Open Access
Issue
Knowl. Manag. Aquat. Ecosyst.
Number 419, 2018
Article Number 10
Number of page(s) 4
DOI https://doi.org/10.1051/kmae/2018001
Published online 16 February 2018

© H. Kokko et al., Published by EDP Sciences 2018

Licence Creative Commons
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Short communication

The narrow-clawed crayfish, Astacus leptodactylus, is the only native freshwater crayfish species in Turkey. In addition to its natural distribution, it has also been widely introduced into lakes, reservoirs and rivers in many parts of the country because of its economic importance and restoration of the crayfish stocks previously devastated by Aphanomyces astaci infections (Harlıoğlu, 2008). The narrow-clawed crayfish has been reported either to have some resistance against A. astaci (Unestam, 1969) or being very susceptible to A. astaci infection (Schikora, 1906; Alderman et al., 1987).

The Turkish narrow clawed crayfish populations got infected with A. astaci in early 1980's (Rahe and Soylu, 1989; Timur, 1990; Alderman, 1996; Kokko et al., 2012; Svoboda et al., 2012) with a devastating effect on the crayfisheries and crayfish export (Harlıoğlu, 2004; Harlıoğlu and Harlıoğlu, 2006; Aydın et al., 2012; Kokko et al., 2012). Later, it was discovered, that some of the collapsed narrow-clawed crayfish populations recovered (Harlıoğlu, 2008; Güner and Harlıoğlu, 2010). However, recent reports indicated, that some populations were chronically infected with A. astaci (Kokko et al., 2012; Svoboda et al., 2012, 2014). Furthermore, it has been reported that both freshwater crayfish and freshwater crabs are A. astaci carriers in Turkish waters (Svoboda et al., 2014).

Initially, some of the Turkish A. leptodactylus populations collapsed and never recovered (e.g., Harlıoğlu, 2008), an indication of limited resistance against A. astaci infection. On the other hand, some Turkish A. leptodactylus populations chronically infected with A. astaci are productive, such as those in Lake İznik (Bursa) and Lake Hirfanlı Dam (Kırşehir) (Kokko et al., 2012). The observed latent infections may indicate past and contemporary partial resistance in the host, as has been argued by Unestam (1969) or even virulence evolution of A. astaci (Jussila et al., 2015).

The aim of this study was to investigate the distribution of A. astaci in wild narrow-clawed crayfish populations in Turkey. We selected narrow-clawed crayfish from eight populations, which were either showing potential gross symptoms of A. astaci infection, i.e., melanisation, necrosis or erosion of carapace, or were reported to be A. astaci infected (Tab. 1). Crayfish (n = 35) were bought from commercial crayfishermen from each location. They were caught during summer 2011 and 2012 from Lake Hirfanlı Dam, Kırşehir (39°11′ N 33°33′ E), Lake Iznik, Bursa (40°43′ N 29°52′ E), Lake Eğirdir, Isparta (38°00′ N 30°53′ E), Lake Çıldır, Ardahan (41°03′ N 43°14′ E), Lake Porsuk Dam, Kütahya (39°38′ N 30°11′ E), Lake Sarımsaklı Dam, Kayseri (38°53′ N 35°44′ E), Lake Yenikarpuzlu Dam, Edirne (40°49′ N 26°19′ E) and Lake Keban Dam, Elazığ (38°38′ N 39°28′ E) (Fig. 1). The A. astaci prevalence in the first two populations was studied earlier (Kokko et al., 2012; Svoboda et al., 2012), while the last six populations' infection status has not been reported earlier.

Samples for the qPCR analyses of A. astaci were taken by cutting a piece of melanised cuticle or, in case of no melanised spots, a uropod from each crayfish. The samples were stored in absolute ethanol (Merck) by the Firat University staff in Turkey. The dissection tools were disinfected after every sampled crayfish tissue. The preserved samples were stored at −21 °C and then shipped by airmail to the University of Eastern Finland, Kuopio campus, for further analyses. In addition, a previous sample set from year 2011 (Kokko et al., 2012) with two locations (Tab. 1) were included into further analyses.

