Spiny-cheek crayfish Orconectes limosus carry a novel genotype of the crayfish plague pathogen Aphanomyces astaci

The oomycete Aphanomyces astaci causes mass mortalities of European crayfish. Different species of North American crayfish, original hosts of this parasite, seem to carry different strains of A. astaci. So far, four distinct genotype groups have been recognised using Random Amplification of Polymorphic DNA (RAPD-PCR). We succeeded in isolating A. astaci from the spiny-cheek crayfish Orconectes limosus, a widespread invader in Europe, and confirmed that this species carries a novel A. astaci genotype. Improving knowledge on the diversity of this parasite may facilitate identification of genotypes in mass mortalities of European crayfish, thus tracing the sources of infection.     

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.     

The effect of fungicides on survival of the crayfish plague fungus,Aphanomyces astaci,Oomycetes, growing on fish scales

An antifungal factor isolated from extracts of Cymbidium (Orchidaceae) roots and infected pseudobulbs was identified as monolinolein.     

Laboratory investigations of the pathogenicity of Aphanomyces astaci for Irish freshwater crayfish

Crayfish plague, caused by the oomycete Aphanomyces astaci, was first diagnosed in Irish stocks of Austropotamobius pallipes from a midlands limestone lake and crayfish farm in October, 1987. Behavioural activity of infected crayfish was monitored and the position at death noted. Crayfish showed no gross, clinical or behavioural signs although they were somewhat lethargic for about 14 days after infection before a rapid deterioration in their condition. Death followed after about 18 and 21 days at 10 ° and 5 °C respectively, with approximately 85% of the animals dying in the open. Spores remained viable and infective in tanks for between 6 and 9 days after death of an infected crayfish at 10 °C. Based on these experiments, suggestions are given for containing the Irish plague outbreak.     

Detection of Aphanomyces astaci in North American crayfish by polymerase chain reaction

We present a PCR based method to detect Aphanomyces astaci in North American crayfish. Primers were designed to specifically amplify parts of the internal transcribed spacer (ITS) regions and the 5.8 rRNA gene of A. astaci. A single round and a semi-nested assay were tested for their sensitivity and specificity. Specificity of the PCR assays was tested against several closely related Aphanomyces species, other Oomycetes and some non-A. astaci DNA that might be found in or on crayfish. The single round assay was fully specific against all DNA tested. In the semi-nested assay, cross-reaction was seen when the equivalent of 40,000 or more genomic units of A. invadans or A. frigidophilus were entered into the PCR reaction. The lower detection limit of both assays lies around 1 genomic unit of A. astaci. Investigation of various parts of the exoskeleton of 3 North American crayfish species revealed that for O. limosus and P. leniusculus the telson and soft abdominal cuticle yielded a positive PCR reaction most frequently. For the third species, Procambarus clarkii, only 1 individual tested positive, so no conclusion as to preferred infestation site(s) could be drawn.     

On the Physiology of Zoospore Production in Aphanomyces astaci

Aphunomgces astaci, Saprolegniaceae, the crayfish plague parasite, grows well in a buffered peptone - glucose - mineral salt medium but does not normally produce spores during growth in this medium. Sporulation is, however, easily induced by transfer to pond water. None of the components of the complete medium inhibited sporulation more than partly when tested solely or in combinations. Neither lack of oxygen, high carbon dioxide concentrations, nor osmotic phenomena could satisfactority exlpain the absence of spore formation in the complete medium. In a peptone - glucose growth medium low in phosphate and metal salts a sporulation inhibiting factor was formed by the mycelium. In this medium both good growth and good spore production could, however, occur simultaneously and spore production was here stimulated by a reduction in the oxygen tension. Liberation of formed spores into the medium was inhibited by mineral salts. It was less sensitive to lack of oxygen, respiratory inhibitors, and a factor formed during growth than was sporangial development. Development and maintenance of spore motility occurred even at very low oxygen tensions but was probably dependent upon an intact respiratory system.     

