Prevalence of the Crayfish Plague Pathogen Aphanomyces astaci in Populations of the Signal Crayfish Pacifastacus leniusculus in France: Evaluating the Threat to Native Crayfish

Aphanomyces astaci, the crayfish plague pathogen, first appeared in Europe in the mid-19^th century and is still responsible for mass mortalities of native European crayfish. The spread of this parasite across the continent is especially facilitated by invasive North American crayfish species that serve as its reservoir. In France, multiple cases of native crayfish mortalities have been suggested to be connected with the presence of the signal crayfish Pacifastacus leniusculus, which is highly abundant in the country. It shares similar habitats as the native white-clawed crayfish Austropotamobius pallipes and, when infected, the signal crayfish might therefore easily transmit the pathogen to the native species. We investigated the prevalence of A. astaci in French signal crayfish populations to evaluate the danger they represent to local populations of native crayfish. Over 500 individuals of Pacifastacus leniusculus from 45 French populations were analysed, plus several additional individuals of other non-indigenous crayfish species Orconectes limosus, O. immunis and Procambarus clarkii. Altogether, 20% of analysed signal crayfish tested positive for Aphanomyces astaci, and the pathogen was detected in more than half of the studied populations. Local prevalence varied significantly, ranging from 0% up to 80%, but wide confidence intervals suggest that the number of populations infected by A. astaci may be even higher than our results show. Analysis of several individuals of other introduced species revealed infections among two of these, O. immunis and P. clarkii. Our results confirm that the widespread signal crayfish serves as a key reservoir of Aphanomyces astaci in France and therefore represents a serious danger to native crayfish species, especially the white-clawed crayfish. The prevalence in other non-indigenous crayfish should also be investigated as they likely contribute to pathogen transmission in the country.     

Absence of the crayfish plague pathogen (Aphanomyces astaci) facilitates coexistence of European and American crayfish in central Europe

1. Most European crayfish species are strongly threatened, mainly as a result of the introduced pathogen, Aphanomyces astaci, transmitted by invasive North American crayfish. Long‐term coexistence of American and European crayfish species is therefore regarded as almost impossible, even though some coexisting populations have been observed. 2. In this study, crayfish were collected from presently coexisting populations of the introduced spiny‐cheek crayfish (Orconectes limosus) and the native noble crayfish (Astacus astacus) from nine standing waters in central Europe. Our aim was to resolve whether the coexistence resulted from reduced virulence in local strains of A. astaci, increased immunity in the native crayfish or an absence of the pathogen in these populations. We used highly sensitive A. astaci‐specific real‐time PCR to evaluate the crayfish latent carrier status, combined with transmission experiments to further validate the molecular results. 3. From the total of 523 crayfish tested (490 spiny‐cheek crayfish, 33 noble crayfish), none positive for A. astaci was detected. Transmission experiments confirmed these results: No abnormal mortality or behavioural changes were seen in noble crayfish kept together with American crayfish from the coexisting populations. If we assume a very low prevalence of A. astaci of 10% in a carrier population, there is a 98% probability of disease being absent in five of the nine coexisting populations tested. Hence, a consistent absence, or an extremely low prevalence, of A. astaci seems to allow the coexistence of European and American crayfish in these central European populations. 4. The results are important for native crayfish conservation and management and demonstrate that disease transmission risk may vary substantially between the different populations of spiny‐cheek crayfish in central Europe.     

First records of the non-indigenous signal crayfish (<Emphasis Type="Italic">Pacifastacus leniusculus</Emphasis>) and its threat to noble crayfish (<Emphasis Type="Italic">Astacus astacus</Emphasis>) populations in Estonia

This study gives an overview of status and distribution of signal crayfish (Pacifastacus leniusculus), the first NICS in Estonia and its influence on native noble crayfish (Astacus astacus) populations. The first specimen of signal crayfish was caught during the monitoring of noble crayfish in North Estonia in 2008. The signal crayfish has since been found in three additional sites. Test fishing has indicated that the abundance of signal crayfish has been fluctuating between years and among localities. It has had strong negative impact on abundance of one noble crayfish population. The disconnected distribution of signal crayfish strongly suggests that these populations are the result of human-assisted introductions. Real-time PCR analyses proved that signal crayfish carry the causative agent of the crayfish plague, an oomycete Aphanomyces astaci, thus contributing to its spread. Mortalities in noble crayfish populations had been caused by A. astaci strains from A, B and E genotype group.     

