High variation of virulence in Aphanomyces astaci strains lacks association with pathogenic traits and mtDNA haplogroups.

Introduced into Europe from North America 150 years ago alongside its native crayfish hosts, the invasive pathogen Aphanomyces astaci is considered one of the main causes of European crayfish population decline. For the past two centuries, this oomycete pathogen has been extensively studied, with the more recent efforts focused on containing and monitoring its spread across the continent. However, after the recent introduction of new strains, the newly-discovered diversity of A. astaci in North America and several years of coevolution with its European host, a new assessment of the traits linked to the pathogen's virulence is much needed. To fill this gap, we investigated the presence of phenotypic patterns (i.e., in vitro growth and sporulation rates) possibly associated with the pathogen's virulence (i.e., induced mortality in crayfish) in a collection of 14 A. astaci strains isolated both in North America and in Europe. The results highlighted a high variability in virulence, growth rate and motile spore production among the different strains, while the total-sporulation rate was more similar across strains. Surprisingly, growth and sporulation rates were not significantly correlated with virulence. Furthermore, none of the analysed parameters, including virulence, was significantly different among the major A. astaci haplogroups. These results indicate that each strain is defined by a characteristic combination of pathogenic features, specifically assembled for the environment and host faced by each strain. Thus, canonical mitochondrial markers, often used to infer the pathogen's virulence, are not accurate tools to deduce the phenotype of A. astaci strains. As the diversity of A. astaci strains in Europe is bound to increase due to translocations of new carrier crayfish species from North America, there is an urgent need to deepen our understanding of A. astaci's virulence variability and its ability to adapt to new hosts and environments.

Host-pathogen coevolution drives innate immune response to Aphanomyces astaci infection in freshwater crayfish: transcriptomic evidence.

BACKGROUND: For over a century, scientists have studied host-pathogen interactions between the crayfish plague disease agent Aphanomyces astaci and freshwater crayfish. It has been hypothesised that North American crayfish hosts are disease-resistant due to the long-lasting coevolution with the pathogen. Similarly, the increasing number of latent infections reported in the historically sensitive European crayfish hosts seems to indicate that similar coevolutionary processes are occurring between European crayfish and A. astaci. Our current understanding of these host-pathogen interactions is largely focused on the innate immunity processes in the crayfish haemolymph and cuticle, but the molecular basis of the observed disease-resistance and susceptibility remain unclear. To understand how coevolution is shaping the host's molecular response to the pathogen, susceptible native European noble crayfish and invasive disease-resistant marbled crayfish were challenged with two A. astaci strains of different origin: a haplogroup A strain (introduced to Europe at least 50 years ago, low virulence) and a haplogroup B strain (signal crayfish in lake Tahoe, USA, high virulence). Here, we compare the gene expression profiles of the hepatopancreas, an integrated organ of crayfish immunity and metabolism. RESULTS: We characterised several novel innate immune-related gene groups in both crayfish species. Across all challenge groups, we detected 412 differentially expressed genes (DEGs) in the noble crayfish, and 257 DEGs in the marbled crayfish. In the noble crayfish, a clear immune response was detected to the haplogroup B strain, but not to the haplogroup A strain. In contrast, in the marbled crayfish we detected an immune response to the haplogroup A strain, but not to the haplogroup B strain. CONCLUSIONS: We highlight the hepatopancreas as an important hub for the synthesis of immune molecules in the response to A. astaci. A clear distinction between the innate immune response in the marbled crayfish and the noble crayfish is the capability of the marbled crayfish to mobilise a higher variety of innate immune response effectors. With this study we outline that the type and strength of the host immune response to the pathogen is strongly influenced by the coevolutionary history of the crayfish with specific A. astaci strains.

Non-destructive method for detecting Aphanomyces astaci, the causative agent of crayfish plague, on the individual level.

The pathogenic oomycete Aphanomyces astaci, transmitted mainly by invasive North American crayfish, causes the crayfish plague, a disease mostly lethal for native European crayfish. Due to its decimating effects on native crayfish populations in the last century, A. astaci has been listed among the 100 worst invasive species. Importantly, detecting the pathogen in endangered native crayfish populations before a disease outbreak would provide a starting point in the development of effective control measures. However, current A. astaci-detection protocols either rely on degradation-prone eDNA isolated from large volumes of water or, if focused on individual animals, include killing the crayfish. We developed a non-destructive method that detects A. astaci DNA in the microbial biofilm associated with the cuticle of individual crayfish, without the need for destructive sampling. Efficiency of the new method was confirmed by PCR and qPCR and the obtained results were congruent with the traditional destructive sampling method. Additionally, we demonstrated the applicability of the method for A. astaci monitoring in natural populations. We propose that the new method should be used in future monitoring of A. astaci presence in endangered European native crayfish individuals as an alternative to eDNA-based monitoring.

