central-eastern regions. Despite the fact that molecular dating analyses provided divergence times that were not statistically supported, the proposed scenarios were congruent with previous studies, which related the origin of these populations with paleogeographic events during the Pleistocene, which suggests an Iberian origin for these WCC. All results generated in this study, indicate that the alternative hypothesis of an introduced origin of the Iberian WCC is highly improbable. The result of this study, therefore, has allowed us to better understand of the genetic diversity, structure patterns, and evolutionary history of the WCC in the Iberian Peninsula, which is crucial for the management and conservation needs of this endangered species.

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Crayfish plague, caused by the oomycete pathogen Aphanomyces astaci, is one of the most devastating of the emerging infectious diseases. This disease is responsible for the decline of native European and Asian freshwater crayfish populations. Over the last few decades, some European crayfish populations were reported to display partial to total resistance to the disease. The immune response in these cases was similar to that exhibited by the natural carriers of the pathogen, North American freshwater crayfish, e.g., weak-to-strong melanization of colonizing hyphae. We tested the degree of resistance displayed by 29 native Iberian populations of Austropotamobius pallipes that were challenged by zoospores of the pathogen. We measured the following parameters: (i) mean survival time, (ii) cumulative mortality, and (iii) immune response, and found that the total cumulative mortality of all the challenged populations was 100%. The integration of the results from these parameters did not allow us to find differences in resistance towards A. astaci among the northern and central populations of the Iberian Peninsula. However, in the southern populations, we could identify four distinct population responses based on an evaluation of a GLM analysis. In the first case, the similar response could be explained by the effect of a pathogen strain with a lower-than-expected virulence, and/or an actual increase in resistance. In the Southern populations, these differences appear to be the consequence of either whole population or individual resistance. Individuals that survived for a longer period than the others showed a stronger immune response, i.e., presence of partially or fully melanized hyphae, which is similar to that of North American crayfish species. This might be the consequence of different mechanisms of resistance or/and tolerance towards A. astaci.

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The fungal pathogens Fusarium falciforme and Fusarium keratoplasticum are responsible for the sea turtle egg fusariosis (STEF) throughout main nesting areas of the world. In this study, we investigated whether eggs of the invasive alien red-eared slider turtle, Trachemys scripta, can carry these fungal pathogens. Using multilocus sequence typing of four nuclear DNA regions, we found that eggs of T. scripta naturally can carry these two Fusarium pathogenic species, as well as other Fusarium species belonging to the Fusarium solani species complex. Physiological studies on F. falciforme and F. keratoplasticum isolates revealed that their optimal growth temperature coincided with the pivotal temperature for T. scripta embryos, ca 29.5 ± 0.5 °C, providing an evidence of a potential advantageous biological property for host colonization and virulence. A host-pathogen interaction network analysis of species of the FSSC and their hosts confirmed that F. falciforme and F. keratoplasticum are generalist pathogens in a wide range of animal hosts of worldwide geographical distribution. Finally, we show that nesting areas of this invasive turtle T. scripta in the Mediterranean freshwater marshes can act as chronic reservoirs of these STEF pathogens, and this invasive species can act as a potential vector for the spread of STEF among wild native species and even to humans.

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The oomycete Aphanomyces astaci is an emerging infectious pathogen affecting freshwater crayfish worldwide and is responsible for one of the most severe wildlife pandemics ever reported. The pathogen has caused mass mortalities of freshwater crayfish species in Europe and Asia, and threatens other susceptible species in Madagascar, Oceania and South America. The pathogen naturally coexists with some North American crayfish species that are its chronic carriers. Presumptions that A. astaci originated in North America are based on disease outbreaks that followed translocations of North American crayfish and on the identification of the pathogen mainly in Europe. We studied A. astaci in the southeastern US, a center of freshwater crayfish diversity. In order to decipher the origin of the pathogen, we investigated (1) the distribution and haplotype diversity of A. astaci, and (2) whether there are crayfish species-specificities and/or geographical restrictions for A. astaci haplotypes. A total of 132 individuals, corresponding to 19 crayfish species and one shrimp species from 23 locations, tested positive for A. astaci. Mitochondrial rnnS and rnnL sequences indicated that A. astaci from the southeastern US exhibited the highest genetic diversity so far described for the pathogen (eight haplotypes, six of which we newly describe). Our findings that A. astaci is widely distributed and genetically diverse in the region supports the hypothesis that the pathogen originated in the southeastern US. In contrast to previous assumptions, however, the pathogen exhibited no clear species-specificity or geographical patterns.

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The causative agent of crayfish plague, Aphanomyces astaci (Saprolegniales, Oomycota), is one of the 100 world's worst invasive alien species and represents a major threat to freshwater crayfish species worldwide. A better understanding of the biology and epidemiology of A. astaci relies on the application of efficient tools to detect the pathogen and assess its genetic diversity. In this study, we validated the specificity of two recently developed PCR-based approaches used to detect A. astaci groups. The first relies on the analysis of mitochondrial ribosomal rnnS (small) and rnnL (large) subunit sequences and the second, of sequences obtained by using genotype-specific primers designed from A. astaci whole genome sequencing. For this purpose, we tested the specificity against 76 selected isolates, including other oomycete species and the recently described species Aphanomyces fennicus, which, when used in nrITS-based specific tests for A. astaci, is known to result in a false positive. Under both approaches, we were able to efficiently and accurately identify A. astaci and its genetic groups in both pure cultures and clinical samples. We report that sequence analysis of the rnnS region alone is sufficient for the identification of A. astaci and a partial characterization of haplogroups. In contrast, the rnnL region alone is not sufficiently informative for A. astaci identification as other oomycete species present sequences identical to those of A. astaci.

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