Search

The introduction of invasive alien species into sensitive aquatic ecosystems contributes to the loss of freshwater biodiversity by altering ecosystem structure and services, and by transferring new, potentially unfamiliar microbial pathogens to native species. However, the microbes in the invaded ecosystem also interact with the microbiome of the invader, which can affect invader’s physiology, immune status, health, and fitness. Additionally, these interactions between microbes can also be affected by changing environmental conditions, further affecting the invader and ultimately the invasion success. We analyzed the bacteriome and mycobiome of the signal crayfish, Pacifastacus leniusculus, one of the most successful freshwater invertebrate invaders in Europe. We collected the signal crayfish individuals, water and sediment samples, along this species' invasion range in the Korana River, Croatia. Using 16S and ITS rRNA amplicon sequencing, we examined the microbiomes of different crayfish tissues (exoskeleton, hemolymph, hepatopancreas, intestine), and environment (water, sediment). We investigated the differences in these microbiomes between two different microenvironments in the Korana River, i.e. upstream and downstream river segments. Our results showed significant differences between all crayfish and environmental microbiomes, confirming their uniqueness. Generally, the environmental microbiomes, and the crayfish exoskeletal microbiome (which is in continuous contact with the environment), exhibited higher taxonomic richness than the microbiomes of internal crayfish tissues. The exoskeletal and hemolymph microbiomes differed between two river segments, indicating that they are at least partly shaped by the environment. Our results provide insight into the microbiome of a successful crayfish invader and report its changes during dispersal through different microenvironments.

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.

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.

Crayfish plague is responsible for decimating populations of native crayfish species in Europe and elsewhere, making Aphanomyces astaci one of the 100 worst invasive species in the world. I will present the results of our research on A. astaci in recent years, including advances in monitoring approaches, new insights into the biology/ecology of the pathogen, and the development of environmentally friendly control strategies. We have shown that A. astaci can be detected in the microbial biofilm collected from the cuticle of individual live crayfish. Thus, we contribute to the growing trend of non-destructive methods for monitoring pathogens in freshwaters. The method has yet to be compared to the direct pathogen detection in water, but it provides results consistent with conventional detection of A. astaci in cuticle samples. For the past five years we have been using the novel method to monitor A. astaci in natural crayfish populations in Croatia. We also investigated the relationship between native crayfish hosts (Austropotamobius torrentium and Pontastacus leptodactylus) and A. astaci group B at different water temperatures. Preliminary data from the infection trials showed lower A. astaci-induced mortality of native crayfish at elevated water temperature (22 vs. 18°C), suggesting that the temperature increase had a greater negative impact on A. astaci than on the crayfish. These results may help predict the spread of crayfish plague in future climate change scenarios. Finally, we are actively developing environmentally friendly methods to control A. astaci as an alternative to the toxic chemicals currently used in astaciculture. We have shown that the application of bioactive plant products such as propolis and essential oils from Mediterranean wild plants can inhibit mycelium and zoospores of A. astaci, while commensal bacterial isolates from the cuticle of crayfish, mainly pseudomonads, can inhibit mycelial growth.

Considering the major anthropogenic pressures affecting marine ecosystems, assessment of the “health status” of the coastal zones is an important topic, especially considering that 50% of the world population inhabits those areas. There has been a number of directives implemented by Mediterranean countries regarding protection and preservation of marine coastal areas. Unfortunately, none of them mention the potential contribution of microorganisms in marine ecosystems health quality assessment. Microorganisms play pivotal role in functioning of the whole marine ecosystem by mediating biogeochemical cycling of all major elements, C sequestration and elimination of pollutant. In the same time microorganisms are characterized by their fast responsiveness to environmental changes, hence anthropogenically-driven pressures in coastal areas could cause changes on the structural and functional level of microbial communities, eventually altering the trophic structure of the whole food web. Through the multidisciplinary and innovative approach, using different molecular tools, MicroLink project, financed by the Croatian Science Foundation, aims to emphasize the need to define potential contribution of microorganisms as quality indicators of marine ecosystems health. Sampling of the sediment material was performed in seven different harbors settled along the Adriatic coast, which represent sites under multiple pollution pressures. Comprehensive physico- chemical characterization of the sediment was used for their pre-classification in different groups thus representing sediments with different “anthropogenic disturbance gradient”. This data sets were further correlated with benthic microbial assemblages data obtained by the multi- layer, multi-trophic molecular-based analyses (structure/diversity/function of the benthic bacterial/archaeal/fungal/protists communities). MicroLink project is going to provide guidelines for future monitoring of anthropogenic pressures in coastal zones in order to improve marine environment policies.

