Jenny Makkonen

Differing virulence of Aphanomyces astaci isolates and elevated resistance of noble crayfish Astacus astacus against crayfish plague.

Crayfish plague epidemics (caused by Aphanomyces astaci) have been causing population collapses among native European crayfish stocks since the late 1800s. Recent indirect and direct evidence has shown that its virulence has been variable, with native European crayfish even acting as carriers. We tested the differences in A. astaci virulence under experimental conditions using both PsI- and As-genotypes with 3 Finnish noble crayfish Astacus astacus populations. We infected crayfish with adjusted quantities of A. astaci zoospores and monitored the symptoms and mortality of the crayfish. The PsI-genotype isolate caused rapid and total mortality among the tested populations, while the As-genotype isolates expressed more variable virulence. In some cases, mortality among the As-genotype-infected crayfish did not exceed the mortality level of the control group. All of the tested noble crayfish stocks showed lower mortality towards the As-genotype of A. astaci isolated from the River Kemijoki epidemic. We conclude that there are clear differences in virulence between different A. astaci genotypes and also differences in virulence within As-genotypes. Furthermore, we observed clear signs of increased resistance in different populations of noble crayfish towards some of the tested strains belonging to the As-genotype of A. astaci.

Monitoring the spore dynamics of Aphanomyces astaci in the ambient water of latent carrier crayfish.

The specialized crayfish parasite Aphanomyces astaci causes the devastating crayfish plague in European crayfish. Even though A. astaci sporulation has been thoroughly studied under pure culture conditions, little is known about the sporulation dynamic from its live host. Our purpose was to investigate the A. astaci spore dynamic in its native parasite-host relationship by monitoring the sporulation from carrier crayfish into the ambient water using agent specific qPCR. American signal crayfish (Pacifastacus leniusculus) with known positive carrier status were housed individually and communally in two experimental set-ups using multiple replicates and different temperatures. Water samples were collected weekly, and spore numbers were quantified. We demonstrate here that live latent carrier crayfish continuously released a moderate number of A. astaci spores (~2700 spores per crayfish/week) in the absence of death and moulting events. In contrast, a pronounced sporulation increase was seen already one week prior to death in moribund crayfish, suggesting a crayfish plague-like condition developing in weakened or stressed individuals. Significantly more spores were produced at 18°C compared to 4°C, while a negative correlation was detected between spore numbers and temperatures rising from 17 to 23°C. This study is the first attempt to quantify the spore release from carrier crayfish on the basis of qPCR applied on water samples, and demonstrate that the approach successfully unravel A. astaci sporulation patterns. The results emphasize that carrier crayfish pose a constant infection risk to highly susceptible crayfish species regardless of crayfish life cycle state.

The diversity of the pathogenic Oomycete (Aphanomyces astaci) chitinase genes within the genotypes indicate adaptation to its hosts

The aim of this work was to evaluate the genetic diversity of the crayfish plague pathogen Aphanomyces astaci (Oomycete) among different isolates and genotypes. Partial chitinase genes were cloned and sequenced from 28 A. astaci isolates including four of the five previously identified RAPD (random amplification of polymorphic DNA)-genotypes. The cloned chitinase sequences (n=176) formed three main groups, CHI1, CHI2 and CHI3, with the CHI2 group then further divided into three subgroups, CHI2A, CHI2B and CHI2C. Some of these chitinases were specific for certain genotypes of A. astaci, as CHI2B and CHI2C were only found from the As-genotype and CHI3 from the Ps-genotypes of A. astaci. Highest diversity rate was observed in the CHI2 group, while the CHI3 group specific for Ps-genotypes was highly homologous. Based on our chitinase data, As- and Pc-genotypes seem to be related, while the two Ps-genotypes were identical to each other, but considerably different from the genotypes As and Pc. These are the first genotype specific differences that are located in the coding region of the chitinase gene of A. astaci and the differences observed here also enable the genotyping of A. astaci. The diversity observed here can also reflect differences in the epidemiological properties of the different genotypes.

