An old unknown: 40 years of crayfish plague monitoring in Switzerland, the water tower of Europe.

The oomycete Aphanomyces astaci is the causative agent of crayfish plague, a disease threatening susceptible freshwater crayfish species in Europe. To detect its spatiotemporal occurrence in Switzerland, we reviewed (1) the literature regarding occurrence of crayfish plague and North American crayfish carrier species and (2) the necropsy report archive of the Institute for Fish and Wildlife Health (FIWI) from 1968 to 2020. In the past, crayfish plague was diagnosed through several methods: conventional PCR, culture, and histology. When available, we re-evaluated archived Bouin's or formalin-fixed, paraffin-embedded samples collected during necropsies (1991-2020) with a recently published quantitative PCR. Literature research revealed putative reports of crayfish plague in Switzerland between the 1870s and 1910s and the first occurrence of three North American crayfish species between the late 1970s and 1990s. Finally, 54 (28.1%) cases were classified as positive and 9 (4.7%) cases as suspicious. The total number of positive cases increased by 14 (14.7%) after re-evaluation of samples. The earliest diagnosis of crayfish plague was performed in 1980 and the earliest biomolecular confirmation of A. astaci DNA dated 1991. Between 1980-1990, 1991-2000 and 2001-2010 crayfish plague spread from one to two and finally three catchment basins, respectively. Similar to other European countries, crayfish plague has occurred in Switzerland in two waves: the first at the end of the 19th and the second at the end of the 20th century in association with the first occurrence of North American crayfish species. The spread from one catchment basin to another suggests a human-mediated pathogen dispersal.

Prevalence of the crayfish plague pathogen in red swamp crayfish populations in western France: How serious is the risk for the native white-clawed crayfish?

The crayfish plague pathogen Aphanomyces astaci has been implicated in a number of mass mortalities and irreversible population declines of native crayfish across Europe. At present, the reservoirs of the pathogen in Europe are mainly populations of invasive North American crayfish species. In southwestern Europe, including France, a particularly widespread invader is the red swamp crayfish Procambarus clarkii. Recent distribution data confirm that P. clarkii is present in at least 75 French departments, i.e. more than 78% of those in metropolitan France. We analysed the prevalence and pathogen load of A. astaci in 42 populations of this species in western France (Nouvelle Aquitaine region), where the species is most densely distributed, particularly in a wide range of environments around the Gironde estuary. The pathogen was detected by two different quantitative PCR assays in more than three quarters of the populations studied (34 out of 42); 163 out of 480 analysed crayfish individuals tested positive for the presence of A. astaci. In most cases, individual infection levels were very low, detectable with quantitative PCR but not sufficient for pathogen genotyping. In seven P. clarkii individuals from four populations, however, we were able to assess A. astaci variation by microsatellite markers and sequencing of mitochondrial markers. All these host specimens carried A. astaci genotype group D, haplotype d1, which has caused the majority of crayfish plague outbreaks in neighbouring Spain. In contrast, the French outbreaks genotyped to date (including eight newly analysed in this study) were mostly caused by strains of genotype group B, specific to the signal crayfish Pacifastacus leniusculus. Haplotype d1 found in P. clarkii was involved in one of the newly characterised outbreaks. Our study confirms that P. clarkii is a potentially important reservoir of the crayfish plague pathogen in France, but not the main source of the pathogen in mass mortalities of A. pallipes, probably due to different ecological requirements of the different invasive host crayfish. However, as P. clarkii continues to spread, the threat posed by this species to native crayfish is likely to increase.

Comparison of exoskeleton microbial communities of co-occurring native and invasive crayfish species.

