Prevalence, molecular identification and genotyping of the crayfish plague pathogen, Aphanomyces astaci in major narrow-clawed crayfish (Pontastacus leptodactylus Eschscholtz, 1823) populations from Türkiye

Freshwater crayfish are represented by two species (Pontastacus leptodactylus and Austropotamobius torrentium) in Türkiye. The narrow-clawed or Turkish crayfish, P. leptodactylus, is the major species and is distributed in most lakes and rivers across Türkiye. However, the stone crayfish Austropotamobius torrentium is distributed only in the Thrace Region (1). The Turkish crayfish is an economically valuable crayfish species indigenous to Türkiye, which was caught commercially from 1970 until 1986 in significant quantities and exported to Europe. By the year 1985, the total crayfish catch had decreased dramatically because of crayfish plague (4, 23). This plague was first reported in crayfish from Lake Çivril in the Denizli Province of Türkiye in 1984, and the sole aetiological agent was identified in 1985 by Baran and Soylu (4) as being Aphanomyces astaci. This pathogen is a fungus-like microorganism belonging to the Oomycota class (Schikora 1906). Timur (27) reported crayfish plague disease from Lake Çivril in Denizli province, Lake Eğirdir in the Isparta province and Lake Karataş in the Burdur province in 1985. In subsequent years, the disease has continued to spread and affect Turkish crayfish populations (13, 14, 26).

Crayfish plague is considered the most important crayfish disease globally. This lethal disease has caused the collapse of the populations of the European, noble or broad-fingered crayfish, Astacus astacus, and the white-clawed or Atlantic stream crayfish, Austropotamobius pallipes in Europe. Although not proven to be caused by crayfish plague, the first mass crayfish mortalities in Europe were reported in northern Italy in the 1860s and in France in 1879; the crayfish plague agent subsequently spread throughout Europe (7). Outbreaks were reported in Spain in the 1960s, and the UK, Türkiye, Greece and Norway in the 1980s (2). Besides the Turkish crayfish (P. leptodactylus), all European crayfish species (Astacus astacus, Austropotamobius torrentium and Austropotamobius pallipes) are also highly susceptible to crayfish plague (5). Infection causes melanisation and tissue erosion in the crayfish cuticle, and in some cases white tufts of oomycete may be visible on the body. Infected crayfish may exhibit unusual behaviour a few days before death, such as appearing during daylight hours, wandering out of the water, and walking strangely because of paralysis (3).

Diagnosis of A. astaci infection is performed by gross observation, wet mount microscopy, histopathology and oomycete culture. Polymerase chain reaction–based identification methods have been commonly used in recent decades because they are rapid and give accurate results. The first PCR identification method for A. astaci was developed by Oidtmann et al. (21), and following this Vrålstad et al. (29) developed a real-time PCR technique for diagnosis of the pathogen. This has been used by many to diagnose A. astaci infection of crayfish populations in different countries: in Astacus astacus from Finland and Czechia (11, 15), Austropotamobius pallipes from Italy (5) and non-indigenous signal crayfish (Pacifastacus leniusculus), spiny-cheek crayfish (Orconectes limosus), calico crayfish (O. immunis) and red swamp crayfish (Procambarus clarkii) from France (7).

There have been several studies performed by conventional (4, 23, 27) and molecular identification methods (13, 14, 26) on crayfish plague in Turkish crayfish populations. However, these studies were conducted at a limited number of locations, and there is currently no information about the prevalence of crayfish plague disease throughout Türkiye. There is also no information on the prevalence of individual A. astaci strains.

In this study, a PCR was used to identify the crayfish plague pathogen (A. astaci) in tissue samples taken from crayfish captured at 41 different locations throughout Türkiye, and the prevalence of A. astaci was determined. For seven of the locations, microsatellite analysis was carried out to determine the genotypes of the A. astaci strains.

Crayfish plague is undoubtedly the most significant factor jeopardising European crayfish populations. Rapid diagnostic methods for this disease with high sensitivity can help inform crayfish management and conservation efforts. In this study we investigated A. astaci from the tissue of infected crayfish by a PCR-based molecular detection method. The single-round PCR with 40 cycles was found to have a detection threshold for A. astaci genomic DNA of 500 fg. This quantity is greater than the 100 fg that Oidtmann et al. (20, 21) reported. Our minimum detection limit, however, is lower than the 1 pg DNA concentration that Phadee et al. (22) reported as the minimum detectable concentration for A. piscicida. This study also supports the use of both abdominal cuticle and telson tissue to detect A. astaci, which aligns well with the findings of Oidtmann et al. (20).