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.

Water hydrodistillation residues produced in parallel with bay laurel, rosemary and sage essential oils contain a remarkable source of bioactive molecules. Despite rich bioactive content, these fractions considered as waste and are often underexploited. To date, there are no studies reporting on the chemical composition of water residues remaining after hydrodistillation of bay laurel and sage leaves, while there are only few reports demonstrating the valuable composition of other hydrodistillation co-products of these plants as a valuable secondary source of polyphenol components. We have used different pretreatments prior to hydrodistillation, i.e. reflux extraction, ultrasound and enzyme assisted extraction. It was described the effect of these pretreatments on the composition of polyphenolic compounds in the water hydrodisillation residues. HPLC analysis showed that the water hydrodistillation residues of bay laurel, rosemary and sage were richest in polyphenols. In bay laurel water hydodistillation residues, procyanidins represented the dominant components, with procyanidin dimer I and II (up to 4.65 mg/g), tetramer II (up to 5.43 mg/g), trimer II, III and IV (up to 4.37, 4.92, 13.15 mg/g, respectively) as major compounds. Among other components, epicatechin-hexoside, epicatechin and epicatechin- 3-O-gallate (up to 3.56, 1.60, 1.07 mg/g, respectively) were also detected in significant quantities. In sage hydrodistillation water residues, rosmarinic acid (up to 5.39 mg/g) was major compounds, followed by caffeic acid methyl ester (up to mg/g) and epicatechin (up to 2.06 mg/g). Finally, in rosemary hydrodistillation water residues rosmarinic acid, galocatehin, p- coumaric acid and syringic acid (up to 7.57, 4.83, 2.19, 3.25 mg/g, respectively) were most represented components. In general, the pretreatments didn’t significantly affect the phenolic composition of water hydrodistillation residues (p

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.

The essential oil manufacture produces a significant quantity of co-products. These potentially valuable hydrodistillation residues include hydrolates contain a remarkable source of bioactive molecules. In this study, we have focused on aromatic Mediterranean plants rich in volatile compounds: bay laurel, rosemary and sage. Their essential oils are frequently used, but hydrodistillation residues produced in parallel with these essential oils are much less explored. Despite rich bioactive content, these fractions are often underexploited or even considered waste. Increasing market demand for essential oils resulted in the development of many innovative methods for improvement of their yield and composition. Traditional extraction technologies prior hydrodistillation mainly related to the high temperature mainted for long extraction periods and the consumption of hazardous organic solvents as well as low extraction efficiency of desired compounds. So, we have analysed different pretreatments prior to hydrodistillation i.e. reflux extraction, ultrasound and enzyme assisted extraction on chemical composition of hydrolates in bay laurel, rosemary and sage. GC-MS analysis showed that the hydrolates of all plants were richest in oxygenated monoterpenes: up to 46%, 42% and 30% of total peak area in bay laurel, rosemary and sage hydrolates, respectively. Sesquiterpenes were also well represented: up to 42%, and 10% of total peak area in rosemary and bay laurel hydrolates, respectively. Dominant compounds were confirmed: 1, 8-cineole, camphor, borneol and berbenone in rosemary hydrolates, 1, 8-cineole, linalool, camphor, α- and β-terpineol, methyleugenol and eugenol in bay laurel hydrolates and 1, 8-cineole, camphor, borneol and berbenone in sage hydrolates. In general, the pretreatments didn’t significantly affect the volatile compounds of hydrolates (p

Saprolegnia parasitica, uzročnik saprolegnioze salmonidnih riba i Aphanomyces astaci, uzročnik račje kuge, oomicetni su patogeni koji uzrokuju značajne ekonomske štete u akvakulturi. Budući da su kemikalije koje se koriste za njihovu kontrolu štetne za ljude i okoliš, cilj ovog istraživanja je istražiti ekološki prihvatljive metode njihove inhibicije. Testiran je inhibicijski potencijal eteričnih ulja odabranog samoniklog mediteranskog bilja, kadulje (Salvia officinalis L.), ružmarina (Rosmarinus officinalis L.) i lovora (Laurus nobilis L.) prema rastu micelija i klijavosti zoospora patogena te su određene EC50 vrijednosti. Testirana eterična ulja bila su bogata hlapivim bioaktivnim komponentama, pretežno monoterpenima, te su pokazala inhibicijsku aktivnost prema rastu micelija i klijavosti zoospora, naročito eterično ulje kadulje koje je imalo najsnažnije djelovanje. To se dijelom može pripisati dominantnim komponentama, primjerice kamforu, ali još vjerojatnije sinergističkom djelovanju velikog broja komponenti. Micelij i zoospore patogena A. astaci bile su osjetljivije od istih razvojnih stadija patogena S. parasitica, gdje je samo eterično ulje kadulje u potpunosti inhibiralo rast micelija. Zaključno, dobiveni rezultati mogu poslužiti kao osnova budućih in vivo istraživanja i razvoja ekološki prihvatljivih metoda kontrole patogenih oomiceta u akvakulturi.