Loss of genetic integrity correlates with stocking intensity in brook charr (Salvelinus fontinalis)

Supportive breeding and stocking performed with non‐native or domesticated fish to support sport fishery industry is a common practice throughout the world. Such practices are likely to modify the genetic integrity of natural populations depending on the extent of genetic differences between domesticated and wild fish and on the intensity of stocking. The purpose of this study is to assess the effects of variable stocking intensities on patterns of genetic diversity and population differentiation among nearly 2000 brook charr (Salvelinus fontinalis) from 24 lakes located in two wildlife reserves in Québec, Canada. Our results indicated that the level of genetic diversity was increased in more intensively stocked lakes, mainly due to the introduction of new alleles of domestic origin. As a consequence, the population genetic structure was strongly homogenized by intense stocking. Heavily stocked lakes presented higher admixture levels and lower levels of among lakes genetic differentiation than moderately and un‐stocked lakes. Moreover, the number of stocking events explained the observed pattern of population genetic structure as much as hydrographical connections among lakes in each reserve. We discuss the implications for the conservation of exploited fish populations and the management of stocking practices.     

Severe and rapid population declines in exotic birds

A particularly vexing phenomenon within invasion ecology is the occurrence of spontaneous collapses within seemingly well-established exotic populations. Here, we assess the frequency of collapses among 68 exotic bird populations established in Hawaii, Puerto Rico, Los Angeles and Miami. Following other published definitions, we define a ‘collapse’ as a decline in abundance of ≥90 % within ≤10 years that lasts for at least 3 years. We show that 44 of the 68 exotic bird populations have exhibited declines at some point within their time series. Sixteen of the populations declined sufficiently to be defined as collapsed. It took on average 3.8 ± 1.8 years for populations to decline into a collapsed state, and this state persisted on average for 7.1 ± 6.3 years across (collapsed) populations. We compared the severity and duration of declines across all 44 declining populations according to taxonomic Order and geographic region. Neither variable explained substantial variation in the metrics of collapse. Our results indicate that severe, rapid, and persistent population declines may be common among exotic populations. We suggest that incorporating the probability and persistence of collapses into management decisions can inform efforts to enact control or eradication measures. We also suggest that applying our approach to other taxa and locations is crucial for improving our understanding of when and where collapses are likely to occur.     

The harvest of freshwater crayfish, <Emphasis Type="Italic">Astacus leptodactylus</Emphasis> (Eschscholtz, 1823) in Turkey

Astacus leptodactylus is naturally and widely distributed in lakes, ponds and rivers throughout of Turkey. It has also been stocked in many freshwater systems in Turkey to establish new populations and to restore crayfish stocks devastated by the plague (Aphanomyces astaci) and some other reasons (i.e., farming and culinary purposes). Human consumption of A. leptodactylushas always been very low in Turkey; therefore, it was exported mainly to Europe until 1986. A plague was observed in 1985 in Eğirdir, Apolyont, Sapanca and Manyas lakes which included the main harvest areas, and the fishery was lost within 2 years. Consequently, crayfish harvest was banned in all populations to control the plague. Because of the plague, over-fishing and pollution, the harvest of A. leptodactylusin Turkey declined from approximately 5000 (1984) to 200 (1991) tons annually. In 2000s, there has been a gradual increase in the rate of harvest of A. leptodactylusin Turkey, reaching approximately 1600–1900 tons annually, but the plague is still observed in this species at certain localities. The present study reveals that A. leptodactylus in Turkey needs professional support for recovery, because the present harvest of A. leptodactylusis still nearly 20% of the crayfish catch in 1980s.     

Genetic structure in alpine brown trout Salmo trutta L. shows that indirect stocking affects native lake populations

Genetic variation of brown trout inhabiting six connected lakes (Krokavatn, Skavatn, Kollsvatn, Litlosvatn, Kvennsjøen and Øvre Krokavatn) of the Hardangervidda mountain plateau was analysed by 11 microsatellites covering a period of 24–35 years. Temporal changes were found and are deemed to be mostly related to stocking in the headwater lakes Krokavatn and Skavatn. A pronounced change was evident in one population (Kollsvatn) and was the result of downstream migration of stocked fish from these headwater lakes. This indirect stocking accounted for up to 50% in the STRUCTURE analysis of Kollsvatn fish and was less pronounced in the other downstream populations of Litlosvatn ( c. 28%), Kvennsjøen ( c. 17%) and Øvre Krokavatn ( c. 13%). Since significant downstream migration of stocked fish affecting native populations occurs, nature management efforts must respond accordingly.     

