A tentative new species Aphanomyces fennicus sp. nov. interferes with molecular diagnostic methods for crayfish plague

Several isolates of an unknown oomycete resembling the genus Aphanomyces were obtained into laboratory culture from samples of noble crayfish (Astacus astacus) in 2016–2017. The crayfish were kept in cages in connection with a study on an eventually persistent crayfish plague infection in a small Finnish lake, following an acute episode of the disease in 2010. Despite the close resemblance of the isolates to the causative agent of crayfish plague, Aphanomyces astaci, and the positive results obtained in OIE recommended A. astaci‐specific ITS‐based conventional PCR and qPCR molecular assays, the isolates can be distinguished from A. astaci by morphological features concerning hyphal structure and chlamydospore formation, as well as using the randomly amplified polymorphic DNA‐polymerase chain reaction (RAPD‐PCR) method, microsatellite‐based genotyping, the pathogenicity test and phylogenetic analysis based on ITS sequencing. The name Aphanomyces fennicus sp. novum is proposed for this close relative of A. astaci. The detection of this tentative novel species giving false‐positive results in existing diagnostic assays for the crayfish plague highlights the importance of careful interpretation of the results from molecular methods, especially concerning crayfish with low‐level infections, excluding the possibility to verify the results from clinical or sequencing data.     

The decline of endangered stone crayfish (Austropotamobius torrentium) in southern Germany is related to the spread of invasive alien species and land‐use change

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Distribution of freshwater crayfish in the British Isles,with particular reference to crayfish plague,alien introductions and water quality

1. The distribution of native and alien crayfish in the British Isles, based on records from 1970–1991, is figured at the 10 km square level and that for Britain is tabulated regionally. 2. Since 1981 crayfish plague has been recorded from six catchments in Britain and one in Ireland, and suspected from an additional four catchments in Britain; 56.2% of native crayfish sites in Britain occur in catchments from which plague has been confirmed. 3. Since the mid-1970s approximately 300 signal crayfish implants have been made in Britain. Only 117 “successful” implants of signals are known to the authors; 39 crayfish farmers were registered in 1986 and 68 by 1990. In 1989 declared production was approximately 7 tonnes. 4. Some mixed populations of natives and disease-free signals exist. Evidence for exclusion of natives is being monitored. 5. Signal crayfish are found in the flowing waters of seven catchments; 58.1% of native crayfish sites are located in catchments which support farmed and wild populations of signals. Some rapidly-expanding Turkish crayfish populations are known, particularly in the Thames catchment. 6. At least 87% of native crayfish records are from good quality waters. 7. Native populations from 88.6% of sites recorded in Britain since 1970 have either been eliminated, or are directly threatened, by crayfish plague infection, and/or habitat invasion by signals, and/or pollution. 8. The native crayfish was added to the list of species protected under the Wildlife and Countryside Act in 1986. 9. Recommendations to assist in the conservation of the native crayfish include a ban on future imports of alien crayfish, a reassessment of records, the setting up of “no-go” areas, the protection of isolated sites, and a restocking programme. 10. There are many restrictedt, healthy populations of native crayfish in areas which could be protected by designating them as Sites of Special Scientific Interest (SSSIs).     

The harvest of the freshwater crayfish <Emphasis Type="Italic">Astacus leptodactylus</Emphasis> Eschscholtz in Turkey: harvest history,impact of crayfish plague,and present distribution of harvested populations

This review focuses on the present distribution of populations of the crayfish Astacus leptodactylus that are harvested in Turkey. It also examines the history of this harvest and the impact that crayfish plague has had on them. Crayfish plague, caused by the fungus-like organism, Aphanomyces astaci Schikora, 1906, is a severe parasite of freshwater crayfish and has caused a lot of damage to A. leptodactylus populations in Turkey since 1984. Turkey was the largest provider of A. leptodactylus to Western Europe from 1970 (or possibly earlier) until 1986. For example, the peak production was reached in the early 1980s, with over 5,000 tonnes being exported in 1984. On the other hand, as a result of the crayfish plague the harvest of A. leptodactylus was reduced severely in most populations in Turkey after 1985. The harvest was only 320 tonnes in 1991. After the occurrence of crayfish plague in Turkey, in order to increase crayfish production uncontrolled A. leptodactylus stockings have been carried out in many waterbodies throughout Turkey. These introductions have caused an increase in the number of A. leptodactylus populations, but exploitation of A. leptodactylus is still under the pressure of the plague, although there has been a steady increase in crayfish production in recent years. The harvest increased to 2,317 tonnes in 2004. Fortunately, among those populations affected by crayfish plague, large amounts of A. leptodactylus can still be harvested from three lakes, ?znik (Bursa), E?irdir (Isparta) and Çivril (Denizli). Thus, it seems that A. leptodactylus has a degree of resistance to crayfish plague. It is therefore interesting to investigate the resistance of A. leptodactylus caught from these populations to crayfish plague.     