Before the DNA extractions, the tissue samples were rinsed in sterile water to remove the ethanol. DNA extractions were conducted with E.Z.N.A Insect DNA isolation kit (Omega Bio-Tek) following manufacturer's instructions. The quantity and quality of the extracted DNA was measured with a NanoDrop-spectrophotometer (Thermo Fisher Scientific). For A. astaci prevalence screening, a quantitative TaqMan® minor groove binder (MGB; Applied Biosystems) real-time PCR assay (qPCR) developed by Vrålstad et al. (2009) was adjusted to LightCycler 480 II qPCR machine (Roche) and the sample volume was adjusted to 10 µL similarly as in Kokko et al. (2012). TaqMan® Environmental Master Mix (Applied Biosystems) was used for the qPCR reactions (Strand et al., 2011) with 2 µL of 1 ×  and 10 ×  diluted DNA. A calibrated standard curve (Vrålstad et al., 2009) was applied to determine the PFU values and agent levels for sampled crayfish tissues. Agent level A0 and A1 (˂5 PFU's) indicated negative samples, A2 (5–50 PFU's) very low level infection, A3 (50–1000 PFU's) low level infection, A4 (103–104 PFU's) moderate infection and agent level A5 (105–106 PFU's) a high level infection.

Three different PCR amplicons of each sample showing agent level A3 or higher in qPCR were sequenced to further characterize the infections. The chitinase gene was amplified and sequenced for selected samples according to Makkonen et al. (2012b) and mitochondrial ribosomal small and large subunits rnnS, and rnnL, according to Makkonen et al. (2018). The obtained PCR amplicons were purified with QiaQuick PCR purification kit (Qiagen) following manufacturer's instructions and sequenced in GATC Biotech (Cologne, Germany). Sequences were submitted to GenBank with access numbers MG596357-MG596378.

Aphanomyces astaci infection was detected in seven of the eight narrow-clawed crayfish populations (Tab. 1), and, in six of those cases, also the haplotype of the disease agent was characterized with at least one sequence. In four cases, i.e., Lake Iznik, Lake Porsuk Dam, Lake Çıldır and Lake Sarımsaklı Dam, haplogroup A (As-genotype) was causing the latent infection. In two cases, i.e., Lake Yenikarpuzlu Dam and Lake Hirfanlı Dam, the infection was caused by the haplogroup B (PsI-genotype) of A. astaci. In Lake Hirfanlı Dam case, the haplogroup B infection was detected from both years' 2011 and 2012 samplings, indicating a latent infection caused by the haplogroup B in this site. In addition to A. astaci, single rnnS sequence similar to Saprolegnia ferax (99.1%) was detected from Lake Porsuk Dam and a sequence similar to Pythium insidiosum (96.5%) from Lake Yenikarpuzlu Dam. In addition, rnnS amplicon showing 97.4% similarity to A. astaci was detected from Lake Eğirdir sample that was showing very low agent level (A2) in qPCR. Lake Keban Dam narrow-clawed crayfish population did not show infection gross symptoms, and also the qPCR detected only trace levels of A. astaci DNA, i.e., agent level A1 considered as negative (Tab. 1).

The detected infections can mainly be defined as latent A. astaci infections (Jussila et al., 2014), as the studied populations have been reported to be productive (Kokko et al., 2012; Svoboda et al., 2012) and show low level of infection gross symptoms (Tab. 1), even though they have variably collapsed since A. astaci spread into Anatolian peninsula (Köksal, 1988; Rahe and Soylu, 1989; Timur, 1990). The latent A. astaci infections might be caused by A. astaci strains of low virulence, while one of the A. astaci strains causing latent infection in Slovenian stone crayfish (Austropotamobius torrentium) has been shown to be virulent against noble crayfish (Jussila et al., 2017). The haplogroups of the observed A. astaci infections were determined according to Makkonen et al. (2018) using mitochondrial rnnS ad rnnL sequences. The grouping of A. astaci haplogroups is similar to the genotypes (Makkonen et al., 2018), and the detected haplogroup B (genotype B/PsI) strains carried by the signal crayfish (Pacifastacus leniusculus) are in most cases considered highly virulent (Aydın et al., 2012, 2014; Makkonen et al., 2012a2014) and no latent infections in European crayfish species caused by this haplogroup have been previously observed.