Re-examination of the prevalence of Aphanomyces astaci in North American crayfish populations in Central Europe by TaqMan MGB real-time PCR

We applied quantitative TaqMan minor groove binder real-time polymerase chain reaction (PCR) on DNA isolates from soft abdominal cuticle of 460 North American crayfish Orconectes limosus and Pacifastacus leniusculus, previously tested for Aphanomyces astaci presence by conventional semi-nested PCR. Both approaches target the internal transcribed spacers of the pathogen nuclear ribosomal DNA, but apply different specific sequence motifs and technologies. The real-time PCR approach seems to provide higher sensitivity; the number of crayfish that tested positive increased from 23 to 32%, and 10 additional crayfish populations were indicated as hosting the disease agent. However, the vast majority of newly recorded positives contained very low agent levels, from 5 to 50 PCR-forming units. An isolate producing a false positive result by the semi-nested PCR (apparently undescribed Aphanomyces related to A. astaci) remained negative using the real-time PCR. The present study shows that previous results based on the semi-nested PCR were not substantially influenced by false positives but might have suffered from some false negatives at low agent levels. Combining alternative methods may therefore provide more reliable conclusions on the pathogen's presence. Further, we found positive correlation between the prevalence of infection carriers in American crayfish populations and the average amounts of A. astaci DNA detected in infected local crayfish individuals.     

Differectial Induction of Zoospore Encystment and Germination in Aphanomyces astaci, Oomycetes

Genmination and encystment of zoosspores of Aphanomyces astaci, the very serious pathogen of European freshwater crayfish, Astacus astacus, were studied in ritro. Encystment of motile zoospores was achieved using mild heating, stirring, or addition of alkali metal chlorides or mannitol. The physical treatments were inhibitory to the subsequent germination, while encytment could be achieved with potassium chloride, indicating that the two processes are differently induced and can be studied separately. The effect of temperature on these processes was also somewhat different. Some ionic substances drastically influenced the final shape of the cyst. but without noticeably influencing the subsequent germination. Germination was induced by a short exposure to the stimulatory substances. A synergistic effect between ionic and non-ionic compounds was found. The receptivity of the zosspores to the triggering substances was low in newly formed spores but increased during the first 3 h of motility. The initiation of germination is suggested to be parltly to be partly due to an osmotic effects on the zoospore. The relevance of the experimental results to in vivo conditions in discuessd.     

Detection and quantification of the crayfish plague agent in natural waters: direct monitoring approach for aquatic environments

Aphanomyces astaci, a specialised parasite of North American freshwater crayfish, is the disease agent of crayfish plague that is lethal to European freshwater crayfish. The life cycle of A. astaci has been inferred from experimental laboratory studies, but less is known about its natural sustainability and ecology. To address such questions, tools for monitoring of A. astaci directly in aquatic environments are needed. Here, we present an approach for detecting and quantifying A. astaci directly from water samples using species-specific TaqMan minor groove binder real-time PCR. Samples of a 10-fold dilution series from approximately 10(4) to approximately 1 spore of A. astaci were repeatedly tested, and reliable detection down to 1 spore was demonstrated. Further, to simulate real-life samples from natural water bodies, water samples from lakes of various water qualities were spiked with spores. The results demonstrated that co-extracted humic acids inhibit detection significantly. However, use of bovine serum albumin or the TaqMan Environmental Master Mix largely removes this problem. The practical application of the approach was successfully demonstrated on real-life water samples from crayfish farms in Finland hosting infected North American signal crayfish Pacifastacus leniusculus. Direct monitoring of A. astaci from aquatic environments may find application in the management of wild noble crayfish Astacus astacus stocks, improved aquaculture practices and more targeted conservation actions. The approach will further facilitate studies of A. astaci spore dynamics during plague outbreaks and in carrier crayfish populations, which will broaden our knowledge of the biology of this devastating crayfish pathogen.     

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.     

Properties of Extracellular Enzymes from Aphanomyces astaci and Their Relevance in the Penetration Process of Crayfish Cuticle

In culture filtrates from the crayfish plague parasite, Aphanomyces astaci, protease and a low level of hyaluronidase activity were found. The hyaluronidase activity was highest at pH 6.5 or above and at about 23°C. The protease activity had a broad pH-optimum, between pH 7 and at least pH 10, and was partially denatured at 30°C. However, when incubated for 30 min with the substrate, casein, the activity increased logarithmically up to about 35–40°C and had an apparent optimum at 45–50°C. The proteases from the parasitic as well as from two less proteolytic, saprophytic Aphanomyces species were predominantly constitutive and were excreted mainly by the older mycelia. Proteases from the parasite and a saprophyte did not reach full activity until 10–30 min after substrate addition. No lipase activity was found in the case of the mycelium of the parasitic species. However, esterase was apparently present inside germinating zoospores. The native enzymes of A. astaci could degrade freeze-dried soft cuticle from crayfish. The relevance of the different enzymes of A. astaci for the penetration process within the cuticle of crayfish is discussed.