The signal crayfish (Pacifastacus leniusculus) in Lake Tahoe (USA) hosts multiple Aphanomyces species.

The genus Aphanomyces (Oomycetes) comprises approximately 50 known species of water molds in three lineages. One of the most notorious is Aphanomyces astaci, the causative agent of crayfish plague. In this study, fresh isolates of Aphanomyces were collected from 20 live specimens of the signal crayfish Pacifastacus leniusculus (Dana, 1852) from Lake Tahoe, California, providing 35 axenic cultures of A. astaci as well as two apparently undescribed Aphanomyces spp. isolates. Based on the results of ITS-, chitinase-, mitochondrial rnnS- and rnnL-sequences and microsatellite markers combined, the Lake Tahoe A. astaci isolates were identical to isolates of A. astaci B-haplogroup commonly detected in Europe, and infection experiments confirmed their high virulence towards noble crayfish. One of the two undescribed Aphanomyces spp. isolates was highly similar to an Aphanomyces lineage detected previously in crustacean zooplankton (Daphnia) in Central Europe, while the other was distinct and most closely related (ITS sequence similarity of 93%) to either A. astaci or to Aphanomyces fennicus isolated recently from Astacus astacus in Finland. Neither of the two Aphanomyces spp. isolates caused crayfish mortality under experimental conditions. Our results indicate that the populations of North American signal crayfish can act as carriers of both pathogenic and non-pathogenic Aphanomyces at the same time. Furthermore, considering that a limited number of crayfish individuals from a single location yielded multiple distinct Aphanomyces isolates, our results suggest that substantial species diversity within this genus remains undescribed.

MtDNA allows the sensitive detection and haplotyping of the crayfish plague disease agent Aphanomyces astaci showing clues about its origin and migration.

The oomycete Aphanomyces astaci, the causative agent of crayfish plague, is listed as one of the 100 worst invasive species in the world, destroying the native crayfish populations throughout Eurasia. The aim of this study was to examine the potential of selected mitochondrial (mt) genes to track the diversity of the crayfish plague pathogen A. astaci. Two sets of primers were developed to amplify the mtDNA of ribosomal rnnS and rnnL subunits. We confirmed two main lineages, with four different haplogroups and five haplotypes among 27 studied A. astaci strains. The haplogroups detected were (1) the A-haplogroup with the a-haplotype strains originating from Orconectes sp., Pacifastacus leniusculus and Astacus astacus; (2) the B-haplogroup with the b-haplotype strains originating from the P. leniusculus; (3) the D-haplogroup with the d1 and d2-haplotypes strains originating from Procambarus clarkii; and (4) the E-haplogroup with the e-haplotype strains originating from the Orconectes limosus. The described markers are stable and reliable and the results are easily repeatable in different laboratories. The present method has high applicability as it allows the detection and characterization of the A. astaci haplotype in acute disease outbreaks in the wild, directly from the infected crayfish tissue samples.

Hidden sites in the distribution of the crayfish plague pathogen Aphanomyces astaci in Eastern Europe: Relicts of genetic groups from older outbreaks?

The crayfish plague agent Aphanomyces astaci is one of the world's most threatening invasive species. Originally from North America, the pathogen is being imported alongside American crayfish species, which are used for various purposes. In this study, we investigated the marginal, currently known distribution area of the pathogen in Eastern Europe by sampling narrow-clawed crayfish (Astacus leptodactylus) and spiny-cheek crayfish (Orconectes limosus) populations. In addition, using specific real-time PCR, we tested several marine decapod species, which also occur in brackish waters of the Danube at the West coast of the Black Sea and the Dniester River basin. By sequencing the nuclear chitinase gene, mitochondrial rnnS/rnnL DNA and by genotyping using microsatellite markers, we identified the A. astaci haplogroups of highly infected specimens. The A. astaci DNA was detected in 9% of the investigated A. leptodactylus samples, both in invaded and non-invaded sectors, and in 8% of the studied O. limosus samples. None of the marine decapods tested positive for A. astaci. The results revealed that narrow-clawed crayfish from the Dniester River carried the A. astaci B-haplogroup, while A. astaci from the Danube Delta belonged to the A- and B-haplogroups. In the invaded sector of the Danube, we also identified the A-haplogroup. Microsatellite analysis revealed a genotype identical to the genotype Up. It might be that some of the detected A. astaci haplogroups are relics from older outbreaks in the late 19th century, which may have persisted as a chronic infection for several decades in crayfish populations.

Aphanomyces astaci isolate from latently infected stone crayfish (Austropotamobius torrentium) population is virulent.