Although we aimed to sample 20 individuals per locality, for various reasons sample sizes differed in some locations, as can be seen in Table 2. Symptomatic crayfish were identified at all except one of the 41 locations and A. astaci was detected in crayfish from 34 of them, confirming the widespread distribution of the disease and the pathogen. We were not able to detect the presence of A. astaci in the crayfish captured from the Altınyazı (n = 20), Karacakılavuz (n = 20), Porsuk (n = 20) or Yenice reservoirs (n = 15) or Lakes Bayramşah (n = 5), Gölmarmara (n = 20) or Manyas (n = 24). However, in all locations except Lake Bayramşah (n = 5) individuals with crayfish plague symptoms were observed. This observation is consistent with that of Schrimpf et al. (25), who demonstrated that it is virtually impossible to prove the absence of the pathogen with such small sample sizes. In contrast to the PCR results in this study, but consistently with the physical examination of the crayfish in this study, Rahe and Soylu (23) reported crayfish plague in Lakes Gölmarmara and Manyas, and recently Kokko et al. (13) provided evidence of the presence of A. astaci in the crayfish population inhabiting Porsuk reservoir.

It is possible that the number of diseased crayfish was underrepresented in this study because of the sampling method; this method also reduced the probability of detecting A. astaci. Given that uncoordinated limb movements, paralysis, muscle weakness, loss of extremities and abnormal behaviour are among the symptoms of crayfish plague (4, 23, 27), and since the crayfish capture for each sample was carried out using fyke nets with bait, it is probable that the crayfish which were suffering these symptoms were not capable of competing for food or had limited mobility, and were thus less likely to be caught. In addition to this, several other factors (i.e. temporal variations and/or detectability) as discussed by Filipova et al. (7) could also affect the detection probability of the pathogen. Therefore, these results do not mean that the seven localities where 0% prevalence resulted are truly free of crayfish plague.

Of the 41 locations reported in this study on the prevalence of A. astaci in Turkish narrow-clawed crayfish populations, 6 were previously studied by Svoboda et al. (26) and Kokko et al. (13, 14); however, the infection status of 35 populations was reported for the first time in this study (Table 2). The test results and calculated prevalence values in PCR-positive populations are low compared to those of other studies. This may be due to the lower diagnostic capability of conventional PCRs compared to real-time PCRs and the random selection of individuals used in the study. However, Svoboda et al. (26) randomly selected healthy and symptomatic samples from 32 individuals in Lake Eğirdir and were able to detect pathogens in 4 of them (12.5%) with real-time and conventional PCR analysis. On the other hand, Kokko et al. (14) reported high prevalence rates (95% and 100%) in Lake Iznik and the Hirfanlı reservoir in symptomatic and asymptomatic individuals from two populations. Kokko et al. (13) reported a relatively lower prevalence of the disease in six populations examined in their study of seven populations from Türkiye. Among these populations, 100% prevalence in the Hirfanlı reservoir (n = 5), 80% in Lake Egirdir (n = 5), 60% in Lake Çıldır (n = 5), 100% in the Porsuk reservoir, (n = 5), 80% in the Sarımsaklı reservoir (n = 5) and 50% in the Yenikarpuzlu reservoir (n = 4) was calculated, while no infected individuals were identified in the Keban reservoir (n = 6). Kuzbikova et al. (15) analysed 28 populations of two invasive Pacifastacus leniusculus and Orconectes limosus by conventional PCR in Czechia. The pathogen was found in 17 of the 28 populations examined. In O. limosus populations, prevalences of positive reactions ranging from 0% to 100% were reported, while in Pacifasticus leniusculus, only 1 of the 124 individuals examined was positive for the pathogen. In another study, Maguire et al. (17) reported similar prevalence rates in Croatia. They reported higher prevalence of infection in samples tested from three native species (Astacus astacus, Austropotamobius pallipes and Austropotamobius torrentium) than in P. leptodactylus samples, at 68%, 38% and 40%, respectively, against 27%. The same researchers reported infection prevalence in samples from two exotic species as 58% in O. limosus and 25% in Pacifastacus leniusculus, but could not detect infected individuals in a third exotic species, the redclaw crayfish (Cherax quadricarinatus).

Among the crayfish populations that tested positive for A. astaci infection, the highest prevalence percentages were in the populations inhabiting Lakes Taşkısığı (68.2%) Işıklı (60%), Eğirdir (42.9%) and Beyşehir (40%) and the Hanoğlu reservoir (40%). In Türkiye, the crayfish plague first appeared in Lake Işıklı in 1984, then in Lake Eğirdir in 1985, and in several others in the following years (4, 23). Since then, various studies have reported the crayfish plague from different locations. Earlier studies postulated that Turkish crayfish populations are recovering, based on the hypothesis that the causative agent has long been persistent in Turkish water bodies and despite the high prevalence of the disease, crayfish populations have been reproductive and have managed to co-exist with the pathogen (13, 14, 26). It has been proposed that this is because P. leptodactylus has developed resistance and/or the virulence of the pathogen has decreased (13, 14, 26). This study supports this notion in some of its findings. In Lake Eğirdir, for example, A. astaci was detected at a high level compared to other locations; from this together with the previous observations in Lake Eğirdir (4, 13 26), it can be inferred that A. astaci has been persistent in the lake for over 30 years. However, despite the high prevalence observed in the present study (42.9%), the recent catch amount of 891 tonnes in 2020 (28) suggests that the crayfish population is indeed recovering.