Host-associated microbial communities are an important determinant of individual fitness and have recently been highlighted as one of the factors influencing the success of invasive species. Invasive hosts introduce their microbes into the new environment, and then both the host and its associated microbes enter into a series of interactions with the native macroscopic and microscopic biota. As these processes are largely unexplored, we aimed to compare the exoskeletal microbial communities of co-occurring and phylogenetically related crayfish: the native narrow-clawed crayfish Pontastacus leptodactylus and the invasive signal crayfish Pacifastacus leniusculus from the recently invaded Korana River, Croatia. The results of high-throughput 16S rRNA sequencing showed that the exoskeletal microbiome of both species is very diverse, significantly influenced by the local environment and dominated by low abundance bacterial families from the phylum Proteobacteria. Furthermore, the exoskeletal microbiomes of the crayfish species differed significantly in the composition and abundance of Amplicon Sequence Variants (ASVs), suggesting that they are to some extent shaped by species-specific intrinsic factors, despite sharing a common habitat. However, over 95% of the bacterial genera associated with the exoskeleton were detected in the exoskeleton samples of both native and invasive crayfish. We paid particular attention to two known crayfish pathogens, Aphanomyces astaci and Saprolegnia parasitica, and find that both species carry low amounts of both pathogens. On the side, we find that a non-standard ddPCR protocol outperforms standard qPCR test for A. astaci under low concentration conditions. Taken together, our results indicate the possibility of bidirectional mixing and homogenisation of exoskeleton microbiome. As such, they can serve as a baseline in future detangling of the processes that act together to shape the microbiomes of co-occuring native and invasive congeners during biological invasions.

eDNA monitoring as a tool for evaluating the reintroduction of Austropotamobius pallipes after a crayfish plague outbreak.

The crayfish plague, a severe disease caused by the oomycete Aphanomyces astaci, is responsible for most population declines of susceptible crayfish in Europe. This pathogen has been devastating native populations of Austropotamobius pallipes since the 1970s in the Iberian Peninsula. In this study, we report a massive mortality event in one of the most important Spanish populations of A. pallipes. We aimed to: (i) identify the cause of the mortality, and (ii) evaluate the reintroduction viability of the species. Over the course of six months, we used environmental DNA (eDNA) and traditional trap-based methods to detect the presence of A. astaci or of native or invasive crayfish in order to evaluate the reintroduction viability of A. pallipes to the affected population. We did not capture any live crayfish or detect the presence of A. astaci in the reservoir water during the six months following the mass mortality event. Our analyses indicated that it was feasible to initiate a reintroduction program at the site, which will continue to be monitored for three to five years and will help improve the conservation status of A. pallipes.

Crayfish plague affects juvenile survival and adult behaviour of invasive signal crayfish.

The spread of invasive, non-native species is a key threat to biodiversity. Parasites can play a significant role by influencing their invasive host's survival or behaviour, which can subsequently alter invasion dynamics. The North American signal crayfish (Pacifastacus leniusculus) is a known carrier of Aphanomyces astaci, an oomycete pathogen that is the causative agent of crayfish plague and fatal to European crayfish species, whereas North American species are considered to be largely resistant. There is some evidence, however, that North American species, can also succumb to crayfish plague, though how A. astaci affects such 'reservoir hosts' is rarely considered. Here, we tested the impact of A. astaci infection on signal crayfish, by assessing juvenile survival and adult behaviour following exposure to A. astaci zoospores. Juvenile signal crayfish suffered high mortality 4-weeks post-hatching, but not as older juveniles. Furthermore, adult signal crayfish with high-infection levels displayed altered behaviours, being less likely to leave the water, explore terrestrial areas and exhibit escape responses. Overall, we reveal that A. astaci infection affects signal crayfish to a much greater extent than previously considered, which may not only have direct consequences for invasions, but could substantially affect commercially harvested signal crayfish stocks worldwide.

Non-destructive method for detecting Aphanomyces astaci, the causative agent of crayfish plague, on the individual level.

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.

Crayfish plague in Czechia: Outbreaks from novel sources and testing for chronic infections.

