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.

Insights from the past: Invasion trajectory and niche trends of a global freshwater invader.

Freshwater ecosystems are invaded by a non-random selection of taxa, among which crayfish stand out with successful examples worldwide. Species distribution models (SDMs) have been used to detect suitable areas for invasive species and predict their potential distributions. However, these prediction exercises assume the stability of realized environmental niches, which is uncertain during invasion. Worldwide evaluations involving cosmopolitan invaders may be particularly useful but have seldom been considered. Focusing on the successful invasion history of the red swamp crayfish, Procambarus clarkii, we assessed its geographic expansion and niche trends over time. Based on global occurrences from 1854 to 2022, multiple sequential SDMs have been implemented based on a set of bioclimatic variables. The environmental suitability for each period was projected through to the next period(s) using an ensemble procedure of commonly used SDM algorithms. As the records of the species are known, it was possible to check whether the modelling projections were concordant with the observed expansion of red swamp crayfish at a global scale. This also permitted analysis of its realized niche, and its dynamics, during different expansion phases. SDM maps based on past species records showed concordance with the known crayfish distributions and yielded similar spatial patterns with outputs overperforming random combinations of cells in term of suitability. The results also reflect the stability of the species niche, which despite some expansions during the invasion process, changed little in terms of main position in functional space over time. SDMs developed in the early stages of invasion provide useful insights but also tend to underpredict the potential range compared to models that were built for later stages. Our approach can be easily transferable to other well-documented taxa and represents valuable evidence for validating the use of SDMs, considering a highly dynamic world where biogeographical barriers are often bypassed.

Diversity and distribution of Aphanomyces astaci in a European hotspot of ornamental crayfish introductions.

Ornamental trade has become an important introduction pathway of non-native aquatic species worldwide. Correspondingly, there has been an alarming increase in the number of established crayfish of aquarium origin in Europe over the previous decade. The oomycete Aphanomyces astaci, the pathogen causing crayfish plague responsible for serious declines of European crayfish populations, is dispersed with introduced North American crayfish. The role of ornamental taxa in introducing and spreading different genotypes of this pathogen in open waters remains unclear. We investigated the distribution, prevalence, and diversity of A. astaci in Budapest, Hungary, which became a hotspot of aquarium crayfish introductions. Their establishment in this area was facilitated by locally abundant thermal waters. We screened for A. astaci in six host taxa from 18 sites sampled between 2018 and 2021: five cambarids (Cambarellus patzcuarensis, Faxonius limosus, Procambarus alleni, P. clarkii, P. virginalis) and one native astacid (Pontastacus leptodactylus). The pathogen was confirmed at five sampled sites in four host taxa: P. virginalis, P. clarkii, F. limosus, and for the first time in European open waters also in P. alleni. Genotyping was successful only in individuals from two different brooks where multiple host species coexisted but revealed unexpected patterns. Mitochondrial B-haplogroup of A. astaci, previously usually reported from Pacifastacus leniusculus or infected European species, was detected in P. virginalis at both sites, and in both F. limosus and P. virginalis sampled from a thermally stable tributary of Barát brook in 2018. In contrast, A-haplogroup of A. astaci was detected in coexisting F. limosus, P. virginalis and P. clarkii sampled in the same watercourse just a few hundred meters downstream in 2020. Additional genotyping methods indicated that a previously unknown A. astaci strain was associated with the latter haplogroup. One P. virginalis individual from 2020 was apparently co-infected by strains representing both mitochondrial haplogroups. The results indicated multiple sources of A. astaci in Budapest, likely directly associated with the introduction of ornamental species, interspecific transmission of this pathogen among ornamental hosts, and potential for a quick spatial or temporal turnover of dominant A. astaci strains at a certain locality. This highlights that in regions with high richness of potential A. astaci hosts, host taxon/pathogen genotype combinations become unpredictable, which might prevent reliable genotyping of pathogen sources in local crayfish mass mortalities.

Invasive Crayfish Faxonius limosus: Meat Safety, Nutritional Quality and Sensory Profile.

