Aphanomyces spp. are water moulds, eukaryotic fungus-like organisms, belonging to the class Oomycota. This genus contains primary pathogens of plants and animals as well as opportunistic and saprotrophic species. One of the animal parasites (A. astaci) is the causal agent of the crayfish plague, a disease listed by the World Organisation for Animal Health (OIE). It is believed that A. astaci was first introduced into Italy from the US in the late 19th century and rapidly spread in Europe causing the decline of native crayfish. It currently threatens to wipe out the UK native white-clawed crayfish (Austropotamobius pallipes). Random amplified polymorphic DNA (RAPD-PCR) on pure isolates of A. astaci distinguished five genotypes (A, B, C, D, and E). This distinction proved to be a useful tool for epidemiological studies aimed at understanding the history and spread of the disease in Europe; furthermore, there are differences in virulence among genotypes. No discriminatory morphological or physiological characters are available and widely used markers such as the internal transcribed spacer (ITS), the divergent domains regions (D1-D2) of nuclear large subunit (LSU) rDNA, and cytochrome c oxidase subunit I (COI) also fail to discriminate between A. astaci genotypes. There are some practical drawbacks to genotype by the currently available genotyping methods. Whole genome sequencing (WGS) was used to catalogue DNA single nucleotide variants and genotype-unique genomic regions that could be exploited as phylogenetic markers. These newly developed molecular markers were tested both on pure cultures and historical samples derived from outbreaks and carrier crayfish available in our laboratories, validating these genotyping methods, which represent new diagnostic tools aiding the detection and prevention of crayfish plague.

Crayfish plague is an emerging disease caused by the oomycete Aphanomyces astaci, a pathogen listed among the 100 World's Worst Invasive Alien Species. It was introduced into Europe in the second half of 19th century from North America and caused collapses of European native crayfish populations. Nowadays, A. astaci is widespread in Europe and has spread also to other parts of the world, threatening all susceptible crayfish of non-North American origin. The aims of this MSc thesis were 1) to provide information about crayfish plague outbreaks from recent years, and by using microsatellite and mtDNA markers reveal A. astaci genotypes involved; 2) to test healthy-looking indigenous crayfish for potential occurrence of chronic infections by A. astaci in Czechia. Six new crayfish plague outbreaks were confirmed from 2016 to 2018, involving at least five distinct pathogen strains. My results provide first evidence of the A. astaci genotype group D causing Astacus astacus and Austropotamobius torrentium mass mortalities in Czechia. MtDNA sequencing revealed two haplotypes of the D haplogroup, indicating two independent sources of infection presumably either from ornamental crayfish or spreading from neighbouring countries. The genotype group A was recorded in two A. astacus mortalities and genotype group...

The crayfish plague pathogen, Aphanomyces astaci, is one of the most serious threats to European indigenous crayfish species, e.g., the noble crayfish (Astacus astacus). The only way to protect susceptible crayfish species from the disease is to prevent the dispersion of the pathogen to their populations. One of the most important sources of the crayfish plague pathogen in Central Europe is the spiny-cheek crayfish (Orconectes limosus), a species of North American origin, which can carry the parasite in its cuticle for years. Some literature sources claimed that the pathogen dispersion from the American vectors is restricted to periods of moulting or to the time before and after the crayfish death. However, experimental evidence for such hypotheses was lacking. The main aim of my thesis was to test these predictions, and the alternative scenario that the crayfish plague pathogen can be transmitted from the infected spiny-cheek crayfish also in other periods. For this purpose, experiments were set up to investigate A. astaci transmission from infected spiny-cheek crayfish to non-infected spiny-cheek or noble crayfish. As expected, the pathogen was transmitted to noble crayfish much more easily than to the uninfected American host. Nevertheless, we succeeded in the pathogen transmission also among spiny-cheek...