Identification of two GH18 chitinase family genes and their use as targets for detection of the crayfish-plague oomycete Aphanomyces astaci

Background  

The oomycete Aphanomyces astaci is regarded as the causative agent of crayfish plague and represents an evident hazard for European crayfish species. Native crayfish populations infected with this pathogen suffer up to 100% mortality. The existence of multiple transmission paths necessitates the development of a reliable, robust and efficient test to detect the pathogen. Currently, A. astaci is diagnosed by a PCR-based assay that suffers from cross-reactivity to other species. We developed an alternative closed-tube assay for A. astaci, which achieves robustness through simultaneous amplification of multiple functionally constrained genes.     

Stocked trout have minimal effects on littoral invertebrate assemblages of productive fish‐bearing lakes: a whole‐lake BACI study

1. Rainbow Trout (Oncorhynchus mykiss [Walbaum]) is commonly stocked as a sport fish throughout the world but can have serious negative effects on native species, especially in headwater systems. Productive fish‐bearing lakes represent a frequently stocked yet infrequently studied system, and effects of trout in these systems may differ from those in headwater lakes. 2. We used a Before‐After Control‐Impact (BACI) design to determine how stocked trout affected assemblage‐level and taxon‐level biomass, abundance and average length of littoral invertebrates in a stocked lake relative to three unstocked control lakes in the boreal foothills of Alberta, Canada. Lakes were studied 1 year before and for 2 years after stocking. Because characteristics of productive fish‐bearing lakes should buffer impacts of introduced fish, we predicted that trout would not affect assemblage‐level structure of littoral invertebrates but might reduce the abundance or average length of large‐bodied taxa frequently consumed by trout. 3. Relative to the unstocked control lakes, biomass, but not abundance, of the littoral invertebrate assemblage was affected indirectly by trout through increases of some taxa after trout stocking. At the individual taxon‐level, trout stocking did not affect most (23 of the 27) taxa, with four taxa increasing in abundance or biomass after stocking. Only one taxon, Chironomidae, showed evidence of size‐selective predation by trout, being consumed frequently by trout and decreasing significantly in average length after stocking. 4. Our results contrast with the strong negative effects of trout stocking on invertebrate assemblages commonly reported from headwater lakes. A combination of factors, including large and robust native populations of forage fish, the generalised diet of trout, overwinter aeration, relatively high productivity and dense macrophyte beds, likely works in concert to reduce potentially negative effects of stocked trout in these systems. As such, productive, fish‐bearing lakes may represent a suitable system for trout stocking, especially where native sport fish populations are lacking.     

The crayfish plague pathogen can infect freshwater‐inhabiting crabs

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The North American crayfish <Emphasis Type="Italic">Procambarus clarkii</Emphasis> is the carrier of the oomycete <Emphasis Type="Italic">Aphanomyces astaci</Emphasis> in Italy

Aphanomyces astaci (Saprolegniales, Oomycetes) is classified among the 100 world’s worst invasive species. This species is endemic to North America and has been introduced into Europe by imports of their hosts, the North American crayfish species. As a consequence, extensive mass mortalities involved several populations of the European crayfish. Here, we checked its occurrence in four Italian populations of Procambarus clarkii, the most widespread alien crayfish in Italy. Digital image analyses and image processing techniques were used to select micro-melanized areas in the subabdominal cuticle of 2–10 crayfish per population. All the selected areas tested positive for A. astaci ITS nrDNA specific primers; moreover, the obtained sequences clearly corresponded to A. astaci, thus revealing that P. clarkii is an active carrier of this oomycete in Italy. Decisions are to be urgently made to control the spread of both P. clarkii and A. astaci for the conservation of the indigenous crayfish biodiversity.     

Stocking impact and temporal stability of genetic composition in a brackish northern pike population (Esox lucius L.), assessed using microsatellite DNA analysis of historical and contemporary samples

During the last decade, brackish northern pike populations in Denmark have been subject to stocking programmes, using nonindigenous pike from freshwater lakes, in order to compensate for drastic population declines. The present study was designed to investigate the genetic impact of stocking freshwater pike into a brackish pike population in Stege Nor, Denmark. We analysed polymorphism at eight microsatellite loci in samples representing the indigenous Stege Nor population prior to stocking (ie from 1956 to 1957), along with a sample of the contemporary Stege Nor population and samples from the three populations used for stocking. Despite large numbers of stocked fry, the results from both individual and population level admixture analyses demonstrated extremely poor performance and <1% introgression of stocked freshwater pike into the brackish pike population. Furthermore, pairwise F(ST) estimates between samples demonstrated close genetic relationship among temporal samples from Stege Nor, indicating temporal stability over the last 45 years. We also estimated the effective population size (N(e)) of pike in Stege Nor and applied a test for recent population bottlenecks. The harmonic mean of N(e) was relatively high (>250), but there were indications of bottlenecks in all samples and populations. We ascribe this finding to historical rather than recent bottlenecks, possibly dating back to founder events associated with postglacial recolonisation.     