The crayfish plague pathogen Aphanomyces astaci, which is among the most studied pathogens of aquatic invertebrates, co-evolved with North American crayfish species but threatens crayfish on other continents. The pathogen causes mass mortalities, particularly in Europe. In this study we document 12 crayfish plague outbreaks that occurred from 2014 to 2019 in Czechia and, by using available molecular techniques (microsatellite and mtDNA markers), we reveal the A. astaci genotypes involved. Our results provide the first evidence of strains from genotype group D, originally associated with the host Procambarus clarkii, causing Astacus astacus and Austropotamobius torrentium mass mortalities in Czechia. Moreover, mtDNA sequencing confirmed two distinct haplotypes of the D haplogroup, indicating two independent sources of infection, presumably originating from ornamental crayfish in the pet trade or spreading from crayfish established in neighbouring countries. Genotype group A was recorded in two As. astacus mortalities, and genotype group E, associated with Faxonius limosus, in two Au. torrentium and three As. astacus mortalities. Microsatellite genotyping also reidentified the unusual genotype SSR-Up in two As. astacus outbreaks, ten years after its first documented occurrence. In addition, we tested healthy-appearing indigenous crayfish from 25 localities for potential chronic infections. No traces of A. astaci DNA were detected; chronic infections in European crayfish species thus do not seem a pervasive phenomenon in Czechia. However, their role as A. astaci latent reservoirs, especially in Pontastacus leptodactylus populations introduced to the country since the late 19th century, cannot be excluded.

Experimental evaluation of the potential for crayfish plague transmission through the digestive system of warm-blooded predators.

The crayfish plague pathogen (Aphanomyces astaci) can be transmitted through the digestive system of fish, but its dispersal through mammalian and bird digestive tracts has been considered unlikely, and direct experimental evidence remains scarce. We present a small-scale transmission experiment with European otter and American mink fed with infected crayfish, and experiments testing survival of cultures of five A. astaci strains at temperatures corresponding to those inside mammal and bird bodies. The pathogen was neither isolated from predator excrements nor transmitted to susceptible crayfish exposed to excrements. In agar-based artificial media, it occasionally survived for 15 min at 40.5°C and for 45 min at 37.5°C, but not so when incubated at those temperatures for 45 min and 75 min, respectively. The five tested strains differed in resistance to high temperatures, two (of genotype groups E and D) being more susceptible than other three (of groups A, B and D). Their survival to some extent varied when exposed to the same temperature after several weeks or months, suggesting that some yet-unknown factors may influence A. astaci resistance to temperature stress. Overall, we support the notion that passage through the digestive tract of warm-blooded predators makes A. astaci transmission unlikely.

Improved method for genotyping the causative agent of crayfish plague (Aphanomyces astaci) based on mitochondrial DNA.

Aphanomyces astaci causes crayfish plague, which is a devastating disease of European freshwater crayfish. The likely first introduction of A. astaci into Europe was in the mid-19th century in Italy, presumably with the introduction of North American crayfish. These crayfish can carry A. astaci in their cuticle as a benign infection. Aphanomyces astaci rapidly spread across Europe causing the decline of the highly susceptible indigenous crayfish species. Random amplified polymorphic DNA-PCR analysis of A. astaci pure cultures characterized five genotype groups (A, B, C, D and E). Current A. astaci genotyping techniques (microsatellites and genotype-specific regions, both targeting nuclear DNA) can be applied directly to DNA extracted from infected cuticles but require high infection levels. Therefore, they are not suitable for genotyping benign infections in North American crayfish (carriers). In the present study, we combine bioinformatics and molecular biology techniques to develop A. astaci genotyping molecular markers that target the mitochondrial DNA, increasing the sensitivity of the genotyping tools. The assays were validated on DNA extracts of A. astaci pure cultures, crayfish tissue extractions from crayfish plague outbreaks and tissue extractions from North American carriers. We demonstrate the presence of A. astaci genotype groups A and B in UK waters.

