Parasite DNA detection in water samples enhances crayfish plague monitoring in asymptomatic invasive populations

Invasive species can facilitate the spread of pathogens by first providing asymptomatic host reservoirs, and then driving disease outbreaks in native populations through pathogen spillover. An example of this are invasive crayfish species in Europe (Faxonius limosus, Pacifastacus leniusculus, Procambarus clarkii), which carry the deadly plague agent (Aphanomyces astaci). Effective disease management requires comprehensive monitoring, however, pathogen detection in carrier populations with low pathogen prevalence and intensities is challenging. We simultaneously collected and analysed crayfish tissue samples of invasive crayfish populations and water samples to compare A. astaci detection in different sample types using quantitative PCR. Combined, the two sampling methods revealed A. astaci presence with DNA concentrations above limit of detection (LOD; the lowest concentration which can be detected with reasonable certainty) in 13 of 23 invasive crayfish populations. In four additional sites, A. astaci DNA concentrations below LOD were found in water. In four populations only were A. astaci concentrations above LOD detected in both sample types and in three populations in concentrations above LOD in tissue but below LOD in water. The likely reason for these discrepancies is the low A. astaci prevalence and concentration in resistant invasive crayfish, which limit detection reliability. Consistency may be improved by timing surveys with seasonal periods of high A. astaci abundance and by increasing water sampling effort. Considering the ease of collecting eDNA samples, compared to crayfish tissue sampling, eDNA methods would facilitate frequent and comprehensive surveys. However, remaining uncertainties in eDNA-based detection reveal the relevance of combining monitoring tools to improve detection of invasive pathogens and their management.

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.

Essential Oils of Sage, Rosemary, and Bay Laurel Inhibit the Life Stages of Oomycete Pathogens Important in Aquaculture

Saprolegnia parasitica, the causative agent of saprolegniosis in fish, and Aphanomyces astaci, the causative agent of crayfish plague, are oomycete pathogens that cause economic losses in aquaculture. Since toxic chemicals are currently used to control them, we aimed to investigate their inhibition by essential oils of sage, rosemary, and bay laurel as environmentally acceptable alternatives. Gas Chromatography–Mass Spectrometry (GC–MS) analysis showed that the essential oils tested were rich in bioactive volatiles, mainly monoterpenes. Mycelium and zoospores of A. astaci were more sensitive compared to those of S. parasitica, where only sage essential oil completely inhibited mycelial growth. EC50 values (i.e., concentrations of samples at which the growth was inhibited by 50%) for mycelial growth determined by the radial growth inhibition assay were 0.031–0.098 µL/mL for A. astaci and 0.040 µL/mL for S. parasitica. EC50 values determined by the zoospore germination inhibition assay were 0.007–0.049 µL/mL for A. astaci and 0.012–0.063 µL/mL for S. parasitica. The observed inhibition, most pronounced for sage essential oil, could be partly due to dominant constituents of the essential oils, such as camphor, but more likely resulted from a synergistic effect of multiple compounds. Our results may serve as a basis for in vivo experiments and the development of environmentally friendly methods to control oomycete pathogens in aquaculture.

Lytic Polysaccharide Monooxygenases as Chitin-Specific Virulence Factors in Crayfish Plague

The oomycete pathogen Aphanomyces astaci, also known as “crayfish plague”, is an obligate fungal-like parasite of freshwater crustaceans and is considered responsible for the ongoing decline of native European crayfish populations. A. astaci is thought to secrete a wide array of effectors and enzymes that facilitate infection, however their molecular mechanisms have been poorly characterized. Here, we report the identification of AA15 lytic polysaccharide monooxygenases (LPMOs) as a new group of secreted virulence factors in A. astaci. We show that this enzyme family has greatly expanded in A. astaci compared to all other oomycetes, and that it may facilitate infection through oxidative degradation of crystalline chitin, the most abundant polysaccharide found in the crustacean exoskeleton. These findings reveal new roles for LPMOs in animal–pathogen interactions, and could help inform future strategies for the protection of farmed and endangered species.