The aim of the present study was to evaluate the safety parameters, nutritional value and sensory profile of the meat from spiny-cheek crayfish (Faxonius limosus), captured from the Danube River in Serbia. To achieve this, we determined their microbiological safety, chemical composition, minerals and heavy metals, fatty acid and amino acid profile, as well as a sensory profile of the meat. The obtained results showed that the meat from crayfish was microbiologically safe. Crayfish meat has a high nutritional quality, high protein content (18.12%) and a total of 17 detected amino acids, of which essential amino acids constituted 6.96 g/100 g sample. Additionally, the crayfish meat was characterized by high levels of essential polyunsaturated fatty acids (PUFA), particularly n-3 PUFA, at an optimal ratio of n-3/n-6 and with low values of atherogenic and thrombogenic indices. Predominant macrominerals in the meat are K, followed by Na, Ca, P and Mg, whereas the content of microminerals was in the following order: Zn > Cu > Fe > Mn. The concentrations of accumulated toxic metals (Cd, Pb, As and Hg) did not exceed the maximum allowed levels. Sensory analysis confirmed that the meat from spiny-cheek crayfish has the potential to become a new food source of essential nutrients.

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.

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 can distinguish between natural and chemical stimuli as assessed by cardiac and locomotor reactions.

In this study, cardiac and locomotor activities of signal crayfish Pacifastacus leniusculus were investigated under exposure to a range of natural (i.e., odors of conspecific crayfish, predatory fish, food, and injured conspecific) and one chemical (i.e., disinfectant chloramine-T) stimuli. Crayfish locomotion was simultaneously initiated with an increase in heart rate only when affected by chloramine-T, while locomotor response was delayed in all cases (or was not manifested at all by some specimens) when disturbed by the natural stressors. The heart rate differences measured before and during the stimulation were arranged as follows: odor of conspecific crayfish (9.2 ± 7.1%) < predator (18.4 ± 13%) < food (33.5 ± 15.7%) < chloramine-T (41.1 ± 14.7%) < injured conspecific (51.8 ± 28.4%). Analysis of the peculiarities of crayfish heartbeat under exposure to the tested stimuli revealed complex cardiac responses as was previously observed by an electrocardiography approach, that is, a slowed heart rate followed by a delayed increase. Evaluation of the intrinsic parameters of crayfish bioindicators remains essential due to the possibility of detection of the substantial ethological responses even in motionless animals. The role and appropriateness of signal crayfish as a bioindicator of water quality is discussed; they seem to be an applicable species for this task due to their sufficient sensitivity and broad availability. In addition to providing a better understanding of stereotypic crayfish behaviors induced by common and chemical stressors, the results of this study may serve as reference data for the evaluation of crayfish suitability for water quality tests.

Microsatellite markers for direct genotyping of the crayfish plague pathogen Aphanomyces astaci (Oomycetes) from infected host tissues.

Aphanomyces astaci is an invasive pathogenic oomycete responsible for the crayfish plague, a disease that has devastated European freshwater crayfish. So far, five genotype groups of this pathogen have been identified by applying random amplified polymorphic DNA analysis on axenic cultures. To allow genotyping of A. astaci in host tissue samples, we have developed co-dominant microsatellite markers for this pathogen, tested them on pure cultures of all genotype groups, and subsequently evaluated their use on tissues of (1) natural A. astaci carriers, i.e., North American crayfish species, and (2) A. astaci-infected indigenous European species from crayfish plague outbreaks. Out of over 200 potential loci containing simple sequence repeat (SSR) motifs identified by 454 pyrosequencing of SSR-enriched library, we tested 25 loci with highest number of repeats, and finally selected nine that allow unambiguous separation of all known RAPD-defined genotype groups of A. astaci from axenic cultures. Using these markers, we were able to characterize A. astaci strains from DNA isolates from infected crayfish tissues when crayfish had a moderate to high agent level according to quantitative PCR analyses. The results support the hypothesis that different North American crayfish hosts carry different genotype groups of the pathogen, and confirm that multiple genotype groups, including the one originally introduced to Europe in the 19th century, cause crayfish plague outbreaks in Central Europe. So far undocumented A. astaci genotype seems to have caused one of the analysed outbreaks from the Czech Republic. The newly developed culture-independent approach allowing direct genotyping of this pathogen in both axenic cultures and mixed genome samples opens new possibilities in studies of crayfish plague pathogen distribution, diversity and epidemiology.