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Saprolegnia parasitica causes saprolegniosis, a disease responsible for significant economic losses in aquaculture and declines of fish populations in the wild, but the knowledge of its distribution and prevalence in the environment is limited. We developed a fast, sensitive and specific S. parasitica droplet digital PCR (ddPCR) assay and demonstrated its applicability for the detection and quantification of the pathogen in environmental samples: swab DNA collected from the host (trout skin, surface of eggs) and environmental DNA extracted from water. The developed assay was used to assess how abiotic (i.e. physico-chemical parameters of the water) and biotic (health status of the host) factors influence the S. parasitica load in the environment. The pathogen load in water samples was positively correlated with some site-specific abiotic parameters such as electrical conductivity (EC) and calcium, while fluorides were negatively correlated, suggesting that physico-chemical parameters are important for determining S. parasitica load in natural waters. Furthermore, skin swabs of injured trout had significantly higher pathogen load than swabs collected from healthy fish, confirming that S. parasitica is a widespread opportunistic pathogen. Our results provide new insights into various environmental factors that influence the distribution and abundance of S. parasitica .

(Oomycota) causes saprolegniosis and poses significant ecological and economic challenges in aquaculture. Experimental research on this pathogen is constrained by the lack of reliable long-term preservation methods, as routine maintenance by serial subculturing is labor-intensive and may result in genetic and phenotypic instability. Deep eutectic solvents (DESs), tunable low-melting mixtures, have recently gained attention as alternative cryoprotectants. However, their application has not been evaluated in oomycetes. Here, twelve glycerol-based two- and multicomponent DESs were assessed for cryopreservation of at -80 °C and compared with glycerol as a conventional cryoprotectant. Cryopreservation efficiency was assessed based on post-thaw survival and mycelial regeneration. Several two-component DESs, particularly glycerol-trehalose, supported 100% survival and high post-thaw mycelial regeneration, performing comparably to glycerol under the tested conditions. Shorter pre-incubation (30 min vs. 1 h and 3 h) and controlled-rate freezing (vs. direct freezing) significantly improved post-thaw growth. Although survival remained 100% under optimized conditions, extending storage from 7 to 32 days significantly reduced mycelial regeneration in the glycerol-trehalose treatment, indicating that survival alone, as done in existing literature, does not reflect physiological recovery. Overall, our results support the use of selected DESs as alternative cryoprotectants in oomycetes and contribute to the development of cryopreservation strategies for .

Crayfish are a keystone species of freshwater ecosystems and a successful invasive species. However, their pathogens, including viruses, remain understudied. The aim of this study was to analyze the virome of the invasive signal crayfish (Pacifastacus leniusculus) and to elucidate the potential differences in viral composition and abundance along its invasion range in the Korana River, Croatia. By the high-throughput sequencing of ribosomal RNA, depleted total RNA isolated from the crayfish hepatopancreas, and subsequent sequence data analysis, we identified novel and divergent RNA viruses, including signal crayfish-associated reo-like, hepe-like, toti-like, and picorna-like viruses, phylogenetically related to viruses previously associated with crustacean hosts. The patterns of reads abundance and calculated nucleotide diversities of the detected viral sequences varied along the invasion range. This could indicate the possible influence of different factors and processes on signal crayfish virome composition: e.g., the differences in signal crayfish population density, the non-random dispersal of host individuals from the core to the invasion fronts, and the 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 sets foundations for understanding the potential risk of virus transmissions as a result of this invader’s dispersal.