Procambarus clarkii Girard as a vector for the crayfish plague fungus, Aphanomyces astaci Schikora

Abstract. Procambarus clarkii Girard, a native freshwater crayfish species of Lousiana, USA, was found to harbour the crayfish plague fungus, Aphanomyces astaci Schikora, in its cuticle as a benign infection. Under certain conditions, P. clarkii dies as a result of this parasite, and the A. astaci infection then becomes acute and can be transmitted to Astacus astacus (L.).
Therefore, it is now shown that at least three different species of North American crayfish ( Pacifastacus leniusculus Dana, Orconectes limosus Raff, and Procambarus clarkii Girard) can carry the crayfish plague fungus, A. astaci , can transfer the disease to other crayfish species, and under certain circumstances can die of its own infection.     

The present situation of freshwater crayfish, Astacus leptodactylus (Eschscholtz, 1823) in Turkey

The only native crayfish species in Turkey, Astacus leptodactylus, is widely distributed in lakes and ponds in many parts of the country. Its distribution area was considerably expanded in Turkey after 1985 because of its commercial importance and declined catches from traditional good fisheries. Although consumption of A. leptodactylus has always been very low in Turkey, it was exported to western Europe until 1986. Due to over-fishing, pollution and a disease (crayfish plague), the total production decreased dramatically to 200 from 5000 tonnes annually. In recent years (1991–1998), there has been a gradual increase in the production of crayfish in Turkey from 320 to 1500 tonnes, but plague is still present.     

Aphanomyces astaci presence in Japan: a threat to the endemic and endangered crayfish species Cambaroides japonicus?

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Prospects for management strategies of invasive crayfish populations with an emphasis on biological control

• 1. The white‐clawed crayfish, Austropotamobius pallipes (Lereboullet), is the only freshwater crayfish indigenous to Great Britain and Ireland. It has a widespread, though declining distribution in England and parts of Wales but does not occur naturally in Scotland.
• 2. The North American signal crayfish, Pacifastacus leniusculus (Dana), is not native to Europe and was introduced to Britain in the 1970s. The signal crayfish out‐competes the native white‐clawed crayfish as it is larger and more aggressive. It is also responsible for the introduction and spread of crayfish plague, which has devastated white‐clawed crayfish populations in Europe.
• 3. Signal crayfish populations are causing significant changes to the equilibrium of native flora and fauna through increased grazing and predation pressures; they also contribute to habitat degradation through burrowing.
• 4. Manual removal of crayfish using traps and pond trials with biocides have met with moderate success in reducing crayfish numbers and containing populations. However, with new populations of signal crayfish being reported each year within the UK, there is now an urgent need to develop a strategy with which to eradicate or contain their spread.
• 5. Signal crayfish have populated many habitat types in the UK, each of which may require a different control strategy; hence no single strategy or universal solution is likely to be attainable.
• 6. Signal crayfish are susceptible to various biocides and microbial pathogens but significant scientific research will be required to develop safe biological control methods and integrated pest management (IPM) strategies to control these invasive organisms. Copyright © 2009 John Wiley & Sons, Ltd.

     

Variation in the hyphal growth rate and the virulence of two genotypes of the crayfish plague organism Aphanomyces astaci

Crayfish plague, a devastating disease of freshwater crayfish, is caused by an oomycete organism, Aphanomyces astaci. Currently five genotypes of A. astaci are known, but variable features between the strains or genotypes have not been studied extensively. This study analysed 28 isolates of the As genotype and 25 isolates of the Ps1 genotype and reveals that the radial growth rate is significantly (P < 0.001) different between these two genotypes, although highly variable inside the genotype As. Two Ps1 genotype isolates and two As genotype isolates with different radial growth rates were tested in an infection trial. Clear differences were detected in the development of mortality in the test groups. The representatives of the Ps1 genotype caused total mortality within a short time span. The As genotype isolates were much less virulent. The slow‐growing As isolate showed higher virulence than the As isolate with a high growth capacity. Although slow growth could be one survival strategy of the pathogen, several other mechanisms are involved in the pathogenicity and warrant further studies.     

