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Microbiome affects various interactions between the invader and its novel environment during the process of biological invasion. In turn, dispersal process and characteristics of the novel environment may affect the composition of invader’s microbiome, directly and indirectly affecting its fitness and invasion success. So far, the majority of studies focus on bacteriome, insufficiently addressing other components of microbiome, such as mycobiome. Microbial fungi are among the most damaging pathogens in freshwater crayfish populations, with both native and invasive crayfish species being susceptible to fungal colonization and possible infection. Using ITS rRNA amplicon sequencing, we have analyzed the mycobiome of a successful invader in Europe, the signal crayfish. We explored the mycobiome of four types of crayfish samples (exoskeletal biofilm, hemolymph, hepatopancreas, intestine), compared them to environmental samples (water, sediment), and examined differences in fungal biodiversity and abundance between upstream and downstream segments of the signal crayfish’ invasion range in the Korana River, Croatia. A small number of amplicon sequence variants was obtained from hemolymph and hepatopancreas samples, pointing to small fungal abundance. Thus, only exoskeleton, intestine, sediment and water samples were analyzed further. Significant differences were recorded between their mycobiomes, confirming their uniqueness. Environmental samples showed higher biodiversity than crayfishassociated samples. Intestinal mycobiome showed significantly lower richness compared to other three mycobiomes. Sediment and exoskeletal mycobiome differed significantly between different river segments, indicating that environment (i.e. sediment mycobiome) at least partly shapes the exoskeletal mycobiome of crayfish. Intestinal mycobiome showed no differences between river segments, which indicates that this internal organ’s mycobiome remains more stable despite environmental changes. Our results present the first metagenomic data on crayfish-associated fungal communities across different tissues, and offer a baseline for assessing how mycobiome contributes to species’ overall health and further invasion success.

Oomycetes are fungal-like microorganisms parasitic towards a large number of plant and animal species. Genera from order Saprolegniales, such as Saprolegnia and Aphanomyces, cause devastating infections in freshwater ecosystems and aquaculture facilities. Saprolegnia parasitica is a widely distributed oomycete pathogen that causes saprolegniosis, disease responsible for significant economic losses in aquaculture, as well as declines of natural populations of fish and other freshwater organisms. Despite its negative impact, no monitoring protocol for S. parasitica has been established to date. Thus, we aimed to develop droplet digital PCR (ddPCR) assay for detection and quantification of S. parasitica DNA in environmental samples. Material and methods Saprolegnia parasitica-specific primers were designed to target internal transcribed spacer region 2 (ITS 2), based on the alignment of ITS sequences of S. parasitica and a range of Saprolegnia spp., as well as other oomycetes. The limit of detection (LOD) of the assay was established by using serial dilutions of the S. parasitica genomic DNA. Specificity of the designed primer pair was tested using genomic DNA of S. parasitica (as positive control) and DNA of non–S. parasitica oomycete isolates, as well as trout/crayfish DNA. Assay performance was further assessed with water and swab samples from aquaculture (trout farms) and natural environments. Water samples were collected from 21 different locations in Croatia, while swab samples were collected from S. parasitica host/carrier species: (i) skin (30 samples) and eggs (15) of rainbow (Oncorhynchus mykiss Walbaum, 1792) and brown trout (Salmo trutta Linnaeus, 1758), and (ii) cuticle (20) of signal (Pacifastacus leniusculus Dana, 1852) and narrow clawed crayfish (Pontastacus leptodactylus Eschscholtz, 1823). Samples were classified into agent levels A0 to A6, depending of the number of S. parasitica ITS copies per ng of total DNA. Results Designed primers specifically amplified a segment of the ITS region of oomycete pathogen S. parasitica, while no amplification (i.e. no positive droplets) was obtained for closely related Saprolegnia spp. (like Saprolegnia sp. 1 and S. ferax) and other more distantly related oomycetes. Sensitivity of the assay was high: LOD was 15 fg of pathogen’s genomic DNA per µL of reaction mixture. The assay performance was further assessed using environmental DNA samples (water and swab samples). Saprolegnia parasitica was detected in 16 out of 21 water samples (76 %) and the range of pathogen’s ITS copies in positive samples was between 0.02 and 14 copies/ng of total DNA (agent levels A1 to A3). Furthermore, S. parasitica was detected in swab samples collected from the host surface (i.e. adult trout, trout eggs and crayfish). Saprolegnia parasitica load was significantly higher in diseased trout samples then in those with healthy appearance: 9375 vs 3.28 S. parasitica copies/ng of total swab DNA (median 8, agent level A6 vs. A2, respectively). Despite the fact that none of the sampled crayfish had sings of infection, the pathogen was detected in all tested cuticle swabs. Swabs of P. leniusculus, a known S. parasitica host, had significantly higher S. parasitica load then swabs of P. leptodactylus, S. parasitica carrier: 390 vs 83 S. parasitica copies/ng (median 63 agent level A5 vs. A4, respectively). Conclusion Our results demonstrate the applicability of the newly developed ddPCR assay in monitoring and early detection of S. parasitica in aquaculture facilities and natural freshwater environment. This would help in better understanding of S. parasitica ecology and its effects on the host populations.