Italian acacia honey exhibits lytic effects against the crayfish pathogens Aphanomyces astaci and Fusarium avenaceum.

This study purpose was to evaluate the in vitro inhibitory properties of Italian acacia honey extracts against pathogenic aquatic oomycete/fungal isolates that cause different diseases in crayfish, resulting in an elevated mortality rate. The antimycotic activity of acacia honey aqueous extracts was evaluated against the strain UEF88662 of Aphanomyces astaci (oomycete) and the strain SMM2 of Fusarium avenaceum (fungus). The extracts preparation was carried out with water by a cheap, not complex and organic solvent-free procedure, with low environmental impact and the higher possibility of large-scale reproducibility. The anti-oomycete and antifungal activities were quantitatively evaluated by growth, survival and sporulation microbiological assays. The extracts displayed a dose-dependent inhibitory efficacy on oomycete and fungal growth and survival, as well as on the production of oomycete and fungal spores. Supported by future in vivo studies, our results encourage the use of natural extracts like honey as innovative tools to counteract mycotic infections. SIGNIFICANCE AND IMPACT OF THE STUDY: The continuous spread of aquatic fungal disease as the 'crayfish plague' and the 'burn spot disease' has severe ecological and commercial repercussions. Critical factor to prevent further spread is the availability of effective antifungals possibility derived from local natural resources to use in innovative strategies of control and eradication of these diseases. This study provides relevant information about the in vitro anti-oomycete and antifungal activity of Italian acacia honey aqueous extracts against two highly infectious and dangerous pathogenic species, Aphanomyces astaci and Fusarium avenaceum, that are responsible for important crayfish diseases.

New genotyping method for the causative agent of crayfish plague (Aphanomyces astaci) based on whole genome data.

The oomycete Aphanomyces astaci causes crayfish plague, the most important disease of European freshwater crayfish species. Presumably introduced into Europe 150 years ago with the import of North American crayfish, A. astaci is highly pathogenic to European crayfish species. Five genotypes (A, B, C, D, and E) have been defined based on random amplified polymorphic DNA analysis (RAPD-PCR) from A. astaci pure cultures. The distinction of genotypes is an essential tool to conduct molecular epidemiological studies on crayfish plague and it has been used to clarify and better understand the history and spread of this disease in Europe. Whereas RAPD-PCR requires DNA from pure culture isolates, the development of genotyping tools that can be applied to DNA extracted from clinical samples allows a much wider application of genotyping studies, including revisiting historic samples. In this study, we present a new approach that adds to currently available methods for genotyping A. astaci strains directly from clinical crayfish samples. Whole-genome sequencing of A. astaci strains representing all currently known genotypes was employed, genomic regions unique to the respective genotype identified, and a PCR-based genotyping assay designed, which focuses on the presence/absence of PCR product after amplification with the genotype-specific primers. Our diagnostic methodology was tested using DNA extracts from pure A. astaci cultures, other Aphanomyces species and additional oomycetes, samples from a recent Italian crayfish plague outbreak and additional historical samples available in the Centre for Environment, Fisheries and Aquaculture Science laboratory. The new markers were reliable for pure culture and clinical samples from a recent outbreak and successfully discriminated genotype A, B, D, and E. The marker for genotype C required an additional sequencing step of the generated PCR product to confirm genotype.

Aphanomyces astaci genotypes involved in recent crayfish plague outbreaks in central Italy.