Oomicetni mikroorganizam Saprolegnia parasitica oportunistički je patogen koji izaziva saprolegniozu, bolest koja uzrokuje velike gubitke u salmonidnoj akvakulturi. Neke od kemikalija koje su se do nedavno koristile za suzbijanje saprolegnioze zabranjene su u Europskoj uniji zbog kancerogenosti i toksičnosti te je potreban razvoj novih, ekološki prihvatljivih tretmana. Plazmom aktivirana voda (engl. Plasma Activated Water, PAW) ima široki raspon primjena zbog dokazanog antimikrobnog učinka i niskog rizika za zdravlje ljudi i okoliš. S obzirom da primjena PAW još nije testirana u akvakulturi, cilj ovog rada bio je po prvi puta istražiti može li PAW inhibirati rast micelija vrste S. parasitica. Micelij je tretiran s PAW kroz 30 i 60 minuta te su rezultati uspoređeni sa tretmanom vodikovim peroksidom kao pozitivnom kontrolom i vodom (engl. Artificial Water, AW) kao negativnom kontrolom. Utvrđeno je da tretiranje micelija s PAW kroz 60 minuta uzrokuje sporiji rast patogena (do 33 % smanjeni rast u odnosu na negativnu kontrolu). U usporedbi s tim, rast micelija bio je potpuno inhibiran nakon tretmana vodikovim peroksidom koncentracije 1000 mg/L u trajanju od 60 minuta. Zaključno, iako utjecaj PAW na micelij S. parasitica nije bio jednako snažan kao djelovanje vodikovog peroksida, ovo istraživanje otvorilo je mogućnost primjene PAW, kao ekološki prihvatljivog tretmana, u akvakulturi.

Mikrobiom ima ključnu ulogu u održavanju zdravlja domaćina i može imati značajan utjecaj na njegovu podložnost bolesti. Sve više dokaza ukazuje na ulogu mikrobioma u biološkim invazijama, budući da mikrobi mogu utjecati na brojne interakcije između domaćina i okoliša te na ekologiju domaćina. S druge strane, proces širenja i karakteristike novog okoliša mogu utjecati na strukturu i sastav mikrobioma invazivne vrste koja se širi, što može izravno i neizravno utjecati na njen fitnes i invazivni uspjeh. Invazivne strane vrste jedan su od najznačajnijih uzroka gubitka biološke raznolikosti, posebice u slatkovodnim ekosustavima. Uspješne invazivne strane vrste, poput signalnoga raka, pokazuju promjene u karakteristikama populacije duž invazivnog areala, što ukazuje da jedinke koje se akumuliraju na rubovima širenja (invazijskim frontama) mogu posjedovati specifične osobine koje olakšavaju širenje i/ili uspostavljanje populacije u novom okolišu, što u konačnici može utjecati na budući invazivni uspjeh vrste. U ovom smo istraživanju analizirali postoje li takve razlike u sastavu mikrobioma vrste, tj. javljaju li se razlike u mikrobiomu zbog karakteristika populacije (invazijsko središte vs. fronta) ili zbog karakteristika mikrostaništa (uzvodni dio rijeke vs. nizvodni). Prikupili smo uzorke egzoskeletnog biofilma, hemolimfe, hepatopankreasa i crijeva rakova sa četiri lokacije duž rijeke Korane u Hrvatskoj i analizirali mikrobni sastav 16S rRNA sekvenciranjem. Naši rezultati otkrivaju da se alfa raznolikost značajno razlikovala između različitih lokacija samo u slučaju mikrobioma hemolimfe. Nadalje, hemolimfa je također pokazala značajno različitu beta raznolikost između uzvodne i nizvodne lokacije, dok su se mikrobiomi hepatopankreasa i crijeva značajno razlikovali između populacija invazijskog središta i fronte. Mikrobiom egzoskeleta pokazao je značajno različitu beta raznolikost među svim lokacijama. Naši rezultati sugeriraju da je egzoskeletni mikrobiom oblikovan i lokalnim okolišnim karakteristikama i gustoćom rakova/strukturom populacije, dok su razlike u mikrobiomu hemolimfe vjerojatnije pod utjecajem karakteristika uzvodnog (ruralnog) i nizvodnog (industrijskog) okoliša rijeke. Nadalje, mikrobiomi hepatopankreasa i crijeva mogu biti određeni gustoćom populacije, budući da ona utječe na režim hranjenja i fiziološko stanje rakova, ali mogu ovisiti i o uvjetima okoliša (tj. tipu dostupne hrane).