The crayfish plague pathogen, the oomycete Aphanomyces astaci, has been decimating populations of European crayfish species for more than 150 years, and is therefore considered one of the 100 worst world's invasive species. A. astaci is highly specialised for a parasitic life, but it can be isolated from moribund crayfish and grown on synthetic media, as it is the case also for several other oomycetes (chapter 7). The life of A. astaci includes three basic forms: mycelium in host's tissues, and the infective units occurring in water, zoospores and cysts. All North American crayfish species tested so far have shown some resistance to A. astaci, i.e., they could carry the infection for long, serving as vectors of the pathogen. Massive sporulation from infected North American crayfish starts when the host is moulting, stressed, or dying (chapter 4). However, I could show in my experiments that some sporulation occurs even from apparently healthy and non-moulting American crayfish hosting A. astaci, so infected North American crayfish must be considered a permanent source of the infection (chapter 4). Five genotype groups of A. astaci have already been distinguished. Strains from a particular genotype group probably share the same original host crayfish species of North American origin. Nevertheless, they can...

- ABSTRACT - The crayfish plague pathogen, Aphanomyces astaci, is responsible for substantial declines and local extinctions of native European crayfish populations. As a consequence, the pathogen is now listed among 100 world's worst invasive alien species. The spread of A. astaci is greatly facilitated by its natural hosts, North American crayfish, that thanks to a long co-evolutionary history with the crayfish plague pathogen evolved efficient defence mechanisms. In contrast, European, Australian and Asian crayfish species are highly susceptible to this disease agent. However, progress of A. astaci infection in native European crayfish was observed to differ between distinct pathogen strains, indicating variability in their virulence. Indeed, we demonstrated a relationship between patterns in crayfish immune response and A. astaci virulence in an experimental infection involving the European noble crayfish and three differently virulent crayfish plague strains. The European continent is currently inhabited by at least eight North American crayfish species. The carrier status was confirmed in six of them, including also Orconectes cf. virilis occurring in the Netherlands and the UK. In this country, we detected Aphanomyces astaci presence in some populations of the non-indigenous crayfish species as well...

<p>The crayfish plague refractory crayfish, <i>Pacifastacus leniusculus</i>, which can harbour the fungal parasite within melanotic sheath, are found to constitutively express the gene encoding for prophenoloxidase (proPO) after mimicking parasite attack. In contrast, the susceptible crayfish, <i>Astacus astacus</i>, responds to the parasite by increased levels of proPO transcript, particularly in the semigranular haemocytes. The upregulation of proPO could confer a temporary resistance towards the fungal infection, suggesting that additional factors are involved in maintaining the balance between host and parasite. The resistant crayfish may have adapted to the parasite by increasing the transcript level of immune genes. The parasite can be considered as a symbiont since it does not harm the host rather than it activates the immune gene and possibly preventing other pathogens to become established.</p><p>Two serine proteinase genes encoding a subtilisin-like (<i>AaSP1</i>) and a trypsin (<i>AaSP2</i>) enzyme were isolated from the crayfish plague fungus, <i>Aphanomyces astaci</i>. These proteinases are prepropeptides and generate mature proteins of 39 kDa and 29 kDa, respectively. Characterisation of <i>AaSP1</i> suggests that the enzyme may be involved in intracellular control mechanisms rather than playing a role in pathogenesis. The <i>AaSP2 </i>transcript was not controlled by catabolic repression, but was induced by crayfish plasma, implying a role in pathogenesis toward the crayfish host. </p><p>Physiology and genetics of five <i>Aphanomyces</i> strains, which were isolated from moribund crayfish, were characterised with regard to their pathogen diversity. These strains are not virulent against crayfish. Some physiological properties of these strains differed from <i>A. astaci</i>, such as growth rate, germination and production of chitinase. Genetic analysis clearly indicated that they are not related to <i>A. astaci</i> and their name are proposed to be <i>Aphanomyces repetans</i>.</p><p>The crayfish <i>P. leniusculus </i>was found to be susceptible to white spot syndrome virus infection. The virus has a significant effect to the population of crayfish haemocyte. The number and proportion of granular cell from virus-infected crayfish were higher than in controls, indicating granular cells are more resistant to and may interact by some means with the virus.</p><p>Two morphotypes of the crayfish parasite <i>Psorospermium haeckeli</i> obtained from different crayfish hosts of different geographical origin were analysed for ribosomal ITS DNA in order to compare their genetic diversity. The sequence difference between them was found largely in ITS 1 and ITS 2 regions, which was variable in length and showed 66% and 58% sequence similarity. Thus, different morphotypes of <i>P. haeckeli</i> are genetically diverse.</p>

