The pathogen Aphanomyces astaci Schikora is responsible for the decline of the native crayfish species of Europe, and their current endangered status. This pathogenic species is native to North America and only colonizes aquatic decapods. The North American crayfish species have a high resistance to this pathogen, while species from other regions are highly susceptible. However, recent field and laboratory observations indicate that there might exist some populations with resistance against this disease. The objective of this study was to test the susceptibility of 8 selected native European crayfish populations of Austropotamobius pallipes Lereboullet from the Pyrenees.
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The pathogen Aphanomyces astaci Schikora is responsible for the decline of the native crayfish species of Europe, and their current endangered status. This pathogenic species is native to North America and only colonizes aquatic decapods. The North American crayfish species have a high resistance to this pathogen, while species from other regions are highly susceptible.
However, recent field and laboratory observations indicate that there might exist some populations with resistance against this disease. The objective of this study was to test the susceptibility of 8 selected native European crayfish populations of Austropotamobius pallipes Lereboullet from the Pyrenees.
We challenged them against the genome sequenced strain AP03 of A. Histological analyses revealed a high immune reaction in tissues examined, i. These results represent the first observation of a native European crayfish population showing high resistance towards the most virulent genotype of this pathogen, i.
The identification of this population is of key importance for the management of these endangered species, and represents a crucial step forward towards the elucidation of the factors involved in the immune reaction against this devastating pathogen. Fungal and fungal-like emerging infectious diseases EIDs are seriously affecting endangered wildlife species and present a major concern for biodiversity conservation. As a result, EIDs are currently attracting an important number of scientific efforts and attention [ 1 — 3 ].
So far, one of the most devastating EIDs is the crayfish plague caused by the organism Aphanomyces astaci Schikora Oomycota. The crayfish plague constitutes a classic example of a disease emergence as consequence of introducing alien species into a new biogeographic region [ 6 ].
This pathogen originates from North America, where it coexists with their natural hosts, i. In North American crayfish, their interaction with A. Thus, the pathogen is usually unable to entirely colonize its host because their immune system can encase the pathogen within the cuticle by increasing its phenoloxidase activity [ 8 ]. This enzyme produces melanin as an end product, which has both fungitoxic and fungistatic activity [ 8 ]. The equilibrium prevents the pathogen from overtaking its host but instead the host becomes chronically infected.
However, in native European crayfish species, the immune system does not respond as efficiently to prevent the spread of A. The hyphae of the pathogen can colonize the host almost without opposition and results in death within a few days [ 10 ]. In this case, no melanization, i. Most of these cases have been reported in the native European species, Astacus astacus , Linnaeus originating from Northern Europe, and these cases seem to be caused by low virulent strains of A.
So far, 5 genotypes of Aphanomyces astaci have been characterized, i. The original hosts of these genotypes are North American crayfish species; however, the original host of the genotype As has not identified found yet [ 14 — 16 ].
The original host of strains corresponding to genotypes PsI and PsII is the crayfish species Pacifastacus leniusculus Dana [ 14 ], while the original hosts of the genotypes Pc and Or are Procambarus clarkii Girard and Orconectes limosus Rafinesque , respectively [ 15 , 16 ]. Isolates corresponding to the genotype As are thought to be responsible for the first outbreaks of the crayfish plague in Europe during the 19th century [ 14 ] as a consequence of deliberated introduction of freshwater crayfish from North America into Europe.
Recent studies indicate that some isolates of this genotype possess low virulence [ 11 ]. The impact of crayfish plague in Europe has been devastating and has decreased not only crayfish populations and their fisheries but also altered the ecology of freshwater ecosystems[ 17 , 18 ]. In Spain, since the introduction in of P. There are no efficient treatments to overcome this disease, and the only efficient measures to control it are, so far, those that aim to prevent the spread of their carriers.