Hosts and transmission of the crayfish plague pathogen Aphanomyces astaci: a review

The crayfish plague pathogen, Aphanomyces astaci Schikora, has become one of the most well‐studied pathogens of invertebrates. Since its introduction to Europe in the mid‐19th century, it has caused mass crayfish mortalities, resulting in drastic declines of local populations. In contrast, North American crayfish usually serve as latent carriers, although they may also be negatively affected by A. astaci infections under some circumstances. Recent research benefiting from molecular tools has improved our knowledge about various aspects of A. astaci biology. In this review, we summarize these advances, particularly with respect to the host range and transmission. We highlight several aspects that have recently received particular attention, in particular newly confirmed or suspected A. astaci hosts, latent A. astaci infections in populations of European crayfish, and the relationship between A. astaci genotype groups and host taxa.     

Freshwater crayfish farming technology in the 1990s: a European and global perspective

This paper aims to describe the state of crayfish farming technology in the USA, Australia and Europe, and to discuss some of the prerequisites for this industry. Data from Europe are partly based on replies from a questionnaire sent out to scientists in all European countries. For other parts of the world, the crayfish literature has been reviewed and data from the August 2000 meeting of the International Association of Astacology are also included. Issues addressed in this review are cultivated species, production and productivity figures, production technique with regard to enclosures, reproduction and feed items, disease problems, predators, pond vegetation and water quality. Fewer than a dozen crayfish species are cultivated. The most attractive ones for culture and stocking in natural waters have been transferred to more than one continent. Pond rearing techniques predominate in all countries, and the technology required to achieve the spawning and rearing of juveniles is relatively simple. Pieces of fish, carrots and potatoes are frequent supplementary feed items; plants, cereals, pieces of meat, zooplankton and pellets are also common. Diseases are not usually a major concern, except in Europe where the American plague fungus, Aphanomyces astaci, has eradicated many European crayfish populations. Predators identified as common include insects and amphibians, as well as fishes, birds and mammals. Many water macrophytes are common in crayfish farms. These may either serve a useful function or cause problems for the crayfish farmer. Water temperature is the crucial factor for crayfish production. Water parameters such as pH and certain inorganic ion concentrations may also be of concern. Acidic waters that occur in some areas are generally detrimental to crayfish. The total yield from crayfish production from farming and fishery is in the order of 120 000–150 000 tonnes, more than four times the quantity given by FAO statistics. The largest crayfish producer is the Peoples’ Republic of China, followed by the USA (70 000 and 50 000 tonnes in 1999, respectively). Of the quantity produced in the USA in 1999, about 35 000 tonnes was farmed. The yield in Europe was about 4500 tonnes in 1994, and of this quantity only 160 tonnes came from aquaculture. There are no official statistics for crayfish fishery production in Australia, but about 400 tonnes came from aquaculture in 1999.     

Oral vaccination trials with crayfish, Procambarus clarkii, to induce resistance to the white spot syndrome virus

The potential of oral vaccination against white spot syndrome virus (WSSV) in crayfish Procambarus clarkii was investigated. The protective effect of binary ethylenimine (BEI)-inactivated WSSV was tested by oral vaccination, followed by an oral challenge with WSSV. The crayfish fed with feed pellets coated with BEI-inactivated WSSV showed a resistance to WSSV on the seventh day post vaccination (dpv). The relative percentage survival values were 60%, 70% and 75% for the vaccinated once, twice and thrice with inactivated WSSV. Following an intramuscular injection experiment, no mortality was recorded in the inactivated WSSV group and the negative control at 17 days post challenge. The cumulative mortalities in the heated WSSV group and WSSV group were 100%. Shrimp that survived the WSSV challenge on the seventh day after cessation of oral vaccination were positive for the presence of WSSV by a polymerase chain reaction assay specific for WSSV. This result indicated that inactivated WSSV could protect crayfish against WSSV by oral delivery.     

Enigmatic hotspot of crayfish diversity at risk: Invasive potential of non‐indigenous crayfish if introduced to New Guinea

1. The large island of New Guinea has a rich indigenous astacofauna represented by numerous parastacids from the genus Cherax. The western half of the island is part of Indonesian territory.
2. Indonesia is known to be the main exporter of ornamental crayfish globally, and certain New Guinean species are exploited as ornamentals within the international pet trade. Moreover, one non‐indigenous species has been previously recorded being cultured in Java, Indonesia. This species, the North American Procambarus clarkii, is a vector of crayfish plague, the disease that is lethal to most parastacids. This population has already tested positive for the disease.
3. As the transport of non‐indigenous crayfish within the Indonesian territory is not restricted, their introduction to New Guinea can be expected. The Indonesian market was therefore surveyed for ornamental crayfish and their environmental suitability evaluated, as represented by temperature during the drought and rainy seasons in New Guinea.
4. Four North American and one Australian species were found advertised for sale. One of them, P. clarkii, was assessed as the most damaging species, followed by other North American species. A total ban on the culture and transport of the highest risk crayfish species in Indonesia and Papua New Guinea is recommended.