The crayfish plague refractory crayfish, Pacifastacus leniusculus, which can harbour the fungal parasite within melanotic sheath, are found to constitutively express the gene encoding for prophenoloxidase (proPO) after mimicking parasite attack. In contrast, the susceptible crayfish, Astacus astacus, responds to the parasite by increased levels of proPO transcript, particularly in the semigranular haemocytes. The upregulation of proPO could confer a temporary resistance towards the fungal infection, suggesting that additional factors are involved in maintaining the balance between host and parasite. The resistant crayfish may have adapted to the parasite by increasing the transcript level of immune genes. The parasite can be considered as a symbiont since it does not harm the host rather than it activates the immune gene and possibly preventing other pathogens to become established. Two serine proteinase genes encoding a subtilisin-like (AaSP1) and a trypsin (AaSP2) enzyme were isolated from the crayfish plague fungus, Aphanomyces astaci. These proteinases are prepropeptides and generate mature proteins of 39 kDa and 29 kDa, respectively. Characterisation of AaSP1 suggests that the enzyme may be involved in intracellular control mechanisms rather than playing a role in pathogenesis. The AaSP2 transcript was not controlled by catabolic repression, but was induced by crayfish plasma, implying a role in pathogenesis toward the crayfish host. Physiology and genetics of five Aphanomyces strains, which were isolated from moribund crayfish, were characterised with regard to their pathogen diversity. These strains are not virulent against crayfish. Some physiological properties of these strains differed from A. astaci, such as growth rate, germination and production of chitinase. Genetic analysis clearly indicated that they are not related to A. astaci and their name are proposed to be Aphanomyces repetans. The crayfish P. leniusculus was found to be susceptible to white spot syndrome virus infection. The virus has a significant effect to the population of crayfish haemocyte. The number and proportion of granular cell from virus-infected crayfish were higher than in controls, indicating granular cells are more resistant to and may interact by some means with the virus. Two morphotypes of the crayfish parasite Psorospermium haeckeli obtained from different crayfish hosts of different geographical origin were analysed for ribosomal ITS DNA in order to compare their genetic diversity. The sequence difference between them was found largely in ITS 1 and ITS 2 regions, which was variable in length and showed 66% and 58% sequence similarity. Thus, different morphotypes of P. haeckeli are genetically diverse.

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- ABSTRACT (IN ENGLISH) - Biological invasions by crustaceans represent a serious threat for native species in Europe. In my thesis I focus on non-indigenous freshwater crayfish introduced to Europe and their parasite Aphanomyces astaci, the pathogen of the crayfish plague. The thesis consists of four already published first-author papers (chapters I, II, IV and V), two first-author manuscripts (chapters III and VI), and one paper which I co-authored (chapter VII). The first part (chapters I-V) focuses on genetic variation in North American crayfish introduced to Europe. We showed that in two crayfish species, both successful invaders in Europe, genetic variation differs significantly, reflecting their different colonization histories on the continent. The spiny-cheek crayfish Orconectes limosus was likely introduced to Europe just once, in small numbers (90 individuals). Variation at the mitochondrial DNA (mtDNA) level in the spiny-cheek crayfish in Europe is much lower compared to North America (chapter I), although some variation was revealed by nuclear markers in its Central European populations (chapter II). In contrast, the signal crayfish Pacifastacus leniusculus was introduced to Europe several times, in large numbers. Its European populations are highly diverse genetically and belong to a single...

The most widespread of invasive crayfish in the Czech Republic is the spiny-cheek crayfish (Orconectes limosus). It is a major disease carrier of crayfish plague, caused by the pathogen Aphanomyces astaci. The infection is lethal to European native species of crayfish. Transmission of this disease is usually associated with the spread of crayfish, which is dependent on their movement. The main aim of this work was to evaluate the differences in crayfish migratory activity in the Pšovka brook among seasons, and assess the factors that may affect it, by the use of radiotelemetry and trapping. We tested the hypothesis that crayfish use brook in the period from spring to autumn and then return back into a pond to overwinter. Furthermore, we evaluated how far from the pond crayfish migrate, and whether there is any relationship between migration and the season or water temperature. The results demonstrate that the activity varies among seasons (being strongly affected by breeding season) but in most studied periods did not significantly depend on water temperature. According to our monitoring, the spiny-cheek crayfish does not spread substantially to upstream parts of the Pšovka. Seasonal migration between the pond and the brook was monitored using two-way flow-through traps installed at the mouth of...