Aphanomyces astaci infection is the cause of crayfish plague in European crayfish. Here the virulence of an A. astaci As strain isolated from apparently healthy stone crayfish (Austropotamobius torrentium) from Slovenia was compared to that of the Psl-Puujärvi A. astaci isolate in 3 crayfish species: noble crayfish (Astacus astacus), signal crayfish (Pacifastacus leniusculus) from Finland and stone crayfish from Slovenia. All 3 crayfish species were challenged with PsI-Puujärvi A. astaci and succumbed to crayfish plague, with both noble crayfish and stone crayfish showing 100% mortality, while 25% of the signal crayfish died during the challenge. In comparison, the As-Slovenia A. astaci isolate was pathogenic for noble crayfish but not for signal crayfish or stone crayfish. This finding suggests that A. astaci virulence could be species specific and a strain from latent A. astaci infection in one native European crayfish species could be detrimental to other native European crayfish species.

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.

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.

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.

Draft Genome Sequence of Lactiplantibacillus plantarum IMI 507026.

Here, we announce the draft genome sequence of Lactiplantibacillus plantarum isolated from corn silage in Nicholasville, KY. L. plantarum IMI 507026 is deposited in the Centre for Agriculture and Bioscience International (CABI) Culture Collection with the accession number IMI 507026.

Genome sequence data of Lactiplantibacillus plantarum IMI 507028.

Here, we report the genome sequencing data for the fermented milk isolate, Lactiplantibacillus plantarum (L. plantarum) IMI 507028. The Bioproject, SRA, and GenBank data were deposited at NCBI under accession numbers PRJNA801616, SRR18323693, and JAKMAX000000000, respectively. The size of the genome was 3,231,321 bp, with a GC% of 44.52. Before sequence trimming, the genome contained 40 contigs, in which 35 contigs were annotated, revealing 2937 coding sequences out of 3052 total genes. The strain was identified as L. plantarum with an average nucleotide identity (ANI) value of 99.9922% between IMI 507028 and L. plantarum JDM1. Genes related to antimicrobial resistance or pathogenic factors were not found during screening.

Pediococcus pentosaceus IMI 507025 genome sequencing data.

The genome sequence data for the pickled cucumbers isolate, Pediococcus pentosaceus IMI 507025, is reported. The raw reads and analysed genome reads were deposited at NCBI under Bioproject with the accession number PRJNA814992. The number of contigs before and after trimming were 17 and 12 contigs, respectively. The total size of the genome was 1,795,439 bp containing 1,811 total genes, of which 1,751 were coding sequences. IMI 507025 identity was determined via average nucleotide identity (ANI), obtaining an identity value of 99.5994% between IMI 507025 and the type strain P. pentosaceus ATCC 33316, identifying the strain as P. pentosaceus. Screening for the antimicrobial resistance (AMR) and virulence genes in the genome of IMI 507025 showed no hits, confirming the safety of the tested strain. Presence of plasmids was not found.

Draft Genome Sequence of Pediococcus pentosaceus IMI 507024.

We report here the draft genome sequence of Pediococcus pentosaceus strain IMI 507024, a lactic acid bacterium isolated from fermented sausage in Kentucky (Nicholasville, KY, USA). The strain is deposited in the Centre for Agriculture and Bioscience International (CABI) Culture Collection with the accession number IMI 507024.

Draft Genome Sequence of Lacticaseibacillus rhamnosus IMI 507023.

We report here the draft genome sequence of Lacticaseibacillus rhamnosus strain IMI 507023, a lactic acid bacterium, isolated from corn silage in Nicholasville, Kentucky, USA. The strain is deposited in the Centre for Agriculture and Bioscience International (CABI) Culture Collection with the accession number IMI 507023.

Whole genome sequence data of Lactiplantibacillus plantarum IMI 507027.

Here we report the draft genome sequence of the Lactiplantibacillus plantarum IMI 507027 strain. The genome consists of 37 contigs with a total size of 3,235,614 bp and a GC% of 44.51. After sequence trimming, 31 contigs were annotated, revealing 3,126 genes, of which 3,030 were coding sequences. The Average Nucleotide Identity (ANI) gave a value of 99.9926% between IMI 507027 and L. plantarum JDM1, identifying the strain as L. plantarum. No genes of concern for safety-related traits such as antimicrobial resistance or virulence factors were found. The annotated genome and raw sequence reads were deposited at NCBI under Bioproject with the accession number PRJNA791753.

Dataset of the de novo assembly and annotation of the marbled crayfish and the noble crayfish hepatopancreas transcriptomes.