Introduction and spread of invasive alien species may result in transmission of pathogens to susceptible native counterparts and lead to their populations decline. We analysed the presence of two aquatic oomycete pathogens, Aphanomyces astaci – causative agent of crayfish plague and Saprolegina parasitica – causative agent of saprolegniasis in fish and an oportunistic crayfish pathogen, in populations of the signal crayfish along its invasion range in the Korana River, Croatia. We collected mixed epibiotic microbial communities from carapace of 110 crayfish by swabs. Next, DNA was isolated and PCR-based pathogen detection conducted. A. astaci was detected by previously developed specific primers in 6.4% of crayfish along the entire invasion range (invasion core, upstream and downstream fronts). To detect S. parasitica, we developed specific primer pairs and demonstrated their specificity and sensitivity using genomic DNA of S. parasitica and related oomycetes. However, S. parasitica was not detected in any of the examined individuals.

Background: Some oomycetes from the genus Saprolegnia are causing saprolegniosis, a disease of salmonid fish important in aquaculture. Saprolegnia species cause significant economic losses in freshwater aquaculture, with Saprolegnia parasitica as the most destructive pathogen. Despite its negative effects, no monitoring protocol of S. parasitica has been established to date. Objectives: We aimed to develop PCR-detection assay for S. parasitica in order to enable its monitoring in freshwater aquaculture facilities. Methods: We constructed multiple sequence alignment of 958 internal transcribed spacer (ITS) sequences belonging to various oomycetes. We have identified S. parasitica specific regions and created three primer pairs, designated A, B and C. Next, we used gradient PCR and S. parasitica (CBS 233.65) genomic DNA as a template to determine optimal annealing temperature for each primer pair. Results: Primer pairs A and B have amplified unspecific bands that could not be eliminated, but primer pair C amplified a single band of expected size. The sensitivity of the assay with primer pair C was high: lower detection limit was < 1 pg. Also, we have demonstrated the specificity of the assay using a range of Saprolegnia species and other, more distantly related Oomycetes. In conclusion, intense fish farming in large- scale aquaculture can lead to a significant increase in saprolegniosis. S. parasitica detection assay developed here will enable early detection of S. parasitica in fish farms and therefore help in improving poor farming conditions.

The Chinese pond mussel Sinanodonta woodiana is an invasive bivalve species present in the flowing and standing waters of most of Europe. In a series of physiological and toxicological experiments, we examined if invasive mussel (S. woodiana) is more tolerant to environmental pressures, in comparison with indigenous mussel (Anodonta anatina). The activity of both electron transport system (ETS), and oxidative stress-related enzymes (catalase, glutathione reductase and cholinesterases), as well as the comparison of the activity of multixenobiotic resistance mechanisms (MXR) in gill tissue, were used to evaluate a physiological response on water temperature extremes, feeding and starving, and zinc contaminations. S. woodiana showed higher tolerance to metal contaminations as well as to increased water temperature which is an important physiological benefit for further invasion success, especially in the context of climate changes.

