Is Aphanomyces astaci Loosing its Stamina: A Latent Crayfish Plague Disease Agent From Lake Venesjärvi, Finland

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Is Aphanomyces astaci Loosing its Stamina: A Latent Crayfish Plague

Disease Agent From Lake Venesjärvi, Finland

Jussila, J

The International Association of Astacology

Tieteelliset aikakauslehtiartikkelit

© 2021 by The Authors

CC BY http://creativecommons.org/licenses/by/4.0/

http://dx.doi.org/10.5869/fc.2021.v26-2.139

https://erepo.uef.fi/handle/123456789/26802

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139 INTRODUCTION

Crayfish plague epidemics, caused by Aphanomyces astaci Schikora, have had detrimental effects on wild native crayfish populations in Europe (Alderman 1996; Souty-Grosset et al. 2006;

Jussila et al. 2015a), until recently. Since the turn of the century, there has been widely debated reports on latent crayfish plague infections among European native crayfish populations (Jussila et al. 2011a; Viljamaa-Dirks et al. 2011), which later have been verified by numerous reports both from wild stock observations and laboratory experiments (Kokko et al. 2012, 2018; Schrimpf et al. 2012; Svoboda et al. 2012, 2017; Kušar et al. 2013; Jussila et al.

2014b; Ungureanu et al. 2020). Now it is obvious that the crayfish plague disease agent, A. astaci, and some populations of the native European crayfish species have obtained a more balanced relationship (e.g., Jussila et al. 2015a). Whether this relationship has long-term effects on the survival of the native European crayfish remains to be seen.

The first reported detection of a latent crayfish plague infection was from Lake Mikitänjärvi (Finland) by Jussila et al. (2011a) with the result being based on qPCR detection without successful isolation of the causative oomycete. It is now obvious that a coevolution of some A. astaci haplogroups and native European crayfish has progressed rather rapidly (Jussila et al. 2014b, 2015a, 2017; Martín-Torrijos et al. 2017) and reached a point where survival of native European crayfish during A. astaci infection is in some cases possible (Kokko et al. 2012, 2018, Svoboda et al. 2012;

Ungureanu et al. 2020; Francesconi et al. 2021). The coevolution has mostly been observed as lowered virulence of A. astaci from haplogroup A, a haplogroup originating from an unknown crayfish host (e.g., Makkonen et al. 2012a). On the other hand, those haplogroups which have their North American host species present in Europe seem to have maintained high virulence (Makkonen et al.

2012b; Jussila et al. 2013, 2014b, 2015a; Svoboda et al. 2017). The coevolution in the case of alien crayfish and A. astaci in Europe has so far resulted in more susceptible alien crayfish stocks, especially

1 Department of Environmental and Biological Sciences, The University of Eastern Finland, P.O. Box 1627, 70211 Kuopio, Suomi-Finland.

Corresponding Author: japo.jussila@uef.fi

2 Natural and Enviromental Sciences, Institute for Environmental Sciences, University of Koblenz-Landau, Campus Landau, Fortstraße 7, 76829 Landau, Germany.

3 LOEWE Centre for Translational Biodiversity Genomics, Senckenberg Biodiversity and Climate Research Centre, Senckenberg Research Institute, Frankfurt, Germany

A B S T R A C T

The crayfish plague disease agent, Aphanomyces astaci, has coevolved with the native European crayfish since its arrival to mainland Europe in the 1860s. There are indications that some of the A. astaci strains are of reduced virulence, while the resistance against A. astaci infection varies among native European crayfish stocks. In Lake Venesjärvi, Finland, a potential case of latent crayfish plague infection was observed in the early 2000s. We have isolated an oomycete from live Astacus astacus originating from Lake Venesjärvi and identified it as A. a staci of haplogroup A by specific qPCR, nuclear ITS and mitochondrial LSU and SSU sequencies, and named the isolate as UEF_VEN5/14. The A. astaci was isolated from a potentially latently infected A. astacus, subsequently identified, genotyped and proven to be of reduced virulence in a separate infection challenge experiment under laboratory conditions. These findings add to speculations of rather rapid coevolution of A. astaci and European native crayfish and support the reports on latent crayfish plague infections among wild native European crayfish.

Copyright © 2021 by The Author(s). Published by the International Association of Astacology.