OBJECTIVES: Crayfish plague disease, caused by the oomycete pathogen Aphanomyces astaci represents one of the greatest risks for the biodiversity of the freshwater crayfish. This data article covers the de novo transcriptome assembly and annotation data of the noble crayfish and the marbled crayfish challenged with Ap. astaci. Following the controlled infection experiment (Francesconi et al. in Front Ecol Evol, 2021, https://doi.org/10.3389/fevo.2021.647037 ), we conducted a differential gene expression analysis described in (Bostjancic et al. in BMC Genom, 2022, https://doi.org/10.1186/s12864-022-08571-z ) DATA DESCRIPTION: In total, 25 noble crayfish and 30 marbled crayfish were selected. Hepatopancreas tissue was isolated, followed by RNA sequencing using the Illumina NovaSeq 6000 platform. Raw data was checked for quality with FastQC, adapter and quality trimming were conducted using Trimmomatic followed by de novo assembly with Trinity. Assembly quality was assessed with BUSCO, at 93.30% and 93.98% completeness for the noble crayfish and the marbled crayfish, respectively. Transcripts were annotated using the Dammit! pipeline and assigned to KEGG pathways. Respective transcriptome and raw datasets may be reused as the reference transcriptome assemblies for future expression studies.

Improved chromosome-level genome assembly of the Glanville fritillary butterfly (Melitaea cinxia) integrating Pacific Biosciences long reads and a high-density linkage map.

BACKGROUND: The Glanville fritillary (Melitaea cinxia) butterfly is a model system for metapopulation dynamics research in fragmented landscapes. Here, we provide a chromosome-level assembly of the butterfly's genome produced from Pacific Biosciences sequencing of a pool of males, combined with a linkage map from population crosses. RESULTS: The final assembly size of 484 Mb is an increase of 94 Mb on the previously published genome. Estimation of the completeness of the genome with BUSCO indicates that the genome contains 92-94% of the BUSCO genes in complete and single copies. We predicted 14,810 genes using the MAKER pipeline and manually curated 1,232 of these gene models. CONCLUSIONS: The genome and its annotated gene models are a valuable resource for future comparative genomics, molecular biology, transcriptome, and genetics studies on this species.

The duration of exposure to 50 Hz magnetic fields: Influence on circadian genes and DNA damage responses in murine hematopoietic FDC-P1 cells.

We investigated the effects of 50 Hz extremely low-frequency magnetic fields (MFs) on gene expression related to the circadian rhythm or DNA damage signaling and whether these fields modify DNA damage repair rate after bleomycin treatment. Murine FDC-P1 hematopoietic cells were exposed for different durations (15 min, 2 h, 12 h, and 24 h) to either 200 µT MFs or sham-exposures. Cells were then collected for comet assay or real-time PCR to determine immediate DNA damage level and circadian rhythm gene expression, respectively. To assess DNA-damage signaling and DNA repair rate, the cells were subsequently treated with 20 µg/mL bleomycin for 1 h and then either assayed immediately or allowed to repair their DNA for 1 or 2 h. We found that circadian rhythm-related genes were upregulated after 12 h of MF exposure and downregulated after 24 h of MF exposure, but none of the affected genes were core genes controlling the circadian rhythm. In addition, we found that the repair rate for bleomycin-induced damage was only decreased after MF exposure for 24 h. In conclusion, our findings suggest that the effects of MFs are duration-dependent; they were observed predominantly after long exposures.

Mapping 15 years of crayfish plague in the Iberian Peninsula: The impact of two invasive species on the endangered native crayfish.

Crayfish plague, caused by the pathogen Aphanomyces astaci, is one of the main factors responsible for the decimation of the native European crayfish species Austropotamobius pallipes. In Spain, two North American freshwater crayfish species, Procambarus clarkii and Pacifastacus leniusculus, were intentionally introduced during the 1970s for aquaculture and fishery purposes. Since then, incidences of crayfish plague have been continually reported. In this work, we evaluated more than 50 diagnosed cases of crayfish plague that have occurred in the Iberian Peninsula since 2004 by performing a microscopic examination of infected specimens and by molecularly identifying and haplotyping the pathogen. Our results showed that (i) the pathogen A. astaci has been active 45 years since the first introductions of the invasive North American crayfish species in the Iberian Peninsula, and (ii) P. clarkii and P. leniusculus are chronic reservoirs of the crayfish plague pathogen. Moreover, our data confirmed a correspondence between pathogen origin and spread and the specific haplotypes carried by the North American invasive crayfish located in the vicinity of each outbreak. We generated a crayfish plague incidence map of the Iberian Peninsula that shows (i) a northern area, mainly inhabited by alien P. leniusculus, where crayfish plague cases are associated with the b-haplotype specific to P. leniusculus, and (ii) southern, central and eastern areas that are basically inhabited by alien P. clarkii, where crayfish plague cases are associated with the d1- and d2-haplotypes specific to P. clarkii. The results presented here are evidence of the long standing and negative impact of the two invasive crayfish species on the native species, indicating the need for more extensive control measures.