The oomycete Aphanomyces astaci is the causative agent of crayfish plague in native European freshwater crayfish. Molecular analyses showed that several distinct genotype groups of this pathogen, apparently associated with different original host taxa, are present in Europe. Tracking their distribution may contribute to understanding the introduction pathways of A. astaci. We used microsatellite markers to genotype the pathogen strains involved in 7 mass mortalities of the endangered indigenous crayfish Austropotamobius pallipes that occurred between 2009 and 2016 in the Abruzzi and Molise regions, central Italy. Three A. astaci genotype groups (A, B, and D, with the latter represented by 2 distinct multilocus genotypes) were identified, suggesting the existence of multiple infection sources even in a relatively small area. Most crayfish plague episodes were due to genotype groups associated with the North American host species Pacifastacus leniusculus and Procambarus clarkii, although these crayfish are not widespread in the study area. A. astaci genotype group A was detected not only in crayfish plague outbreaks but also in apparently healthy Astacus leptodactylus imported for human consumption from Armenia and kept alive in an aquaculture facility. Imports of chronically infected A. leptodactylus from Armenia, Turkey, and possibly Eastern Europe are an underestimated introduction pathway for A. astaci. Although we cannot exclude the presence of latently infected native populations of A. pallipes in the region, A. astaci infections in legally imported crayfish species considered vulnerable to crayfish plague may represent further reservoirs of A. astaci; this should be reflected in the policies regulating the trade of live crayfish.

Crayfish plague Aphanomyces astaci detected in redclaw crayfish, Cherax quadricarinatus in Taiwan.

Between December 2013 and January 2014, five outbreaks of an unknown disease with moderate to high cumulative mortality were observed among the freshwater redclaw crayfish (Cherax quadricarinatus) populations at four crayfish farms in Miaoli and Changhua counties (northern Taiwan) and at one crayfish farm in Pingtung County (southern Taiwan). Polymerase chain reaction (PCR) analysis allowed the detection of Aphanomyces astaci DNA in dead crayfish. Histopathological examination revealed an infection of host tissue by fungal hyphae that presented as typical non-septate hyphae within the soft abdominal cuticle from the first to second segment and in the tail fan. In PCR assays completed for the detection of crayfish plague, an expected 568-bp product, specific for the A. astaci ITS gene, was obtained from all sub-adults and adults examined. In a comparison of our strains with the known strains of A. astaci in Europe, nucleotide sequence identities were very similar, with 99.8-100% sequence similarity in that gene region. Positive reactions to in situ hybridization, using a digoxigenin (DIG)-labelled DNA probe, further confirmed A. astaci as the causative agent. This is the first report concerning natural infection of A. astaci in freshwater redclaw crayfish in Asia.

Variation in the hyphal growth rate and the virulence of two genotypes of the crayfish plague organism Aphanomyces astaci.

Crayfish plague, a devastating disease of freshwater crayfish, is caused by an oomycete organism, Aphanomyces astaci. Currently five genotypes of A. astaci are known, but variable features between the strains or genotypes have not been studied extensively. This study analysed 28 isolates of the As genotype and 25 isolates of the Ps1 genotype and reveals that the radial growth rate is significantly (P < 0.001) different between these two genotypes, although highly variable inside the genotype As. Two Ps1 genotype isolates and two As genotype isolates with different radial growth rates were tested in an infection trial. Clear differences were detected in the development of mortality in the test groups. The representatives of the Ps1 genotype caused total mortality within a short time span. The As genotype isolates were much less virulent. The slow-growing As isolate showed higher virulence than the As isolate with a high growth capacity. Although slow growth could be one survival strategy of the pathogen, several other mechanisms are involved in the pathogenicity and warrant further studies.

Resistance to the crayfish plague pathogen, Aphanomyces astaci, in two freshwater shrimps.