Our study shows a geographically widespread distribution of A. astaci among Turkish narrow-clawed crayfish populations. As those populations are still productive and have thus been commercially exploited, it seems that the Turkish narrow-clawed crayfish could have considerably high resistance against both haplogroup A and B haplogroup A. astaci infections, as has already been indicated (Unestam, 1969; Kokko et al., 2012; Svoboda et al., 2012, 2014, 2017), whilst there are also studies reporting significant susceptibility (Schikora, 1906; Alderman et al., 1987). This host-parasite co-evolution and adaptation possibility opens avenues for further studies on the relationship between A. astaci, other co-infecting pathogens and its native European crayfish hosts (e.g., Edgerton et al., 2004).

Regardless of the implications of a possibly elevated resistance of the Turkish narrow-clawed crayfish, the conservation of the native European crayfish requires swift actions against the spreading of different strains of A. astaci among native European crayfish populations. On the other hand, our finding together with the recent latent crayfish plague observations from native European crayfish populations (Jussila et al., 2011; Viljamaa-Dirks et al., 2011; Kokko et al., 2012; Svoboda et al., 2012; Kušar et al., 2013; Jussila et al., 2017) might be indicating a brighter future for the European crayfish under the pressures from the crayfish plague disease.

Table 1

Aphanomyces astaci in Turkish narrow-clawed crayfish (A. leptodactylus) populations from eight lakes. Agent level A0 and A1 (>5 PFU's) indicates negative samples, A2 (5–50 PFU's) very low level infection, A3 (50–1000 PFU's) low level infection, agent level A4 (103–104 PFU's) moderate infection and agent level A5 (105–106 PFU's) a high level infection. Abbreviations As = As genotype A. astaci, commensurate to mitochondrial haplogroup A; PsI = PsI-genotype A. astaci, commensurate to mitochondrial haplogroup B.

thumbnail Fig. 1

Locations of the sampled Turkish narrow-clawed crayfish (Astacus leptodactylus) populations: Lake Çıldır, Lake Eğirdir, Lake Hirfanlı Dam, Lake İznik Dam, Lake Keban Dam, Lake Porsuk Dam, Lake Sarımsaklı Dam and Lake Yenikarpuzlu Dam. Lake Hirfanlı Dam and Lake İznik Dam were analyzed earlier with qPCR by Kokko et al. (2012). Red stars indicate the proximate locations of the sampled water bodies.

 Acknowledgements

The manuscript preparations were supported by LIFE + CrayMate project (LIFE12 INF/FI/233).

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Cite this article as: Kokko H, Harlioglu MM, Aydin H, Makkonen J, Gökmen G, Aksu Ö, Jussila J. 2018. Observations of crayfish plague infections in commercially important narrow-clawed crayfish populations in Turkey. Knowl. Manag. Aquat. Ecosyst., 419, 10.

All Tables

Table 1

Aphanomyces astaci in Turkish narrow-clawed crayfish (A. leptodactylus) populations from eight lakes. Agent level A0 and A1 (>5 PFU's) indicates negative samples, A2 (5–50 PFU's) very low level infection, A3 (50–1000 PFU's) low level infection, agent level A4 (103–104 PFU's) moderate infection and agent level A5 (105–106 PFU's) a high level infection. Abbreviations As = As genotype A. astaci, commensurate to mitochondrial haplogroup A; PsI = PsI-genotype A. astaci, commensurate to mitochondrial haplogroup B.

All Figures

thumbnail Fig. 1

Locations of the sampled Turkish narrow-clawed crayfish (Astacus leptodactylus) populations: Lake Çıldır, Lake Eğirdir, Lake Hirfanlı Dam, Lake İznik Dam, Lake Keban Dam, Lake Porsuk Dam, Lake Sarımsaklı Dam and Lake Yenikarpuzlu Dam. Lake Hirfanlı Dam and Lake İznik Dam were analyzed earlier with qPCR by Kokko et al. (2012). Red stars indicate the proximate locations of the sampled water bodies.

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