Dose-dependent mortality of the noble crayfish (Astacus astacus) to different strains of the crayfish plague (Aphanomyces astaci)

Several reports of the European crayfish species carrying a latent infection of the crayfish plague (Aphanomyces astaci) have emerged and the discussion has focused especially on the lowered virulence of As-genotypes behind decreased mortality. The aim of this study was to compare the killing rate of different A. astaci strains in controlled infection experiments. Two separate infection experiments with three A. astaci strains (UEFT2B (As), Evira6462/06 (As) and UEF8866-2 (PsI)) were made to compare the noble crayfish populations from the Lake Viitajärvi, Tervo, (Expt I) and the Lake Mikitänjärvi, Hyrynsalmi (Expt II). In the Expt III, the Lake Koivujärvi population noble crayfish were infected with A. astaci strains UEF8866-2 (PsI) and Evira6462/06 (As) using different dosages (1, 10, 100 and 1000sporesml(-1)) of A. astaci zoospores. The results confirmed that PsI-genotype strain is highly virulent and kills all the crayfish within a few days. The tested two As-genotype strains caused the mortalities more slowly, and part of the challenged crayfish survived until the end of the follow-up period. Our results also confirmed the variance of virulence among A. astaci strains within the As-genotype and demonstrated that the mortality is dependent on the number of zoospores used in the infections. It also appeared, that some noble crayfish populations show increased resistance towards the crayfish plague, especially against the As-genotype of A. astaci.

De Novo assembly and annotation of the freshwater crayfish Astacus astacus transcriptome

We generated RNA-seq data to assemble the transcriptome of the noble crayfish (Astacus astacus) from four combined tissues (abdominal muscle, hepatopancreas, ovaries, green glands). A total of 194 million read pairs with a length of 100 bp were generated. The transcriptome was assembled de novo using Trinity software, producing 158,649 non-redundant transcripts. Lowly expressed transcripts were filtered out leaving 45,415 transcripts of which 14,559 were found to contain open reading frames with predicted gene function. The Transrate software revealed that 91% of the total reads were realigned to the assembly. Furthermore, BUSCO analysis indicated that our assembly is 64% complete. A total of 13,770 transcripts were assigned at least one GO term. This first de novo transcriptome assembly is an important foundation for future genomic research on the noble crayfish and adds to the general knowledge and further characterization of transcriptomes of non-model organisms.

Timing and quantifying Aphanomyces astaci sporulation from the noble crayfish suffering from the crayfish plague

Aphanomyces astaci sporulation is crucial for the spreading potential of this disease agent. For the first time, we are reporting timing and quantity of A. astaci spores released from noble crayfish (Astacus astacus) suffering from crayfish plague under practical aquatic conditions. We infected nine noble crayfish with A. astaci PsI-genotype and maintained them in individual 8L tanks. Spores (zoospores and cysts) were quantified from water samples (3 × 1 mL) taken every 12h over 10 d using A. astaci specific qPCR. A clear sporulation trend was found, together with a high individual spore estimate variation. The median spore counts from two days before death to 12h post mortem were from ~500 to ~2000 spores L(-1). A significant sporulation increase occurred after 24h post mortem (~12,000 spores L(-1)) and reached a peak after two days (~65,000 spores L(-1)) before declining to or below pre mortem levels from the fourth day. The single most sporulating crayfish released from ~75,000 to ~400,000 spores L(-1) during the mass sporulating period, yielding a maximum estimate of ~3,200,000 spores released from a single crayfish if we assume homogeneous spore distribution. The results confirm a mass A. astaci spore release from moribund and recently dead infected noble crayfish, with a sporulation peak one to three days post mortem. The acute crayfish mortality only three days after zoospore exposure confirm the lethal potential of the PsI-genotype. The powerful sporulation potential observed here may be one of the key virulence factors of this genotype.

Eroded swimmeret syndrome in female crayfish Pacifastacus leniusculus associated with Aphanomyces astaci and Fusarium spp. infections.