Aphanomyces astaci, the causal agent of the crayfish plague, has recently been confirmed to infect also freshwater-inhabiting crabs. We experimentally tested the resistance of freshwater shrimps, another important decapod group inhabiting freshwaters, to this pathogen. We exposed individuals of two Asian shrimp species, Macrobrachium dayanum and Neocaridina davidi, to zoospores of the pathogen strain isolated from Procambarus clarkii, a known A. astaci carrier likely to get into contact with shrimps. The shrimps were kept in separate vessels up to seven weeks; exuviae and randomly chosen individuals were sampled throughout the experiment. Shrimp bodies and exuviae were tested for A. astaci presence by a species-specific quantitative PCR. The results were compared with amounts of A. astaci DNA in an inert substrate to distinguish potential pathogen growth in live specimens from persisting spores or environmental DNA attached to their surface. In contrast to susceptible crayfish Astacus astacus, we did not observe mortality of shrimps. The amount of detected pathogen DNA was decreasing steadily in the inert substrate, but it was still detectable several weeks after zoospore addition, which should be considered in studies relying on molecular detection of A. astaci. Probably due to moulting, the amount of A. astaci DNA was decreasing in N. davidi even faster than in the inert substrate. In contrast, high pathogen DNA levels were detected in some non-moulting individuals of M. dayanum, suggesting that A. astaci growth may be possible in tissues of this species. Further experiments are needed to test for the potential of long-term A. astaci persistence in freshwater shrimp populations.

Survey of the crayfish plague pathogen presence in the Netherlands reveals a new Aphanomyces astaci carrier.

North American crayfish species as hosts for the crayfish plague pathogen Aphanomyces astaci contribute to the decline of native European crayfish populations. At least six American crayfish species have been reported in the Netherlands but the presence of this pathogenic oomycete with substantial conservational impact has not yet been confirmed in the country. We evaluated A. astaci prevalence in Dutch populations of six alien crustaceans using species-specific quantitative PCR. These included three confirmed crayfish carriers (Orconectes limosus, Pacifastacus leniusculus, Procambarus clarkii), two recently introduced but yet unstudied crayfish (Orconectes cf. virilis, Procambarus cf. acutus), and a catadromous crab Eriocheir sinensis. Moderate levels of infection were observed in some populations of O. limosus and P. leniusculus. Positive results were also obtained for E. sinensis and two Dutch populations of O. cf. virilis. English population of the latter species was also found infected, confirming this taxon as another A. astaci carrier in European waters. In contrast, Dutch P. clarkii seem only sporadically infected, and the pathogen was not yet detected in P. cf. acutus. Our study is the first confirmation of crayfish plague infections in the Netherlands and demonstrates substantial variation in A. astaci prevalence among potential hosts within a single region, a pattern possibly linked to their introduction history and coexistence.

The Crayfish Plague Pathogen Aphanomyces astaci in Ireland.

Crayfish plague is a devastating disease of European freshwater crayfish and is caused by the oomycete Aphanomyces astaci (Ap. astaci), believed to have been introduced to Europe around 1860. All European species of freshwater crayfish are susceptible to the disease, including the white-clawed crayfish Austropotamobius pallipes. Ap. astaci is primarily spread by North American crayfish species and can also disperse rapidly through contaminated wet gear moved between water bodies. This spread, coupled with competition from non-indigenous crayfish, has drastically reduced and fragmented native crayfish populations across Europe. Remarkably, the island of Ireland remained free from the crayfish plague pathogen for over 100 years, providing a refuge for A. pallipes. However, this changed in 1987 when a mass mortality event was linked to the pathogen, marking its introduction to the region. Fortunately, crayfish plague was not detected again in Ireland until 2015 when a molecular analysis linked a mass mortality event in the Erne catchment to Ap. astaci. Since then, the pathogen has appeared across the island. Between 2015 and 2023, Ap. astaci was detected in 18 water catchments, revealing multiple genotypes. Intriguingly, the pathogen in Ireland is present without its natural host species. The uneven distribution of various genetic lineages strongly suggests the human-mediated transport of zoospores via contaminated water equipment as a primary cause of spread. This review details the timeline of these events, Ap. astaci's introduction into Ireland, and its rapid spread. As well, this review references the genotypes that have been determined, and discusses the issue of non-indigenous crayfish species in Ireland and management efforts.

Crayfish population size under different routes of pathogen transmission.

