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.     

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.     

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.     

Molecular identification of a bronopol tolerant strain of Saprolegnia australis causing egg and fry mortality in farmed brown trout, Salmo trutta

Some species of the genus Saprolegnia, such as Saprolegnia diclina and Saprolegnia ferax are responsible for devastating infections on salmonid eggs. Members of this group cause saprolegniasis, a disease resulting in considerable economic losses in aquaculture. Although both S. diclina and S. ferax have received much attention, the role of other Saprolegnia species in infecting fish eggs is less known. For this purpose, we have investigated the aetiology of chronic egg mortality events occurring in farmed brown trout, Salmo trutta. A total of 48 isolates were obtained from eggs with signs of infection as well as from water samples. A molecular analysis based on nrDNA internal transcribed spacer (ITS) operational taxonomic units indicated that the majority of the isolates correspond to Saprolegnia australis. All isolates of S. australis exhibited the same random amplified polymorphic DNA (RAPD) band patterns suggesting that a single strain is implicated in egg infections. The isolates followed Koch postulates using trout eggs and fry. Under standard concentrations of bronopol commonly used in farms, these isolates could grow, but not sporulate. However, both growth and sporulation were recovered when treatment was removed. This study shows that S. australis can infect and kill salmon eggs, and helps in defining oomycetes core pathogens.     

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.     

DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer

Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes.     

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.     

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.     

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.     

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.     

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.     

Reprint of: Saprolegnia strains isolated from river insects and amphipods are broad spectrum pathogens

Saprolegnia species are destructive pathogens to many aquatic organisms and are found in most parts of the world. Reports based on phylogenetic analysis suggest that Saprolegnia strains isolated from aquatic animals such as crustaceans and frogs are close to Saprolegnia strains isolated from infected fish or fish eggs and vice versa. However, it has often been assumed that host specificity occurs for each individual isolate or strain. Here we demonstrate that Saprolegnia spp. can have multiple hosts and are thus capable of infecting different aquatic organisms. Saprolegnia delica, Saprolegnia hypogyna, and 2 strains of Saprolegnia diclina were isolated from aquatic insects and amphipods while S. delica, Saprolegnia ferax, Pythium pachycaule, and a Pythium sp. were isolated from the water of a medium to fast flowing river. The ITS region of the rRNA gene was sequenced for all isolates. In challenge experiments, all four isolates from insects were found to be highly pathogenic to eggs of Atlantic salmon (Salmo salar) and embryos of the African clawed frog (Xenopus laevis). We found that Saprolegnia spp. isolated from salmon eggs were also able to successfully establish infection in nymphs of stonefly (Perla bipunctata) and embryos of X. laevis). These results suggest that Saprolegnia spp. are capable of infecting multiple hosts, which may give them an advantage during seasonal variation in their natural environments.     

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.     

Non-indigenous signal crayfish <Emphasis Type="Italic">Pacifastacus leniusculus</Emphasis> are now common in Danish streams: preliminary status for national distribution and protective actions

North American signal crayfish (Pacifastacus leniusculus) are invasive in Europe and pose a serious threat to indigenous European crayfish such as the noble crayfish (Astacus astacus). This is mainly because signal crayfish is the carrier of crayfish plague agent, Aphanomyces astaci, which freshwater crayfish from all other continents are highly susceptible to. Until recently, the distribution of signal crayfish in Danish streams has been considered local and restricted to a small geographical area. Here we present data demonstrating that signal crayfish are now widespread in Denmark, including the largest Danish river, River Gudenå. For one of the rivers where co-existing signal crayfish and indigenous noble crayfish were documented, sensitive molecular tests could not detect the crayfish plague agent Aphanomyces astaci in either species. Hence, it seems that not all signal crayfish are chronic carriers of the disease. For the remaining freshwater systems with the introduced signal crayfish, the infection status is presently unknown. Large areas of the freshwater systems in Denmark also remain unexplored with respect to presence/absence of signal crayfish and noble crayfish. Nevertheless, our preliminary data that covers about 14% of the Danish rivers, strongly suggests that signal crayfish should be considered as a common invader that poses an increased threat to the biota in Danish streams, in particular for the indigenous noble crayfish.     

Population collapses in introduced non-indigenous crayfish

Invasive species often have instable population dynamics and are known to collapse or oscillate heavily after passing through the initial lag/growth phases. Long-term data-series documenting these fluctuations are however rare. We use long-term (starting in the early 1960s), semi-quantitative data on the invasive signal crayfish (Pacifastacus leniusculus), capturing its population development after introduction in 44 Swedish lakes. In total 18 (41 %) of these populations had experienced a collapse. A stepwise discriminant function analysis including 20 different ecological or physicochemical characteristics identified three variables explaining collapses in the following order: stocking year, population age and mean air temperature. Populations stocked in the 1980s were more likely to collapse than populations stocked in the 1970s. Lakes with collapses were located in areas with 0.4 °C higher yearly mean air temperatures than the still viable populations. Collapses also depended on the time phase of the population and started to occur 12 years after stocking and were most frequent in the interval 16–20 years after stocking and after 11–15 years duration of the established phase with harvestable densities. An analysis of prevalence and pathogen load of Aphanomyces astaci was conducted in eight of the studied populations. A. astaci was present in all populations but neither the level of prevalence nor the pathogen load in infested specimens differed significantly between lakes with collapses and lakes without. Our results highlight the potential sensitivity and instability of introduced crayfish. The importance of density-dependence and temperature suggest that both climate variability and/or fisheries can influence these processes.     

Phoma glomerata, a potential new threat to Italian inland waters

Non-indigenous crayfish species, carrier of the oomycete Aphanomyces astaci, are the most important threats to European crayfish species. However, other ectoparasite species have been reported to be the pathogen of aquatic organisms. As little is known about other potential pathogens carried by the red swamp crayfish Procambarus clarkii, a total of 86 individuals of this species was collected in Lake Trasimeno from June 2007 to 2008. Phoma glomerata, never recorded in Italy, colonised more than 65% of the captured crayfish. No significant differences in prevalence were recorded between sexes, among life stages and between soft- and hard-shelled crayfish. This Coelomycetes is the causal agent of many diseases in plants, animals and humans. These results may contribute to a better understanding of the implications of epibiontic microfungi on the invasive crayfish P. clarkii, for both ecological and sanitary purposes.     

Purification and properties of a protease inhibitor from crayfish hemolymph

A protease inhibitor from the hemolymph of crayfish, Astacus astacus, has been purified by differential centrifugation, acid precipitation and preparative isoelectric focusing. The inhibitor was apparent homogenous in SDS-electrophoresis and had a molecular weight of 23,000. pI was determined to be 4.7 by isoelectric focusing. No inhibitory activity was lost when the inhibitor was incubated in a pH range of 1–11.5. The purified inhibitor was heat stable. Urea (6 m) had no effect upon the inhibitor. The inhibitor was active against subtilisin and a partly purified protease from the fungus Aphanomyces astaci. Pronase was slightly inhibited whereas trypsin, chymotrypsin, papain, Arthrobacter protease, and extracellular proteases from the fungi Aphanomyces stellatus and A. laevis were unaffected. The importance of protease inhibitors in pathogenesis between the parasitic fungus, A. astaci, and its crayfish host, A. astacus is discussed.     

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.