This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

A R T I C L E I N F O Article History:

Submitted: 24 MAR 2021 Accepted: 17 NOV 2021 Published Online: 31 DEC 2021 Published Print: 31 DEC 2021

Keywords:

A. astacus;

adaptation;

co-evolution;

invertebrate pathogen;

Is Aphanomyces astaci Losing its Stamina: A Latent Crayfish Plague Disease Agent From Lake Venesjärvi, Finland

Japo Jussila,

* Caterina Francesconi,

Kathrin Theissinger,

Harri Kokko

and Jenny Makkonen

140

Freshwater Crayfish

1987; Aydin et al. 2014; Jussila et al. 2014a; Sandström et al. 2014;

Edsman et al. 2015). Thus, virulence adaptation among these very virulent A. astaci haplogroups is now a possibility.

The virulence adaptation of A. astaci in Europe might allow for improved survival among native European crayfish stocks experiencing A. astaci epidemics (e.g., Jussila et al. 2015a). The possible resistance of native European crayfish against some A.

astaci haplogroup strains could also increased resistance towards any strain of A. astaci. In that case, the achieved resistance would allow a better conservation outlook for the native European crayfish stocks (e.g., Makkonen et al. 2012b, 2014; Jussila et al. 2015b) and could also result in increased overall condition and possibly enhanced resistance against other pathogens and parasites, similar to how the signal crayfish (Pacifastacus leniusculus (Dana)) has been reported to resist A. astaci infection (e.g., Persson and Söderhäll 1983; Söderhäll and Cerenius 1999; Söderhäll and Söderhäll 2002; Cerenius et al. 2003). On the other hand, as observed among wild P. leniusculus stocks in Fennoscandia, decreased resistance against A. astaci might have allowed opportunistic pathogens to cause infections, such as eroded swimmeret syndrome (ESS) caused by Fusarium SC (Edsman et al. 2015). The secondary and multiple infections would complicate further the relationship between A. astaci and crayfish species inhabiting European aquatic ecosystems.

We isolated and identified a potential latent crayfish plague infection disease agent, presumably A. astaci, from a wild Astacus astacus (Linnaeus) population in Lake Venesjärvi. Based on our results, and an infection challenge experiment by Francesconi et al. (2021), we report the practical case of a latent crayfish plague infection among a wild A. astacus population in Finland and introduce the identified A. astaci isolate causing the latent crayfish plague infection.

MATERIALS AND METHODS Lake Venesjärvi A. astaci Epidemics

The Lake Venesjärvi, Kankaanpää, Finland (61°4’41”N, 22°10’26”E) A. astacus population has experienced at least three crayfish plague epidemics, namely one with few details known about it during the 1970s, then again in 1994, and the most recent around the year 2000. After the first epidemic, A. astacus were decimated from the surrounding water courses in a 10 km radius.

The A. astacus has slowly recovered from the most recent crayfish plague epidemic, and trappers are catching mature A. astacus at a low rate. There are no documented stockings of the A. astacus in Lake Venesjärvi prior to 2013, while stockings have occurred from an apparently healthy A. astacus population in 2013 and 2014 from Lake Koivujärvi in Kiuruvesi, Finland (Hovi 2020, personal communication).

Detection of Aphanomyces astaci Infection by PCR and qPCR The first batch of A. astacus were obtained from Lake Venesjärvi in autumn 2013. The set consisted of 10 dead crayfish that had died in a holding cage in Lake Venesjärvi during the collection of crayfish samples, and an additional 18 A. astacus that were alive when transferred to the University of Eastern Finland laboratory.

These crayfish were collected before the first introduction of A.

astacus into Lake Venesjärvi from Lake Koivujärvi.

The sampling followed the protocol described in Vrålstad et al. (2009). From the dead crayfish, telson and abdominal cuticles were prepared as samples and the DNA from three different tissues from each crayfish were isolated using an E.Z.N.A. Insect DNA purification Kit. From the live crayfish, DNA was isolated from walking legs using the Zymo Insect Kit. Both extraction methods followed the protocols provided by the kit manufacturer in question. The success of the DNA extraction was monitored using Nanodrop and the DNA samples from different tissues were then analysed by qPCR according to Vrålstad et al. (2009) using the annealing temperature of 62°C according to Strand et al. (2014).

The DNA from the tissues showing positive detection in qPCR were further analysed using chitinase-PCR (Makkonen et al. 2012a) and ITS-PCR with primers ITS39F and ITS640R (Oidtmann et al. 2004). The PCR amplicons were confirmed in agarose gel electrophoresis (1.5% agarose gel containing 0.5 ug/

mL ethidiumbromide) and obtained amplification products were cleaned using the Qiaquick Spin PCR Cleanup Kit. The PCR amplicons were sequenced in GATC Biotech (Cologne, Germany) using both F and R primers.