In this study, the strains of A. astaci present in the tissue samples were determined for seven of the populations sampled. Genotype B was present at six of the locations (Lakes Eğirdir, Karababa, Karaidemir and Mamasin and the Hanoğlu and Atikhisar reservoirs), while genotype A was present only at Lake Suğla. Previously, five different genotype groups (A, B, C, D and E) of A. astaci isolates from various populations of crayfish were identified using the random amplified polymorphic PCR technique (6, 9, 16). Among these groups, genotype A is known as the As genotype and was isolated from Astacus astacus and P. leptodactylus (9). Genotypes B, C, D and E were first noted in North American crayfish species. Genotype B is known as the PsI genotype and genotype C as PsII and they were detected in Pacifastacus leniusculus from California and Canada (9). Genotype D is known as Pc and was reported from Procambarus clarkii (6), and finally, genotype E is known as Or and was identified in O. limosus (16). Huang et al. (9) first classified an isolate from Türkiye as genotype A (AsI). In another study, Kokko et al. (13) reported both genotype A (Lake Çıldır and the Porsuk and Sarimsakli reservoirs) and genotype B (the Yenikarpuzlu and Hirfanlı reservoirs) in some crayfish populations different to the populations used in this study. Similarly, in a study conducted by Maguire et al. (17) on the Croatian P. leptodactylus population, A. astaci isolates were reported as both genotypes A and B. In this study, most of the A. astaci samples obtained from Turkish crayfish populations were genotype B. This genotype has been reported in the Pacifastacus leniusculus population (6, 9). Rezinciuc et al. (24) reported all A. astaci isolates as genotype B in Austropotamobius pallipes populations from the Iberian Peninsula of Spain. Grandjean et al. (8) reported genotype B in Pacifastacus leniusculus, Astacus astacus and Austropotamobius pallipes from France, Finland, Norway and Czechia, whereas genotype A was found in different French and Czech populations of noble crayfish.

Although crayfish plague is a highly destructive phenomenon in susceptible crayfish species, some A. astaci strains appear to show signs of lower virulence (11, 19). Therefore, knowing the genotype of the plague pathogen can be of great benefit for the management of crayfish stocks. Some studies on virulence variation of A. astaci genotypes in different outbreaks documented that genotypes and isolates varied in virulence (10, 12, 18, 19). During the spread of this agent in Europe, some genotypes have reportedly adapted to their hosts (12), and two genotypes, A (As) and B (Psl) detected in some water bodies in Türkiye in this study, are currently causing crayfish plague with variable mortality rates in Europe (12, 19). There is a lack of information on the virulence of these genotypes in controlled laboratory conditions on crayfish from P. leptodactylus populations from Türkiye. However, it was reported that genotype A caused very variable mortality among native European crayfish in laboratory trials (19) as well as in wild populations (11, 14). Makkonen et al. (18) reported that genotype B virulence was higher in Finnish Astacus astacus populations, whereas that of genotype As was more variable and lower in groups infected with these isolates. Furthermore, the researchers reported signs of increased resistance to some of the tested strains belonging to the As genotype of A. astaci in different noble crayfish populations. Therefore, it is of great importance to determine the virulence of the A. astaci strains causing crayfish plague in Türkiye.

Crayfish plague poses a significant threat to crayfish populations, necessitating the development of rapid, highly sensitive diagnostic methods. This study has yielded significant insights into the prevalence, detection sensitivity and genetic diversity of A. astaci in Turkish crayfish populations. The impressive sensitivity limit of 500 fg/μL which resulted underscores the effectiveness of PCR assays, particularly the 40-cycle PCR, in detecting A. astaci DNA. Tissue examinations revealed a variable prevalence of crayfish plague across different locations, with the highest (68.2%) observed in Lake Taşkısığı. Additionally, microsatellite analysis unveiled the presence of distinct genetic strains (genotypes A and B) of A. astaci in various locations, genotype A being exclusively identified in Lake Suğla and genotype B predominating in the remaining sampled locations. These findings emphasise the need for continuous monitoring and conservation measures in Turkish crayfish populations to protect them from the detrimental effects of crayfish plague. The high sensitivity of PCR assays and the identification of genetic strains offer valuable tools for disease management and population conservation strategies. An in-depth understanding of crayfish plague dynamics is crucial for preserving the biodiversity of Turkish crayfish and their ecosystems. The needs for future research and action have become evident, as expanding the geographical scope of sampling, especially those water bodies previously unexamined, and concomitant analysis would provide a more comprehensive picture of the distribution of crayfish plague in Türkiye.