A better understanding of the mechanism of disease resistance represents one of the first steps to designing new strategies to prevent disease development. Increased activation of the innate and adaptive immune defenses by pathogen exposure has recently been described for diverse species, e. Nichols [ 22 , 23 ], and some Australian rabbits have increased their resistance towards hemorrhagic disease virus RHDV by implementing of a whole complex of inter-related defenses [ 24 ].
Recent studies on native European crayfish appear to show that some Finnish wild populations might have an increased resistance towards the less virulent genotype of A. During the last decade we have observed a number of crayfish plague events involving native Pyrenean populations of A.
Interestingly, Pc infected specimens from these populations show melanization and have longer survival [ 21 ] Jokin Larumbe and Joan Montserrat, personal communication. However, there are no comparative studies regarding pathogen resistance in European crayfish populations towards the A.
Lack of dedicated studies precipitated our investigation into a number of potential resistant European populations against A. Therefore, the objective of this study is to test the susceptibility and resistance of geographically isolated native populations of A.
All experimental procedures and animal manipulations, as well as field sampling, were performed according to the EU and Spanish legislation. Furthermore, the crayfish specimens used in this study were provided by Gobierno de Navarra 2 populations from the Western Pyrenees and by Generalitat de Catalunya 6 populations from the Eastern Pyrenees which are the Authorities entitled to collect samples of endangered species, i.
No additional permits were required for the described field or laboratory studies, since the ethics approval in the Spanish law is not required for working with arthropod invertebrates. Moreover, this study was carried out in strict accordance with the recommendations and the protocols stablished in previous studies to minimize suffering, i.
Specimens of the native European crayfish, A. None of the selected populations included in this study cohabit with any North American crayfish species. Some of these populations, i. Populations were obtained from Eastern six and Western two Pyrenees Fig 1B and 1C and Table 1 and only males were used in the experiments. Each population was marked by clipping with scissors the lateral tips of selected dorsal abdomen segments.
Specimens from each population were first kept in separated 0. The specimens were maintained for two weeks to ensure the acclimatization and were fed weekly with potato and monitored daily to remove dead crayfish or excess dirtiness. A Distribution of the eight selected populations of Austropotamobius pallipes from the Spanish Pyrenees.
Five individuals from each population were transferred and pooled into four experimental aquaria 40 specimens each, five specimens from each population maintained under the same conditions as described above. In order to ensure a suitable temperature, oxygen levels and water quality, the aquaria were checked daily before and during the experiment.
Water was replaced weekly to remove excess dirtiness and to avoid cannibalism, dead individuals were removed once they were found. During the acclimatization period some individuals died.
For this reason, the experiments had different number of individuals Table 1. For the challenge experiments, the AP03 strain of A. This strain was isolated from specimens of a population of P. The number of crayfish from each population is listed in Table 1.
A control was also run in a separated aquarium that had 40 crayfish five specimens from each population. No zoospores were added into this aquarium Table 1.
The experiment was followed for days and the aquaria were examined on a daily basis. For further microscopic and molecular analyses, surviving crayfish from replicates 1, 2 and 3, as well as the crayfish from control aquarium were euthanized by exposure to chloroform vapors [ 26 ] after days from the challenge experiments.
The crayfish did not show any melanization before the experiments. All crayfish were checked upon arrival to the laboratory and before being challenged with zoospores. Crayfish were checked daily for disease symptoms and dead crayfish were removed and examined for the presence of melanized areas both macroscopically and microscopically. Light micrographs were captured using a Qimaging Micropublisher 5.
Digital image analysis was performed using the software Syncroscopy-Automontage Microbiology International Inc. After days of the challenge experiments, live crayfish were euthanized [ 26 ] and examined as described above. All the crayfish were tested for the presence of A. Genomic DNA was extracted by using an E. DNA extractions and A. Each sequence strand was assembled and edited using the program Geneious v6. We ran a BLAST search to check the nature of the generated sequences in order to verify the coincidence with the strain used in the zoospore challenge, i.