We present an epidemiological model for the crayfish plague, a disease caused by an invasive oomycete Aphanomyces astaci, and its general susceptible freshwater crayfish host. The pathogen shows high virulence with resulting high mortality rates in freshwater crayfishes native to Europe, Asia, Australia, and South America. The crayfish plague occurrence shows complicated dynamics due to the several types of possible infection routes, which include cannibalism and necrophagy. We explore this complexity by addressing the roles of host cannibalism and the multiple routes of transmission through (1) environment, (2) contact, (3) cannibalism, and (4) scavenging of infected carcasses. We describe a compartment model having six classes of crayfish and a pool of crayfish plague spores from a single nonevolving strain. We show that environmental transmission is the decisive factor in the development of epidemics. Compared with a pathogen-free crayfish population, the presence of the pathogen with a low environmental transmission rate, regardless of the contact transmission rate, decreases the crayfish population size with a low risk of extinction. Conversely, a high transmission rate could drive both the crayfish and pathogen populations to extinction. High contact transmission rate with a low but nonzero environmental transmission rate can have mixed outcomes from extinction to large healthy population, depending on the initial values. Scavenging and cannibalism have a relevant role only when the environmental transmission rate is low, but scavenging can destabilize the system by transmitting the pathogen from a dead to a susceptible host. To the contrary, cannibalism stabilizes the dynamics by decreasing the proportion of infected population. Our model provides a simple tool for further analysis of complex host parasite dynamics and for the general understanding of crayfish disease dynamics in the wild.

Variations in the Sporulation Efficiency of Pathogenic Freshwater Oomycetes in Relation to the Physico-Chemical Properties of Natural Waters.

Oomycete pathogens in freshwaters, such as Saprolegnia parasitica and Aphanomyces astaci, are responsible for fish/crayfish population declines in the wild and disease outbreaks in aquaculture. Although the formation of infectious zoospores in the laboratory can be triggered by washing their mycelium with natural water samples, the physico-chemical properties of the water that might promote sporulation are still unexplored. We washed the mycelia of A. astaci and S. parasitica with a range of natural water samples and observed differences in sporulation efficiency. The results of Partial Least Squares Regression (PLS-R) multivariate analysis showed that SAC (spectral absorption coefficient measured at 254 nm), DOC (dissolved organic carbon), ammonium-N and fluoride had the strongest positive effect on sporulation of S. parasitica, while sporulation of A. astaci was not significantly correlated with any of the analyzed parameters. In agreement with this, the addition of environmentally relevant concentrations of humic acid, an important contributor to SAC and DOC, to the water induced sporulation of S. parasitica but not of A. astaci. Overall, our results point to the differences in ecological requirements of these pathogens, but also present a starting point for optimizing laboratory protocols for the induction of sporulation.

A Comprehensive Review on Crustaceans' Immune System With a Focus on Freshwater Crayfish in Relation to Crayfish Plague Disease.

Freshwater crayfish immunity has received great attention due to the need for urgent conservation. This concern has increased the understanding of the cellular and humoral defense systems, although the regulatory mechanisms involved in these processes need updating. There are, however, aspects of the immune response that require clarification and integration. The particular issues addressed in this review include an overall description of the oomycete Aphanomyces astaci, the causative agent of the pandemic plague disease, which affects freshwater crayfish, and an overview of crustaceans' immunity with a focus on freshwater crayfish. It includes a classification system of hemocyte sub-types, the molecular factors involved in hematopoiesis and the differential role of the hemocyte subpopulations in cell-mediated responses, including hemocyte infiltration, inflammation, encapsulation and the link with the extracellular trap cell death pathway (ETosis). In addition, other topics discussed include the identity and functions of hyaline cells, the generation of neoplasia, and the emerging topic of the role of sessile hemocytes in peripheral immunity. Finally, attention is paid to the molecular execution of the immune response, from recognition by the pattern recognition receptors (PRRs), the role of the signaling network in propagating and maintaining the immune signals, to the effector elements such as the putative function of the Down syndrome adhesion molecules (Dscam) in innate immune memory.