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Crayfish are keystone species of freshwater ecosystems and successful invasive species, however, their pathogens, including viruses, remain understudied. The aim of this study was to analyze the virome of one of the most successful invader in European freshwaters, the signal crayfish (Pacifastacus leniusculus), and shed light on the potential differences in viral sequence composition along its invasion range in the Korana River, Croatia. 120 hepatopancreas tissues were dissected from signal crayfish sampled along its invasion range at four sites: upstream invasion front and core and downstream invasion front and core. Total RNA was isolated, depleted of ribosomal RNA and used for high- throughput sequencing. Subsequent bioinformatics analysis based on protein similarity search of de novo assembled contigs identified novel RNA viruses belonging to reo-like, hepe-like, toti-like and picorna-like viral clades. Sequences of novel signal crayfish associated viruses had relatively low levels of similarity to known viral sequences, however they were phylogenetically related to viruses previously associated with crustacean hosts. The patterns of viral reads abundance and calculated nucleotide diversities of the detected viral sequences varied between the sampling locations along the invasion range, which could indicate the possible influence of different factors and processes on signal crayfish virome composition: e.g., the differences in signal crayfish population density and transfer of viruses from the native co-occurring and phylogenetically related crayfish species. The study reveals a high, previously undiscovered diversity of divergent RNA viruses associated with signal crayfish and set foundations for understanding the potential risk of virus transmissions as a result of this invader’s dispersal.

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.

Tomato is important crop, hosting at least 312 viruses, satellites or viroids, and a pivotal research species in the Croatian-Slovenian project “Nanopore high throughput sequencing for the resolution of problems in the plant pathogen epidemiology and diagnostics“. Main sampling sites are greenhouses in the continental Croatia (Vidovec, Sedlarica). In 2021, tomatoes from the coastal region (Turanj) and symptomatic peppers neighboring tomatoes were also collected. Leaves were used for lateral flow tests (LFT) and nucleic acids extraction. For nanopore high throughput sequencing (HTS) library preparation, representative tomato DNase treated CTAB-extracts were pooled, rRNA depleted and polyA tailed. Based on HTS and LFT results, tomato (31) and pepper (3) samples were individually tested by RT-PCR with primers for pospiviroids, potato virus S (PVS), potato virus Y (PVY), tomato spotted wilt virus (TSWV), cucumber mosaic virus (CMV) and CMV satellite. PVS consensus genome from a tomato in Vidovec (ON468562) was highly similar to a potato isolate from the Netherlands (MF418030) belonging to the ordinary (PVSO) strain. That tomato was also infected with PVY (ON505007), as were others from Vidovec and Sedlarica (ON505008). Their genomes were most similar to a Slovakian tomato isolate (MW685827) clustering with PVY-NTN strains. A severely diseased tomato from Turanj harbored only TSWV with segments S and M highly similar to isolates from Italy (DQ376178 and MH756624, respectively) and L to a Spanish isolate (KP008130). Peppers were either infected by CMV (two from Vidovec) or by potyviruses (positive in LFT for potyvirus group, Agdia, species pending identification) and a pospiviroid (one from Sedlarica and Vidovec). A pospiviroid amplicon was also obtained for one of the Sedlarica PVY+ tomatoes. Partial sequences suggest citrus exocortis viroid (CEVd) is distributed in tomatoes and peppers but this needs to be confirmed. This first glimpse into the Croatian tomato and pepper viromes suggests PVY was predominant in the continental tomatoes with a virus or a viroid occasionally in coinfections. A few peppers tested so far had viromes constituting of CMV, potyviruses and pospiviroids.