Background The microbiome plays an important role in biological invasions, since it affects various interactions between host and environment. However, most studies focus on the bacteriome, insufficiently addressing other components of the microbiome such as the mycobiome. Microbial fungi are among the most damaging pathogens in freshwater crayfish populations, colonizing and infecting both native and invasive crayfish species. Invading crayfish may transmit novel fungal species to native populations, but also, dispersal process and characteristics of the novel environment may affect the invaders’ mycobiome composition, directly and indirectly affecting their fitness and invasion success. This study analyzes the mycobiome of a successful invader in Europe, the signal crayfish, using the ITS rRNA amplicon sequencing approach. We explored the mycobiomes of crayfish samples (exoskeletal biofilm, hemolymph, hepatopancreas, intestine), compared them to environmental samples (water, sediment), and examined the differences in fungal diversity and abundance between upstream and downstream segments of the signal crayfish invasion range in the Korana River, Croatia. Results A low number of ASVs (indicating low abundance and/or diversity of fungal taxa) was obtained in hemolymph and hepatopancreas samples. Thus, only exoskeleton, intestine, sediment and water samples were analyzed further. Significant differences were recorded between their mycobiomes, confirming their uniqueness. Generally, environmental mycobiomes showed higher diversity than crayfish-associated mycobiomes. The intestinal mycobiome showed significantly lower richness compared to other mycobiomes. Significant differences in the diversity of sediment and exoskeletal mycobiomes were recorded between different river segments (but not for water and intestinal mycobiomes). Together with the high observed portion of shared ASVs between sediment and exoskeleton, this indicates that the environment (i.e. sediment mycobiome) at least partly shapes the exoskeletal mycobiome of crayfish. Conclusion This study presents the first data on crayfish-associated fungal communities across different tissues, which is valuable given the lack of studies on the crayfish mycobiome. We demonstrate significant differences in the crayfish exoskeletal mycobiome along the invasion range, suggesting that different local environmental conditions may shape the exoskeletal mycobiome during range expansion, while the mycobiome of the internal organ (intestine) remained more stable. Our results provide a basis for assessing how the mycobiome contributes to the overall health of the signal crayfish and its further invasion success.

Essential oils from Mediterranean wild plants are widely used, but the hydrodistillation residues produced in parallel with these essential oils are significantly understudied and underexploited. Since there are only fragmentary data in the literature, we have, for the first time, systematically analyzed the chemical composition of the by-products obtained after hydrodistillation of sage, bay laurel, and rosemary leaves, i.e., hydrolates, water residues, and solid residues. The chemical composition of the hydrolates changed compared to their respective essential oils towards the dominance of more hydrophilic, oxygenated compounds, such as camphor in sage, 1,8-cineole in bay laurel, and berbenone in rosemary. However, some compounds, mostly sesquiterpenes, which were present in considerable amounts in essential oils, were absent or only present in very small amounts in the hydrolates. Furthermore, both the water and the solid residues were rich in polyphenols, such as procyanidins in bay laurel and rosmarinic acid in rosemary and sage. In conclusion, we demonstrate the valuable chemical composition of sage, rosemary, and bay laurel hydrodistillation by-products and discuss a wide range of their possible applications.

We have investigated the changes in the microbial communities on the surface of trout eggs and the skin of adult trout in relation to the presence of Saprolegnia parasitica. This pathogen causes saprolegniosis, a disease responsible for significant losses in salmonid farms and hatcheries. It is known from other disease systems that the host-associated microbiome plays a crucial role in the defence against pathogens, but if the pathogen predominates, this can lead to dysbiosis. However, analyses of the effects of S. parasitica on the diversity, composition, and function of microbial communities on fish skin and eggs are scarce. Thus, we have collected skin swabs from injured and healthy trout (N = 12), which differed in S. parasitica load, from three different fish farms in Croatia (Kostanjevac, Radovan, and Solin), while trout egg samples (N = 12) were infected with S. parasitica in the laboratory. Illumina sequencing of the V4 region of the 16S rRNA marker gene showed that infection with S. parasitica reduced the microbial diversity on the surface of the eggs, as evidenced by decreased Pielou’s evenness and Shannon’s indices. We further determined whether the bacterial genera with a relative abundance of >5.0% in the egg/skin samples were present at significantly different abundances in relation to the presence of S. parasitica. The results have shown that some genera, such as Pseudomonas and Flavobacterium, decreased significantly in the presence of the pathogen on the egg surface. On the other hand, some bacterial taxa, such as Acinetobacter and Janthinobacterium, as well as Aeromonas, were more abundant on the diseased eggs and the injured trout skin, respectively. Finally, beta diversity analyses (weighted UniFrac, unweighted UniFrac, Bray–Curtis) have shown that the sampling location (i.e., fish farm), along with S. parasitica infection status, also has a significant influence on the microbial communities’ composition on the trout skin and eggs, demonstrating the strong influence of the environment on the shaping of the host surface microbiome. Overall, we have shown that the presence of S. parasitica was associated with changes in the diversity and structure of the trout skin/egg microbiome. The results obtained could support the development of new strategies for the management of saprolegniosis in aquaculture.