Mycoviruses, mostly dsRNA or +ssRNA subcellular pathogens of fungi, often cause asymptomatic, latent infections of their hosts. Some of them, however, have debilitating effect, reducing fitness and/or reproductive capability of the infected fungus. This might be effective in biological control of pathogenic fungi, such as Cryphonectria parasitica, a phytopathogenic Ascomycota native to eastern Asia which causes the chestnut blight disease. Cryphonectria hypovirus 1 (CHV1) is a mycovirus which lacks an extracellular phase and reduces the fitness of its host, as well as its aggressiveness against the chestnut post CHV1 infection, a phenomenon called hypovirulence. It is transmitted vertically via asexual spores and horizontally over the anastomoses between genetically compatible mycelia (heterokaryonic or vegetative compatibility). This is important because the only way to successfully control chestnut blight disease is via biological control of the fungus, utilizing CHV1. Approximately a decade ago, CHV1 prevalence in Croatia was determined to be between 13% (in coastal regions) and up to about 60% in some continental populations. Recently, we have revisited three populations and surveyed them for CHV1 prevalence and genetic diversity (vegetative compatibility – vc) of the fungus, which is a major determinant of the successful horizontal spread of the CHV1 and biological control of the disease. CHV1 prevalence increased in coastal population Buje from 12, 7% to 19, 5% and decreased in continental populations: from 50, 8% to 29, 9% in Hrvatska Kostajnica and from 44, 1% to 33, 3% in Ozalj. At the same time vc type diversity increased in all populations: previously between five and eight vc types were observed in the three mentioned populations, while recently as many as 19 different vc types have been recorded, Shannon diversity index reflecting that increase. In our continued effort to monitor the disease development with shorter sampling intervals, in the last three years we have revisited the same locations and observed somewhat quicker pace of canker area increase in population with lower CVH1 prevalence (Buje) than in populations with higher CHV1 prevalence. This obviously has a major impact on the success of biological control, since CHV1 seems to decrease in prevalence in both continental populations. All this might indicate collapse of the established biological control in those populations. The reason behind this is the increase in genetic diversity of the C. parasitica which negatively affects successful horizontal transfer of the virus.

Viruses represent an important biotic stress and are known to induce epigenetic changes in plants and animals, but knowledge about their effect on fungal genome methylation levels is still scarce. To study epigenetic changes in a fungus infected with a mycovirus we used a model pathosystem for mycovirus-host interactions: Cryphonectria hypovirus 1 (CHV1) and its host, phythopathogenic fungus Cryphonectria parasitica. This system is very important since C. parasitica causes chestnut blight, a devastating disease of chestnut trees which can be biologically controlled with CHV1. In C. parasitica deregulation of methylation cycle enzymes upon virus infection was observed previously. In order to study if those changes are reflected on global cytosine methylation levels of infected fungus, we used methylation-sensitive amplification polymorphism (MSAP) method. Furthermore, to assess if there is a difference in the effects of different CHV1 strains and/or subtypes on different fungus genotypes, we used six CHV1 strains of different virus subtypes transferred into three different C. parasitica isolates. Following the infection, we have observed an increase in the number and diversity of methylated, hemi-methylated, and total MSAP markers compared to virus-free controls, indicating that CHV1 infection affects the methylation pattern of the C. parasitica genome. Since the increase in methylation levels correlated with previously observed CHV1-induced reduction of fungal growth in vitro, we hypothesize that CHV1-induced increase in C. parasitica genome methylation is likely to be the virulence determinant of the virus, rather than being the defensive mechanism of the fungus. Furthermore, CHV1 subtypes did not consistently show either strong or weak effect on all studied fungal isolates. Rather, the severity of CHV1 effect on methylation levels of infected C. parasitica isolates depended both on individual CHV1 strain, and on the combination of host and virus genomes. These novel findings broaden our knowledge about CHV1 biology and practicality of different viral strains to be used in human-mediated biocontrol.