Isolation of the Lake Venesjärvi A. astaci

The second batch of A. astacus (n = 36, 23 females and 13 males) were obtained from Lake Venesjärvi in 2014. After collection, A. astacus were transported live to the University of Eastern Finland laboratory. They were stored individually in maintenance tanks for up to 3 months (10°C). These crayfish were selected to be big enough not to belong to the introduced batch from Lake Koivujärvi in the previous year.

The A. astaci carrier status was monitored from individually coded live crayfish by collecting three pieces of uropod and telson tips from each crayfish, extracting the DNA from those using Zymo Insect Kit and analyzing the obtained DNA samples in qPCR as previously outlined.

Cultures for A. astaci isolation were prepared and monitored as described in Makkonen et al. (2010) when mortalities occurred in the maintenance tanks. The isolates (n = 3) originated from one crayfish. The species of the obtained isolates were confirmed by ITS-PCR (White et al. 1990), chitinase-PCR (Makkonen et al.

2012a) and sequencing the mitochondrial SSU and LSU sequences (Makkonen et al. 2018).

RESULTS Lake Venesjärvi A. astaci Identification

From the sample set obtained in 2013, several crayfish showed high infection levels in qPCR, up to A5 and A6 (c.f., Vrålstad et al. 2009). The agent levels were higher among the dead crayfish (N = 10; telson tissue sample: A0 for 1 crayfish, A5 for 7 crayfish and A6 for 2 crayfish), while they were mostly negative among the

live crayfish (N = 18; walking leg joint tissue sample: A0 for 12 crayfish, A1 for 4 crayfish and A2 and A6, 1 crayfish each).

In the 2014 sample set, no A. astaci infection was detected via qPCR (i.e., outcome below detection level). In spite of that, a total of six attempts to isolate A. astaci from potential carrier crayfish were carried out with one being successful during the three month maintenance period. The isolated Lake Venesjärvi A. astaci was named UEF_VEN5/14 LSU and SSU, with NCBI accession codes MF975954.1 and MF973125.1, respectively.

The ITS sequences of all fresh A. astaci isolates from Lake Venesjärvi A. astacus were identical and fully matched several A. astaci sequences available in GenBank, with their GenBank accession numbers being MW221492 and MW221493.

DISCUSSION

The suspected latent crayfish plague infection in Lake Venesjärvi was succesfully shown to be caused by Aphanomyces astaci belonging to haplogroup A (UEF_VEN5/14 LSU). It is an A. astaci isolate from the latently infected and wild caught A.

astacus, which is further verifying causes of the suspected and also reported cases of latent crayfish plague infections among native European crayfish (e.g., Jussila et al. 2011a).

This isolate was later tested in a controlled infection experiment (e.g., Francesconi et al. 2021) with A. astacus from Lake Rytky, Finland, a population previously shown to be susceptible to the crayfish plague disease agent (Jussila et al. 2011b; Makkonen et al.

2012b). The infection challenge experiment by Francesconi et al.

(2021) showed no mortalities among Lake Rytky A. astacus within the 45-day experiment. The qPCR analyses indicated, that only 30% of the challenged crayfish showed transmission as A. astaci DNA in the sampled tissues, while the control group crayfish did not show A. astaci DNA in the sampled tissues (Francesconi et al.

2021). This provides evidence that the Venesjärvi A. astaci strain is of very low virulence even under stressing laboratory conditions.

Previously, we observed that A. astaci from haplogroup A during the 2006 River Kemijoki epidemic caused very low mortality in a challenge experiment (Makkonen et al. 2012b), with those A.

astacus surviving the A. astaci challenge experiment showing negative (A0 – A1) or low (A2) infection levels (cf. Vrålstad et al. 2009). Thus, we conclude that one of the features of the low virulence A. astaci strain from the Lake Venesjärvi epidemic could be its lowered ability to germinate, resulting in lower infection of the host crayfish.

The A. astacus population in Lake Venesjärvi has experienced several crayfish plague epidemics. We are aware of three such events, and most probably, this population has at least partially survived these epidemics. There are no official records of introductions of A. astacus from other populations into Lake Venesjärvi prior to our investigation, while this could be suspected, as numerous introductions have been made in Finland without proper documentation. Further studies are needed to investigate the possibility of a unique genetic mixture being behind the suspected elevated A. astaci resistance among Lake Venesjärvi A. astacus, even though our data indicates that lowered A. astaci virulence would explain the success of this A. astacus population.