     

Low genetic variability of the white‐clawed crayfish in the Iberian Peninsula: its origin and management implications

• 1. A study on the genetic variability of the white‐clawed crayfish was carried out based on mitochondrial cytochrome oxidase subunit I gene sequences. The sequences applied were more informative regarding white‐clawed crayfish genetic variability than others previously used.
• 2. Two haplotypes were found to exist in the Iberian Peninsula. The haplotypes exhibit a strong geographic subdivision (Φ_ST=0.83). One of the Iberian haplotype s was similar to north Italian haplotypes and the second differed in only one mutation. This pattern of genetic variability contrasts with those found in glacial refugial areas of France, Italy and the Balkan Peninsula.
• 3. Two hypotheses on the origin of the white‐clawed crayfish in the Iberian Peninsula are discussed: (i) one based on an anthropogenic origin, and (ii) a second based on a successive number of postglacial ancient and recent bottlenecks, i.e. the disjunction between Iberian and Italian populations of white‐clawed crayfish species is due to competition between A. italicus and A. pallipes, in addition to the impact of crayfish plague and human translocations.
• 4. New references for the white‐clawed crayfish in the Iberian Peninsula were found in medieval and Arabic texts. The results show that this species has been thriving in this peninsula since ancient periods and that its indigenous status should not be questioned.
• 5. Conservation action and plans should consider the low genetic diversity as a limitation for farm‐raising specimens more adapted and resistant to changing environments and diseases.

Copyright © 2007 John Wiley & Sons, Ltd.     

Colonization history and human translocations explain the population genetic structure of the noble crayfish (Astacus astacus) in Fennoscandia: Implications for the management of a critically endangered species

1. The noble crayfish (Astacus astacus) is an endangered freshwater species in Europe. The main threat is from lethal crayfish plague, caused by the oomycete Aphanomyces astaci that has been spread over Europe by introduced North American crayfish species, acting as chronic carriers of the disease.
2. Most of the remaining noble crayfish populations are found in the Baltic Sea area, and there is an urgent need to implement conservation actions to slow down or halt the extinction rate in this region. However, limited knowledge about the genetic structure of populations in this area has so far precluded the development of conservation strategies that take genetic aspects into consideration.
3. Key objectives of this large-scale genetic study, covering 77 locations mainly from northern Europe, were to describe the contemporary population genetic structure of the noble crayfish in the Fennoscandian peninsula (Sweden, Norway, and Finland), taking postglacial colonization history into account, and to evaluate how human activities such as stocking have affected the genetic structure of the populations.
4. Analyses of 15 microsatellite markers revealed three main genetic clusters corresponding to populations in northern, middle, and southern Fennoscandia, with measures of genetic diversity being markedly higher within populations in the southern cluster. The observed genetic structure probably mirrors two main colonizations of the Baltic Sea basin after the last glaciation period. At the same time, several deviations from this pattern were observed, reflecting past human translocations of noble crayfish.
5. The results are discussed in relation to the conservation and management of this critically endangered species. In particular, we recommend increased efforts to protect the few remaining noble crayfish populations in southern Fennoscandia and the use of genetic information when planning stocking activities, such as reintroductions following local extinctions.

     

Natural history and development of the introduced signal crayfish,Pacifastacus leniusculus,in a small,isolated Finnish lake,from 1968 to 1993

Signal crayfish (Pacifastacus leniusculus) originating from Lake Tahoe, California, were introduced into Karisjärvi, a small (11 ha) lake in central Finland (61° 58'N, 25° 32'E), in 1968 and 1969. Since then, the population has been monitored regularly by trap catches. The stocked signals (3–5 years old) were caught until 1973, by which time they had reached ages of 7–9 years. Catches have increased slowly since the early 1980s, the peak occurring in 1991 at 2.0 individuals per trap per night. The population size was estimated as 60 trappable specimens in 1974, 95 in 1981 and 420 in 1988. The mean density of adult population in suitable biotopes was low, 0.07 specimens per m^–2, or 0.3 per shore metre, in 1988. The slow development of the P. leniusculus population has been attributed to environmental factors, mainly the limited area of good crayfish habitat. More than 90 % of all signal crayfish were caught in one-third of the shore area, i.e. in steeply sloping lake beds suitable for burrowing or rich in shelters such as rocks and submerged trees. The signals avoided flat, soft bottoms. Fifty per cent of the female P. leniusculus matured at 90 mm TL (smallest 64 mm), i.e. half of the females entered the breeding population in the autumn of their fourth year. The mean size of newly hatched (stage 2) juveniles was 9.7 mm TL and of one-summer olds 30.3 mm. The largest specimen trapped measured 159 mm. The signal crayfish imported into Finland and stocked were infected with crayfish plague (Aphanomyces astaci), but no mortality has been recorded. Two Branchiobdellidae (Annelida, oligochaeta) epibiont species new to Finland were imported from North America with P. leniusculus. The continuous occurrence of these commensals in the signal crayfish population indicates that they have adapted to Finnish conditions.     