Introduction Saprolegnia parasitica and Aphanomyces astaci (Oomycetes) are pathogens with negative impact in freshwater aquaculture. Saprolegnia parasitica causes saprolegniosis, a disease affecting mostly salmonid fishes, while A. astaci causes crayfish plague. Chemicals harmful to humans and the environment are being used globally in aquaculture facilities to prevent the spread of these pathogens. Thus, the development of new, ecologically acceptable methods for their control is urgently needed. The aim of this study was to examine whether propolis, known for its antimicrobial properties and stimulatory effect on the host immune system, can inhibit the life stages of A. astaci and S. parasitica in vitro. Materials and methods Two propolis formulations were used: P1 (pure propolis in ethanol), and P2 (propolis in ethanol with the addition of sage and pepper mint). Their chemical composition was determined by ultra- performance liquid chromatography-tandem mass spectrometry (UPLC- MS/MS) and gas chromatography/mass spectrometry (GC-MS). Two main life stages of the oomycete pathogens, namely mycelium and zoospores, were treated with propolis formulations and their main components, pinocembrin and chrysin. The effect of these compounds on mycelial growth was assessed using the disk diffusion assay and radial growth inhibition test, followed by determination of EC50 values. To test the inhibition of zoospore germination, sporulation was induced by washing the mycelium grown in liquid PG1 with natural water. Test compounds were added to the resulting zoospore suspension in a range of concentrations, and their effect was assessed by comparing the percentage of germinated zoospores with the results of control experiments. Malachite green, with known toxicity towards A. astaci and S. parasitica, was used as a positive control. Results Both propolis samples were rich in volatile and polyphenol compounds. As shown by UPLC-MS/MS analysis, chrysin was most abundant in both propolis samples (up to app. 50 µg/mL), followed by pinocembrin in P2 (4 µg/mL). Significantly higher number of different volatile components was found by GC-MS in P2 than in P1 propolis sample, probably because of addition of sage and pepper mint. In case of S. parasitica, both propolis formulations moderately inhibited the mycelial growth: EC50 (P1) = 206.2 µg/mL, EC50 (P2) = 206.6 µg/mL for P2, when compared with a known inhibitory compound malachite green (EC50 = 0.1 µg/mL). In contrast, EC50 values for P1, P2, and malachite green for mycelial growth inhibition of A. astaci were up to 36 times lower (5.6, 8.6, and 0.02 µg/mL, respectively). Considering zoospore germination, the minimal concentration that caused complete inhibition of S. parasitica germination (minimum inhibitory concentration, MIC) was 62.0 and 39.0 µg/mL for P1 and P2 propolis samples, respectively, while for malachite green it was 0.08 µg/mL. Similar results were obtained for Aphanomyces astaci zoospores: MIC was 31.3 and 39.0 µg/mL for P1 and P2, respectively, while malachite green inhibited zoospore germination at a concentration of 0.04 µg/mL. The inhibitory potential of main propolis components, chrysin and pinocembrin, was also tested but these compounds didn´t show significant anti-oomycete activity when applied in concentrations determined in propolis samples. This suggests that the observed anti-oomycete activity of propolis formulations was probably due to synergistic activity of a number of minor bioactive components. Conclusions Our results demonstrate the inhibitory activity of propolis towards life stages of pathogenic oomycetes S. parasitica and A. astaci. Interestingly, S. parasitica mycelium was relatively resistant to propolis in comparison to mycelium of A. astaci, but zoospores of both species (as main infection agents) were highly susceptible. Future in vivo testing is needed to explore the suggested host- protective effects of propolis during the infection process and to demonstrate its applicability as a feed additive in the fish farms.