It could also be that the previous crayfish plague epidemics could have selected a more resistant A. astacus subpopulation to survive in Lake Venesjärvi.

Latent crayfish plague infection in a viable, even robust, native European crayfish population, namely A. astacus, was first reported by Jussila et al. (2011a). Since then, there has been lively debate regarding A. astaci virulence adaptation and the overall possibility for the native European crayfish to be able to resist A. astaci infections (e.g., Jussila et al. 2015b; Svoboda et al.

2017). In our laboratory experiments (e.g., Makkonen et al. 2012b, 2014), challenging A. astacus with different A. astaci isolates, we could show that even under simplified and stressing laboratory conditions, some crayfish could survive a challenge by A. astaci from haplogroup A, and sometimes even by the highly virulent haplogroup B. This, supported by the observations from the wild A. astacus populations, suggested early that coevolution between A. astaci and native European crayfish could be driving towards less virulent A. astaci strains and more resistant European crayfish stocks (e.g., Jussila et al. 2015a, 2015b).

So far, native European crayfish have only shown a selective resistance in challenge experiments towards certain A. astaci strains belonging to haplogroup A (Makkonen et al. 2012b, 2014;

Francesconi et al. 2021), but we have not observed a general resistance against the full range of A. astaci strains from haplogroup A. Some of the A. astaci strains from haplogroup A are clearly showing decreased virulence, as indicated by observations from the wild (Viljamaa-Dirsk et al. 2011; Jussila et al. 2017) and also from controlled challenge experiments (Makkonen et al. 2012b, 2014). Even though the observed coevolution between A. astaci and native European crayfish is evident, there are still haplogroups among A. astaci, which are high virulent, namely those that do have their original North American hosts present in Europe (e.g., Jussila et al. 2013, 2015a, 2015b). The first clear steps toward a balanced relationship between native European crayfish and A.

astaci have been taken, but a true balance lies in the distant future.

This requires strict and coordinated conservation efforts to ensure a lively existence for the native European crayfish.

It has been discovered lately that a small and regionally limited P. leniusculus population exists in Lake Venesjärvi and local crayfisherpersons have started attempts to eradicate the alien P. leniusculus population (Hovi 2020, personal communication).

The two species are inhabiting different regions within Lake Venesjärvi, but still would be influenced by each other’s diseases, especially A. astaci. It thus remains to be seen, and hopefully also monitored, whether the possible A. astaci infections would have an impact on the P. leniusculus population in Lake Venesjärvi (e.g., Aydin et al. 2014).

Our latest finding, adding to several previous reports of a similar nature (Jussila et al. 2011a; Viljamaa-Dirks et al. 2011;

Kokko et al. 2012, 2018; Svoboda et al. 2012, Francesconi et al. 2021), gives insight into the possible balanced relationship between native European crayfish and A. astaci. This is still early days and, especially in the case of the A. astacus, the indications of the positive outcome of the coevolution between the host and parasite can be considered only for the A. astaci from haplogroup

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which should focus on preventing the spreading of A. astaci carrying alien crayfish in Europe (e.g., Ruokonen et al. 2018).

These preventative actions should be an essential part of national alien species and crayfisheries strategies, one example being those in Finland (e.g., Niemivuo-Lahti 2012; Erkamo et al. 2019).

We have here shown that latent crayfish plague infections among wild A. astacus stocks are a reality and that a coevolution of A. astaci and native European crayfish has resulted within some 150 years to occassional balanced relationships (e.g., Jussila et al.

2015b; Svoboda et al. 2017). We have also shown that the virulence of A. astaci can be lowered to a point which enables a full survival of A. astacus stock, as is indicated by a infection challenge experiment (Francesconi et al. 2021). These findings allow speculation on a brighter future for the native European crayfish under pressure from crayfish plague epidemics caused by A. astaci.

ACKNOWLEDGMENTS

We thank Lake Venesjärvi crayfisherpersons, especially Jaakko Hovi and Kalle Koskela, for their interest in the native A. astacus, as it boosts hopes for the successful conservation of this species. Erasmus+ has kindly provided support during the preparation of this manuscript by offering the opportunity for face- to-face meetings.

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