Experimental evaluation of the potential for crayfish plague transmission through the digestive system of warm-blooded predators

The crayfish plague pathogen (Aphanomyces astaci) can be transmitted through the digestive system of fish, but its dispersal through mammalian and bird digestive tracts has been considered unlikely, and direct experimental evidence remains scarce. We present a small-scale transmission experiment with European otter and American mink fed with infected crayfish, and experiments testing survival of cultures of five A. astaci strains at temperatures corresponding to those inside mammal and bird bodies. The pathogen was neither isolated from predator excrements nor transmitted to susceptible crayfish exposed to excrements. In agar-based artificial media, it occasionally survived for 15 min at 40.5°C and for 45 min at 37.5°C, but not so when incubated at those temperatures for 45 min and 75 min, respectively. The five tested strains differed in resistance to high temperatures, two (of genotype groups E and D) being more susceptible than other three (of groups A, B and D). Their survival to some extent varied when exposed to the same temperature after several weeks or months, suggesting that some yet-unknown factors may influence A. astaci resistance to temperature stress. Overall, we support the notion that passage through the digestive tract of warm-blooded predators makes A. astaci transmission unlikely.     

Genetic structure of Spanish white‐clawed crayfish (Austropotamobius pallipes) populations as determined by RAPD analysis: reasons for optimism

• 1. Spanish populations of the white‐clawed crayfish have declined sharply over the last three decades. Although Austropotamobius pallipes was once widely distributed and very abundant in most of the limestone basins of the country, outbreaks of crayfish plague since 1978 have reduced its populations, and now only some 500–600 small populations are left.
• 2. Consequently, the species now enjoys protection under national legislation. Management decisions regarding the conservation of a threatened species require an understanding of the genetic structure of its populations.
• 3. Using random amplified polymorphic DNA (RAPD) fingerprinting the genetic variability of 11 populations of A. pallipes was assessed over the species' range in Spain, and their phylogenetic relationships determined.
• 4. Substantial genetic differentiation was detected among the populations tested; no clear relationship was found between patterns of genetic variability and hydrological basin. The RAPD markers showed the degree of genetic variability of these populations to be similar to, and in some cases slightly higher than, that reported in previous studies on other Spanish and European populations of A. pallipes.
• 5. The results offer hope for the recovery of this species in Spain, and provide information that might be useful in the management of crayfish reintroduction programmes.

Copyright © 2007 John Wiley & Sons, Ltd.     

Per os challenge of Litopenaeus vannamei postlarvae and Farfantepenaeus duorarum juveniles with six geographic isolates of white spot syndrome virus

White spot syndrome virus (WSSV) is one of the most important pathogens of penaeid shrimp. It is widely distributed in most Asian countries where penaeid shrimp are cultured, as well as in the Gulf of Mexico and SE USA. The virulence of six geographic isolates of WSSV was compared using Litopenaeus vannamei postlarvae and Farfantepenaeus duorarum juveniles. The six geographic isolates of WSSV originated from China, India, Thailand, Texas, South Carolina, as well as from crayfish maintained at the USA National Zoo. For challenge studies, virus infected tissues were given per os to L. vannamei postlarvae and Fa. duorarum juveniles. Resultant WSSV infections were confirmed by histological examination. The cumulative mortality of L. vannamei postlarvae reached 100% after challenge with each of the six geographic isolates of WSSV. However, the Texas isolate caused mortalities more rapidly than did the other shrimp isolates; the crayfish WSSV isolate was the slowest. In marked contrast, cumulative mortalities of juvenile Fa. duorarum reached only 35–60%, and varied among the geographic isolates of WSSV. Interestingly, in Fa. duorarum, the Texas WSSV isolate was also the most virulent, while the crayfish WSSV was the least virulent. The findings suggest that slight differences in virulence exist among geographic isolates of WSSV, and that susceptibility may vary with species and lifestages of the host.