The conservation of gray wolf (Canis lupus) and its coexistence with humans presents a challenge and requires continuous monitoring and management efforts. One of the non-invasive methods that produces high-quality wolf monitoring datasets is camera trapping. We present a novel monitoring approach where camera traps are positioned on wildlife crossing structures that channel the animals, thereby increasing trapping success and increasing the cost-efficiency of the method. In this way we have followed abundance trends of five wolf packs whose home ranges are intersected by a motorway which spans throughout the wolf distribution range in Croatia. During the five-year monitoring of six green bridges we have recorded 28 250 camera-events, 132 with wolves. Four viaducts were monitored for two years, recording 4914 camera-events, 185 with wolves. We have detected a negative abundance trend of the monitored Croatian wolf packs since 2011, especially severe in the northern part of the study area. Further, we have pinpointed the legal cull as probable major negative influence on the wolf pack abundance trends (linear regression, r2 > 0.75, P < 0.05). Using the same approach we did not find evidence for a negative impact of wolves on the prey populations, both wild ungulates and livestock. We encourage strict protection of wolf in Croatia until there is more data proving population stability. In conclusion, quantitative methods, such as the one presented here, should be used as much as possible when assessing wolf abundance trends.

The effect of different hydrodistillation pretreatments, namely, reflux extraction, reflux extraction with the addition of cell wall-degrading enzymes, and ultrasound, on the yield and chemical composition of essential oils of sage, bay laurel, and rosemary was examined. All pretreatments improved essential oil yield compared to no-pretreatment control (40–64% yield increase), while the oil quality remained mostly unchanged (as shown by statistical analysis of GC-MS results). However, enzyme-assisted reflux extraction pretreatment did not significantly outperform reflux extraction (no-enzyme control), suggesting that the observed yield increase was mostly a consequence of reflux extraction and enzymatic activity had only a minute effect. Thus, we show that ultrasound and reflux extraction pretreatments are beneficial in the production of essential oils of selected Mediterranean plants, but the application of enzymes has to be carefully re-evaluated.

Green bridges are used to decrease highly negative impact of roads/highways on wildlife populations and their effectiveness is evaluated by various monitoring methods. Based on the 3-year monitoring of four Croatian green bridges, we compared the effectiveness of three indirect monitoring methods: track-pads, camera traps and active infrared (IR) trail monitoring system. The ability of the methods to detect different species and to give good estimation of number of animal crossings was analyzed. The accuracy of species detection by track-pad method was influenced by granulometric composition of track-pad material, with the best results obtained with higher percentage of silt and clay. We compared the species composition determined by track-pad and camera trap methods and found that monitoring by tracks underestimated the ratio of small canids, while camera traps underestimated the ratio of roe deer. Regarding total number of recorder events, active IR detectors recorded from 11 to 19 times more events then camera traps and app. 80% of them were not caused by animal crossings. Camera trap method underestimated the real number of total events. Therefore, an algorithm for filtration of the IR dataset was developed for approximation of the real number of crossings. Presented results are valuable for future monitoring of wildlife crossings in Croatia and elsewhere, since advantages and disadvantages of used monitoring methods are shown. In conclusion, different methods should be chosen/combined depending on the aims of the particular monitoring study.

Saprolegnia parasitica, the causative agent of saprolegniosis in fish, and Aphanomyces astaci, the causative agent of crayfish plague, are oomycete pathogens that cause economic losses in aquaculture. Since toxic chemicals are currently used to control them, we aimed to investigate their inhibition by essential oils of sage, rosemary, and bay laurel as environmentally acceptable alternatives. Gas Chromatography–Mass Spectrometry (GC–MS) analysis showed that the essential oils tested were rich in bioactive volatiles, mainly monoterpenes. Mycelium and zoospores of A. astaci were more sensitive compared to those of S. parasitica, where only sage essential oil completely inhibited mycelial growth. EC50 values (i.e., concentrations of samples at which the growth was inhibited by 50%) for mycelial growth determined by the radial growth inhibition assay were 0.031–0.098 µL/mL for A. astaci and 0.040 µL/mL for S. parasitica. EC50 values determined by the zoospore germination inhibition assay were 0.007–0.049 µL/mL for A. astaci and 0.012–0.063 µL/mL for S. parasitica. The observed inhibition, most pronounced for sage essential oil, could be partly due to dominant constituents of the essential oils, such as camphor, but more likely resulted from a synergistic effect of multiple compounds. Our results may serve as a basis for in vivo experiments and the development of environmentally friendly methods to control oomycete pathogens in aquaculture.