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.     

Invasive crayfish and crayfish plague on the move: first detection of the plague agent Aphanomyces astaci in the Romanian Danube

Native European crayfish, such as Astacus leptodactylus, are threatened, among other factors, by the crayfish plague agent Aphanomyces astaci, dispersed by invasive North American crayfish. Two of these invaders, Pacifastacus leniusculus and Orconectes limosus, have extended their distribution in the River Danube catchment; the latter was detected for the first time in Romania in 2008. We monitored, at monthly intervals for over 2 yr, occurrence of native A. leptodactylus and invasive O. limosus at 6 sites on the Romanian Danube and checked for the invasive species in 4 of its tributaries. Between January 2009 and March 2011, the relative abundances of O. limosus steadily increased with time, while the native A. leptodactylus dramatically decreased in abundance. O. limosus expanded downstream at a rate of ca. 15 km yr-1; in August 2011, it was already present in the upper 105 km of the Romanian Danube. An agent-specific real-time PCR analyses demonstrated the presence of A. astaci DNA in at least 32% of the analysed invasive (n = 71) and 41% of the native (n = 49) crayfish coexisting in the Danube. Furthermore, A. astaci was also detected in A. leptodactylus captured about 70 km downstream of the O. limosus invasion front (at the time of sampling). Assuming a steady rate of expansion, O. limosus may invade the sensitive Danube delta area in the mid-2060s, even without long-distance dispersal. The crayfish plague agent, however, may reach the delta substantially earlier, through dispersal downstream among populations of native crayfish.     

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.     

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.     

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.     

Physiological Variation in Strains of Aphanomyces astaci

Physiological variation was found both between and within strains of the crayfish plague fungus, Aphanomyces astaci, in culture. The loss of capacity to produce spores was irreversible while losses of motility and virulence to the crayfish were reversible changes. From the tests with motility it was concluded that mutations of hidden or de novo type readily appear in the phenotype of this fungus which belongs to a taxonomic group that is considered genetically very conservative. The authors gratefully acknowledge the assistance of Mrs. Anita Grandin. This research was supported by grants from the Swedish Natural Science Research Council and the Fishery Board of Sweden.     

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.     

Isolation and properties of β-glucan synthetase from the aquatic fungus, Aphanomyces astaci

A β-glucan synthetase was isolated from a membrane fraction of the crayfish parasitic fungus Aphanomyces astaci Schikora, strain Si. [¹⁴C]-UDP-glucose was incorporated linearly for about 1 h at 30°C into an acid insoluble product. The apparent K_m for UDP-glucose was found to be approximately 4.5 m M and the apparent K_i for UDP, a competitive inhibitor of the reaction, was 1 m M . The acid insoluble product obtained after incubating this glucan synthetase with[₁₄C]-UDP-glucose was partially characterized by glucanase treatment. This product mainly consisted of β-1,3-linked glucosyl units. Synthetase activity was not stimulated by MgCl₂, but cellobiose as well as GTP and EDTA in combination or ATP alone enhanced enzyme activity. A high proportion of the A. astaci synthetase was probably already activated during preparation and not accessible to further stimulation by nucleotide additions as found for glucan synthetase of Saccharomyces cerevisiae and Candida albicans. No synthetase activity or any factors affecting this enzyme was present in the cytosol. An exudate prepared from the cuticle of the crayfish host, did not inhibit glucan synthetase activity in vitro.     

Studies on the Crayfish Plague Fungus Aphanomyces astaci

A mycelial suspension of the crayfish plague fungus, Aphanomyces astaci, was able to produce large numbers of zoospores, when transferred to redistilled water, at 20°C, even after storage for months at 2°C. Spore production was greater in redistilled water than in tap water and heavier under shake conditions than under stationary ones. In buffered redistilled water sporulation occurred between pH 5 and 8 and the optimal range was about pH 5 to 7. Of the tested aliphatic alcohols, aldehydes, and carboxylic acids, the long analogues were more toxic to spore formation than the shorter ones. Ethylenediamine-tetraacetic acid (EDTA) prevented sporulation probably by removing some essential metal (s) with an affinity for EDTA near that of calcium. Calcium protected against the toxic effect of lithium, sodium, and potassium. Magnesium, only tested against lithium, had no such protecting effect. Cu²⁺, Ni²⁺, Zn²⁺, Co²⁺, K⁺ Mn²⁺, NH₄⁺, Li⁺, Na⁺, Ca²⁺, Mg²⁺ was the approximate order among tested cations in their ability to stop the swimming stage of the zoospores, the first mentioned being the most effective ones. Nitrate and acetate were more active in the same respect than sulphate, chloride, phosphate, or bicarbonate. The optimal pH range for swimming seemed to be pH 6–7.5, and the maximal range 4.5–9.0. The zoospores showed no chemotactic response to tested substances. The germination ability was as high in horse blood as in crayfish blood. A spore suspension stored for 2 months at 2°C still contained viable spores.     

Protein and nucleic acid synthesis during germination of the asexual spores of the aquatic fungus, Aphanomyces astaci

Macromolecular synthesis of asexual spores of Aphanomyces astaci Schikora, strain is, was studied during the germination phase. Germination of the spores was completely inhibited by aclinomycin D (20 ug/ml) or cycloheximide (100 ug/ml) as was incorporation of labeled uridine and leucine, respectively. During spore germination protein synthesis was almost linear, whereas incorporation of [3Hl-uridine and [3H1-thymidine showed lag phases. Initial protein synthesis is therefore suggested to take place without concomitant RNA-synthesis in this species. Germlings were not developmentally competent to sporulate before 8 h of germination.     

Chitinase and protease activities in germinating zoospore cysts of a parasitic fungus,Aphanomyces astaci,Oomycetes

In germinating spores of the parasitic fungus, Aphanomyces astaci, chitinase was first demonstrated shortly before the germ-tube began to branch, in contrast to protease which was present in both ungerminated and germinated spores. The time at which chitinase would be required when this fungus penetrates the crayfish cuticle is correlated with that of the in vitro production of chitinase.