We describe a novel syndrome in crayfish, eroded swimmeret syndrome (ESS), affecting wild female signal crayfish Pacifastacus leniusculus. ESS causes partial or total swimmeret erosion. We observed ESS only in female signal crayfish larger than 40 mm carapace length, i.e. sexually mature and probably having carried eggs at least once. The eroded swimmerets were melanised, indicating a crayfish immune system response. We isolated Fusarium tricinctum species complex (SC), F. sambucinum SC, Saprolegnia parasitica and S. australis from the melanised tissue of the eroded swimmerets. ESS includes chronic Aphanomyces astaci infection and a secondary infection by Fusarium sp. In Sweden, we found female signal crayfish with ESS in 6 out of 11 populations with a prevalence below 1% in lakes with commercially productive signal crayfish populations and higher than 29% in lakes with documented signal crayfish population crashes. In Finland, the ESS prevalence was from 3.4 to 6.2% in a commercially productive population. None of the sampled male signal crayfish showed signs of ESS. A caging experiment indicated that females with at least 1 lost swimmeret carried on average 25% fewer fertilized eggs compared to females with intact swimmerets. ESS could significantly reduce individual female fecundity and thus could also affect fecundity at the population level. The decline in reproductive success due to ESS could be among the factors contributing to fluctuations in wild signal crayfish populations.

Variation in Resistance to the Invasive Crayfish Plague and Immune Defence in the Native Noble Crayfish

Emerging diseases, such as the crayfish plague, are a worldwide problem with serious ecological and economic impacts. Under the framework of ecological immunology, we investigated whether variation in crayfish plague resistance, the indicators of immune defence (encapsulation response, phenoloxidase and lytic activity), and the exploration behaviour among four subpopulations of noble crayfish is explained by potential local adaptation through differences in crayfish plague history, or alternatively by geographical divergence in a large watershed. We examined whether the strength of immune defence is associated with survival and exploration behaviour. Survival time after experimental crayfish plague infection and phenoloxidase activity differed among the subpopulations of the watershed but did not reveal local adaptation to the disease. Increased investment in immune defence (i.e. encapsulation response) compromised survival time after infection, suggesting the self-reactivity costs of mounting a strong immune response. Exploration behaviour was negatively associated with phenoloxidase activity before and after immune challenge.

Fusarium avenaceum causes burn spot disease syndrome in noble crayfish (Astacus astacus).

Burn spot disease has been causing epidemics both in the Estonian mainland and in Saaremaa Island in the threatened noble crayfish (Astacus astacus) stocks. To study the cause of the disease, we isolated several Fusarium spp. from Estonian noble crayfish (A. astacus) populations suffering from burn spot disease syndrome. We first identified fungi directly from melanised cuticle by their ITS sequences. Then we isolated Fusarium spp. from melanised spots of crayfish showing burn spot disease symptoms, such as melanisation and shell erosion, from two different crayfish populations and watercourses in Estonia. The isolates were then identified based on ITS and EF1α-gene sequences. Isolates of Fusarium spp. taken from two separate Estonian noble crayfish populations were used in infection studies. Koch postulates confirmed that the studied agent was causing burn spot disease symptoms including shell erosion in the noble crayfish, which were significantly more severe after molts. After the infection period, an identical Fusarium spp. was re-isolated from carapace lesions and was thus shown to be the disease agent causing burn spot disease syndrome and shell erosion in noble crayfish. Based on GenBank database searches, the isolates causing burn spot disease symptoms were identified as Fusarium avenaceum in mainland Estonia and F. solani in Saaremaa crayfish.

Mitochondrial genomes and comparative genomics of Aphanomyces astaci and Aphanomyces invadans

The genus Aphanomyces (Saprolegniales, Oomycetes) includes species with a variety of ecologies from saprotrophs to plant and animal parasites. Two important species in this genus are A. astaci, the cause of crayfish plague and its close relative, A. invadans, which causes the epizootic ulcerative syndrome on fish. In this study, we have assembled and annotated the mitochondrial (mt) genomes of A. astaci and A. invadans from the whole genome shotgun sequence reads (PRJNA187372; PRJNA258292, respectively). The assembly was generated from A. astaci Pc-genotype strain APO3 and A. invadans strain NJM9701. The sizes of the mtDNAs were 49,489 bp and 49,061 bp for A. astaci and A. invadans, respectively. The species shared similar genetic content and organization encoding 35 proteins, two ribosomal RNAs, three putative open reading frames and 33 transfer RNAs of 19 amino acids for peptide synthesis. Both species also had a large inverted repeat region (LIR) of approximately 12 kb, the LIR contained large and small ribosomal RNAs and eight protein coding genes. These annotated mt genomes serve as a valuable genetic backbone for further development of diagnostic methods and phylogenetic and migration studies of the animal parasitic species of Aphanomyces.