Money Kills Native Ecosystems: European Crayfish as an Example

Native European crayfish conservation was triggered by invasion of crayfish plague disease agent, Aphanomyces astaci, starting 1860s in Northern Italy. Resulting crayfish plague epidemics quickly spread over Continental Europe, then to Finland, Sweden and finally, after running amok around Europe, A. astaci was discovered also in Iberian Peninsula, Norway, Ireland, and United Kingdom in 1970s and 1980s. By that time significant proportion of native crayfish stocks had been lost, and while crayfish plague epidemics were still recorded, also industrialization and waterways construction were causing damage to remaining native crayfish stocks. While alien crayfish introductions, at least Faxonius limosus, already gave rise to first wave of crayfish plague epidemics in late 19th century, later in 1960s it was decided that introductions of alien Pacifastacus leniusculus should be initiated to replace native European crayfish populations. Decisions were based on presumed advantages for fishery, suitable habitat requirements and supposed immunity against A. astaci. Furthermore, conservation of native European crayfish species was sidelined and focus shifted toward alien crayfish stocking routine and consumption. Alien crayfish species introductions resulted in repeated waves of crayfish plague epidemics among remaining native crayfish stocks. It was soon discovered that alien crayfish of North American origin were, as suspected, permanent reservoirs for A. astaci, that some of those alien species were losing their resistance against selected strains of A. astaci and struggled in European aquatic ecosystems. In this article, we introduce numerous motives behind grand mistake of introducing alien crayfish species to Europe and then promoting their stocks instead of focusing on conservation of native crayfish species. We outline how false economical, biological and ecologic assumptions were used to justify a hasty introduction of alien crayfish, which has further devastated native crayfish and also permanently changed European aquatic ecosystems, both with disastrous consequences. Lesson to be learnt is that science-based warnings about alien species damage to native ecosystems and native crayfish must be taken with utmost caution. Protection of native European crayfish should be core issue, not commercial activities. Finally, we summarize main threats and actions needed to protect remaining native freshwater crayfish fauna in Europe.

Real-Time PCR Assays for Rapid Identification of Common Aphanomyces astaci Genotypes

The oomycete Aphanomyces astaci is the etiologic agent of crayfish plague, a disease that has seriously impacted the populations of European native crayfish species. The introduction of non-indigenous crayfish of North American origin and their wide distribution across Europe have largely contributed to spread of crayfish plague in areas populated by indigenous crayfish. Tracking A. astaci genotypes may thus be a useful tool for investigating the natural history of crayfish plague in its European range, as well as the sources and introduction pathways of the pathogen. In this study, we describe the development of real-time PCR TaqMan assays aiming to distinguish the five genotype groups of A. astaci (A–E) previously defined by their distinct RAPD patterns. The method was evaluated using DNA extracts from pure A. astaci cultures representing the known genotype groups, and from A. astaci-positive crayfish clinical samples collected mostly during crayfish plague outbreaks that recently occurred in Central Italy and Czechia. The assays do not cross-react with each other, and those targeting genotype groups A, B, D, and E seem sufficiently specific to genotype the pathogen from infected crayfish in the areas invaded by A. astaci (particularly Europe). The unusual A. astaci genotype “SSR-Up” documented from crayfish plague outbreaks in Czechia and chronically infected Pontastacus leptodactylus in the Danube is detected by the group B real-time PCR. The assay originally developed to detect group C (one not yet documented from crayfish plague outbreaks) showed cross-reactivity with Aphanomyces fennicus; the A. astaci genotype “rust1” described in the United States from Faxonius rusticus is detected by that assay as well. Analyses of additional markers (such as sequencing of the nuclear internal transcribed spacer or mitochondrial ribosomal subunits) may complement such cases when the real-time PCR-based genotyping is not conclusive. Despite some limitations, the method is a robust tool for fast genotyping of A. astaci genotype groups common in Europe, both during crayfish plague outbreaks and in latent infections.

First Detection of the Crayfish Plague Pathogen Aphanomyces astaci in Costa Rica: European Mistakes Should Not Be Repeated

The crayfish plague pathogen Aphanomyces astaci is one of the main factors responsible for the decline in European and Asian native crayfish species. This pathogen was transported to these regions through its natural carriers, North American crayfish species, which were introduced during the last century. Since then, the carrier species and the pathogen have spread worldwide due to globalization and the highly invasive nature of these species. In Europe, five carrier species have been categorized as high-risk as they are responsible for the loss of provisioning services, which endangers freshwater ecosystems. The red swamp crayfish Procambarus clarkii, in particular, is currently one of the most concerning species as its spread threatens crayfish biodiversity and freshwater ecosystems worldwide. In this study, we describe the first detection of A. astaci in an introduced population of P. clarkii in Central America, specifically in Costa Rica. Using molecular approaches, we analyzed 48 crayfish samples collected from Reservoir Cachí and detected the presence of A. astaci in four of these samples. The introduction of P. clarkii and the incorrect management of the species (related to its fishery and the commercialization of live specimens) over the past decades in Europe are mistakes that should not be repeated elsewhere. The detection of the pathogen is a warning sign about the dangerous impact that the introduction of this invasive crayfish may have, not only as a carrier of an emerging disease but also as a direct risk to the invaded ecosystems. Our results may serve to (1) assess current and future consequences, and (2) direct future research activities, such as determining the potential impacts of A. astaci on native decapod species, or on other introduced crayfish species that are used for aquaculture purposes, such as Cherax quadricarinatus.

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.

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.

Tracing the origin of the crayfish plague pathogen, Aphanomyces astaci, to the Southeastern United States

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.