Aphanomyces astaci mtDNA: insights into the pathogen’s differentiation and its genetic diversity from other closely related oomycetes

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.     

AFLP-PCR and RAPD-PCR evidences of the transmission of the pathogen Aphanomyces astaci (Oomycetes) to wild populations of European crayfish from the invasive crayfish species, Procambarus clarkii

Aphanomyces astaci (Oomycetes) is responsible for the crayfish plague disease. This species is endemic of North America and five genotypes have been described using RAPD-PCR. The red swamp crayfish, Procambarus clarkii, is one of the most widely spread North American species and invasive in the world. However, no outbreaks on its specific genotype, i.e., genotype D, have ever been described in nature. We investigated three major series of crayfish plague outbreaks in indigenous crayfish populations of Austropotamobius pallipes, located in the areas of influence of P. clarkii. All samples collected tested positive for A. astaci using a rnDNA ITS-PCR test. We also performed an AFLP-PCR analysis on 19 isolates, and found that all isolates belong to genotype D. These isolates exhibited similar properties, i.e., adaptation to warm temperatures. We demonstrate, for the first time, the transmission of A. astaci genotype D to indigenous European populations of crayfish, and confirm that the properties of adaptation to warm water temperatures seem to be a specific character of genotype D. The results of this work emphasize once more the need of controlling invasive species and its trade, since they can carry harmful pathogens with specific adaptations or increased virulence in new environments.     

The impact of stocking on the genetic integrity of Arctic charr (Salvelinus) populations from the Alpine region

There is a long tradition of artificially stocking lakes and rivers with fish in the hope to increase the quality and amount of fish that can be harvested. The animals used for stocking often originate in distant hatcheries or hatcheries that breed fish from remote regions. This stocking practice could have effects on the genetic integrity of resident populations. We have analysed here a case of the influence of stocking on Alpine populations of the Arctic charr (usually included into Salvelinus alpinus, but revised to Salvelinus umbla Kottelat 1997 ) within a unique geographical and historical setting. The Königssee in the Bavarian Alps (Berchtesgaden) was heavily stocked several times during the last century. However, a sample of the ancestral Königssee population still exists in the Grünsee, which lies close to Königssee, but 1000 m higher. To trace the influence of stocking in Königssee we have analysed more than 300 individuals from 10 lake populations, including the source populations of the fish used for stocking. From these we have sequenced a part of the mitochodrial control region and have typed them at six microsatellite loci. The differential distribution of haplotypes, as well as assignment tests, show that the influence of stocking on the genetic integrity of the Königssee population has been negligible. However, our data reveal that in another lake included in our study (Starnberger See), the ancestral population was apparently replaced completely by the populations used for stocking. The major difference between the lakes is the relative preservation of ecological integrity. Königssee was ecologically stable in the past, whereas Starnberger See was heavily polluted at one point, with charr approaching extinction. Interestingly, in a lake neighbouring Starnberger See, the Ammersee, which was also subject to strong pollution but not stocked, the ancestral population has recovered. Our data suggest that the practice of artificial stocking should be reconsidered, or at least monitored for effectiveness.     

Stocking may increase mitochondrial DNA diversity but fails to halt the decline of endangered Atlantic salmon populations

Over the last 50 years, Spanish Atlantic salmon (Salmo salar) populations have been in decline. In order to bolster these populations, rivers were stocked with fish of northern European origin during the period 1974–1996, probably also introducing the furunculosis-inducing pathogen, Aeromonas salmonicida. Here we assess the relative importance of processes influencing mitochondrial (mt)DNA variability in these populations from 1948 to 2002. Genetic material collected over this period from four rivers in northern Spain (Cantabria) was used to detect variability at the mtDNA ND1 gene. Before stocking, a single haplotype was found at high frequency (0.980). Following stocking, haplotype diversity (h) increased in all rivers (mean h before stocking was 0.041, and 0.245 afterwards). These increases were due principally to the dramatic increase in frequency of a previously very low frequency haplotype, reported at higher frequencies in northern European populations proximate to those used to stock Cantabrian rivers. Genetic structuring increased after stocking: among-river differentiation was low before stocking (1950s/1960s Φ _ST = –0.00296–0.00284), increasing considerably at the height of stocking (1980s Φ _ST = 0.18932) and decreasing post-stocking (1990s/2002 Φ _ST = 0.04934–0.03852). Gene flow from stocked fish therefore seems to have had a substantial role in increasing mtDNA variability. Additionally, we found significant differentiation between individuals that had probably died from infectious disease and apparently healthy, angled fish, suggesting a possible role for pathogen-driven selection of mtDNA variation. Our results suggest that stocking with non-native fish may increase genetic diversity in the short term, but may not reverse population declines.     