Cumulative mortality rates were followed over a period days after challenging the crayfish with A. Cumulative mortality rates were calculated as the number of dead crayfish per total crayfish challenged. In order to minimize secondary infections from diseased dead crayfish, these were removed from the aquarium immediately after death and the water was changed every week.
Crayfish specimens tested from all populations challenged with zoospore of A. However, all specimens from the population of La Muga Pop 6 and the control survived for the whole experimental period of days.
One main mortality event occurred between day 7 and 14 after zoospore challenge. In this period, a total of 66 crayfishes died 16 crayfish from replicate 1, 30 crayfishes from replicate 2, and 30 crayfishes from replicate 3.
Subsequent isolated deaths were observed till the end of the experiment 8 from replicate 1 and one individual from replicate 2 Fig 2.
At day after zoospore challenge, three crayfishes from the population of Falgars, two from population of Doneztebe, and all from population of La Muga and the control survived. No crayfish from the control died during the experimental period of days. The control is not represented in the figure.
Macroscopic examination of dead crayfish from all periods and all populations did not reveal the presence of melanized spots on the cuticle. Microscopic observations showed the presence of abundant non-melanized hyphae with round tips and homogenous diameter, ca 10 um, characteristic of A. Macroscopic examinations of specimens from La Muga Pop 6 showed the presence of melanized patches on the joints of the walking legs, carapace, uropods and soft ventral cuticle Fig 3C and 3D.
Microscopically, the subabdominal cuticle of these specimens possessed strongly melanized hyphae characteristic of an A. Different stages of hyphal encapsulation by haemocytes Fig 3F were seen, ranging from weak melanized to completely melanin-covered hyphae Fig 3G and 3H.
Micrographs of susceptible crayfish dying after being challenged with A. Macroscopic signs of infection in crayfish with increased-resistance against A. Micrographs showing the immune response process against A. Surviving crayfish from Falgars Pop5 and Doneztebe populations Pop7 did not show macroscopic signs of melanization. At microscopic level, melanized spots were often seen, and when hyphae were observed, they always appeared partially melanized.
All the crayfish tested negative for the presence of A. Tissues from dead and sacrificed crayfish of the 3 replicates exposed to A. Tissues of the control crayfish always tested negative. Alignment of obtained DNA sequences from positive crayfish revealed that all were identical to the isolate of A.
Crayfish plague (Aphanomyces astaci)
The pathogen Aphanomyces astaci Schikora is responsible for the decline of the native crayfish species of Europe, and their current endangered status. This pathogenic species is native to North America and only colonizes aquatic decapods. The North American crayfish species have a high resistance to this pathogen, while species from other regions are highly susceptible. However, recent field and laboratory observations indicate that there might exist some populations with resistance against this disease.
Research shows that chitinase is expressed at a high level during vegetative growth of the crayfish pathogen Andersson and Cerenius, Recently, Oidtmann et al. Notes Ways of transmission only involves movements of chronic carriers, i. The chances of transmission by other means such as items that have been in contact with contaminated water, fish or birds are very low and can only occur during the short period of survival of the Aphanomyces spores, i. Habitat Description Aphanomyces astaci is an oomycete or water mould, which only parasitises freshwater crayfish Unestam,
Crayfish plague Aphanomyces astaci is a water mold that infects crayfish , most notably the European Astacus which dies within a few weeks of being infected. When experimentally tested, species from Australia , New Guinea and Japan were also found to be susceptible to the infection. Crayfish plague first arrived in Europe in Italy in , either with imported crayfish from North America,  or in ballast water. In , to bolster dwindling stocks of native crayfish, the signal crayfish was introduced to Sweden. This species was studied and named by the German Mycologist Friedrich Schikora — , from a type specimen in Germany in Implantations of the signal crayfish were the reason for the spread of the disease to United Kingdom and Ireland. Transport of signal crayfish, red swamp crayfish and infected native European freshwater crayfish between waters is the main cause for contamination.