The diversity of oomycetes on crayfish: Morphological vs. molecular identification of cultures obtained while isolating the crayfish plague pathogen

Numerous oomycetes colonise the crayfish cuticle, the best known being the crayfish plague pathogen Aphanomyces astaci. Although other oomycetes associated with crayfish complicate the isolation and molecular detection of A. astaci, their diversity is little known. To improve this knowledge, we analysed 95 oomycete isolates obtained during attempts to isolate A. astaci from crayfish presumably infected by this pathogen. We characterized the isolates morphologically and by sequencing of the nuclear internal transcribed spacer (ITS) region. We identified 13 taxa by molecular analysis. Ten of them were assigned to five genera; the remaining three were affiliated with the order Saprolegniales but could not be reliably assigned to any genus. Morphological identification to species level was only possible for 15 % of isolates; all corresponded to Saprolegnia ferax, which was confirmed by ITS sequencing. The most frequently isolated species were S. ferax and Saprolegnia australis. Only seven isolates of A. astaci were obtained, all from one disease outbreak. We show that oomycete cultures obtained as by-products of parasite isolation are valuable for oomycete diversity studies, but morphological identification may uncover only a fraction of their diversity. Further, we show that crayfish may be frequently associated with potentially serious parasites of other organisms.     

Reprint of: The diversity of oomycetes on crayfish: Morphological vs. molecular identification of cultures obtained while isolating the crayfish plague pathogen

Numerous oomycetes colonise the crayfish cuticle, the best known being the crayfish plague pathogen Aphanomyces astaci. Although other oomycetes associated with crayfish complicate the isolation and molecular detection of A. astaci, their diversity is little known. To improve this knowledge, we analysed 95 oomycete isolates obtained during attempts to isolate A. astaci from crayfish presumably infected by this pathogen. We characterized the isolates morphologically and by sequencing of the nuclear internal transcribed spacer (ITS) region. We identified 13 taxa by molecular analysis. Ten of them were assigned to five genera; the remaining three were affiliated with the order Saprolegniales but could not be reliably assigned to any genus. Morphological identification to species level was only possible for 15 % of isolates; all corresponded to Saprolegnia ferax, which was confirmed by ITS sequencing. The most frequently isolated species were S. ferax and Saprolegnia australis. Only seven isolates of A. astaci were obtained, all from one disease outbreak. We show that oomycete cultures obtained as by-products of parasite isolation are valuable for oomycete diversity studies, but morphological identification may uncover only a fraction of their diversity. Further, we show that crayfish may be frequently associated with potentially serious parasites of other organisms.     

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.     

Comparison of peptidase activities in some fungi pathogenic to arthropods

Peptidases, highly specific toward several synthetic chromogenic peptides, were found in the mycelia of four arthropod pathogenic fungi: Aphanomyces astaci, Beauveria bassiana, Metarrhizium anisopliae, and Paecilomyces farinosus. A. astaci peptidases had high hydrolyzing activities toward most of the peptides, especially those with arginine in the P1 position, while those of B. bassiana and P. farinosus readily hydrolyzed peptides with valine and arginine, as well as proline and tyrosine in the P2 and P1 positions, respectively. The hydrolyzing capacities of M. anisopliae peptidases were similar to A. astaci, but showed lower specific activities. Casein or azocoll was only hydrolyzed by A. astaci peptidases. B. bassiana and M. anisopliae had a very low hydrolyzing capacity toward casein and could not degrade azocoll. P. farinosus had no hydrolyzing activity toward casein or azocoll. Only peptidases from the crayfish pathogen A. astaci could degrade the crayfish cuticle. The peptidase preparations of A. astaci and B. bassiana hydrolyzing MeO-Suc-Arg-Pro-Tyr-pNA or Bz-Phe-Val-Arg-pNA were of the serine type. The possible importance of peptidase activity of arthropod pathogenic fungi in the infection process is discussed.