Solid hydrodistillation residues produced in parallel with essential oils of bay laurel, rosemary and sage are much less explored or even considered waste despite rich polyphenolic content. Prior to hydrodistillation, plant material is often treated with ultrasound, microwaves or enzymes, in order to disrupt the cell wall and improve the access of solvent to the cell content, thus enhancing the release of bioactive compounds. When comparing different extraction procedures used to process solid hydrodistillation residues like enzyme assisted or reflux extraction, ultrasound application is considered gentler in comparison with other extraction procedures, working at lower temperature and for shorter time, which makes it more suitable to preserve polyphenols from thermal degradation. HPLC analysis showed that the solid hydrodistillation residues of all plants were richest in polyphenols. In bay laurel solid hydrodistillation residues extract, procyanidins represented the dominant components, with procyanidin trimer, dimer and tetramer as major compounds. Among other components, epicatechin, epicatechin-hexoside and epicatechingallate were also detected in significant quantities. In sage solid hydrodistillation residues extract, salvianolic acid I and K were major compounds, followed by luteolin-3-O-glucuronide. Finally, in rosemary solid hydrodistillation residues extract, rosmarinic acid was the most represented component. Applied solvents didn’t show significant variations (p

Invasive species are among the major drivers of biodiversity loss and impairment of ecosystem services worldwide, but our understanding of their invasion success and dynamics still has many gaps. For instance, although it is known that host-associated microbial communities may significantly affect an individual’s health and fitness, the current studies on invasive species are mainly focused on pathogenic microbes. In contrast, the effects of the remaining majority of microbial communities on the invasion process are almost completely unexplored. We have analyzed the microbiome of one of the most successful crayfish invaders in Europe, the signal crayfish in the Korana River, Croatia. Using a 16S rRNA gene amplicon sequencing approach, we analyzed the microbiomes of different crayfish tissues (exoskeleton, hemolymph, hepatopancreas, and intestine) and explored their changes along the invasion range. Exoskeletal, hepatopancreatic, and intestinal microbiomes varied between invasion core and invasion front populations, indicating that they may be partly determined by population density, which was higher in the invasion core than in the invasion front. The highly diverse microbiome of exoskeletal biofilm was partly shaped by the environment (due to the similarity with the sediment microbiome) and partly by intrinsic crayfish parameters (due to the high proportion of exoskeleton-unique amplicon sequence variants [ASVs]). Our findings offer an insight into microbiome changes during dispersal of a successful invader and present a baseline for assessment of their contribution to an invader’s overall health and its further invasion success.

Crayfish plague is one of the most lethal diseases for native freshwater crayfish, while invasive crayfish are tolerant but may be chronically infected. In chronically infected populations the disease can cause direct (mortaility) but also indirect effects such as reduced growth due to increased energy investment in immune response.The aim of this study was to examine the direct (mortality) and indirect (reduction of growth) effects of chronic infection by crayfish plague pathogen (Aphanomyces astaci strain Psl) on individually kept juvenile marbled crayfish (Procambarus virginalis) under laboratory conditions. We conducted an infection trial using two concentrations of A.astaci spores (7500 and 15000 spores/ml) to test if repeated infection by A. astaci (at least two infections) leads to change in growth of infected compared to non- infected individuals (control group). We recorded mortality and measured length and weight of control group and infected groups before each succeeding infection. At the end of the trial the presence of the pathogen was analysed using previously developed A. astaci specific PCR assay. Direct effects of infection by A. astaci were observed at a concentration of 15000 spores/ml. Mortality was recorded in 20 % of infected individuals and additional 15 % of infected individuals had reduced viability and visible signs of infection. Indirect effects in terms of significantly lower length and weight increments of repeatedly infected individuals compared to control group were observed.

Projekt PlasmaArt: upotreba plazmenog mlaza za dezinfekciju drvenih umjetnina Nevena Krstulović1, Domagoj Mudronja2, Ana Bielen3, Ivana Bošnjak4 i Nikša Krstulović5 1Hrvatski restauratorski zavod, Restauratorski odjel Rijeka, Užarska 26, 51000 Rijeka 2Hrvatski restauratorski zavod, Prirodoslovni laboratorij, Zmajevac 8, 10000 Zagreb 3Prehrambeno-biotehnološki fakultet, Laboratorij za biologiju i genetiku mikroorganizama, Pierottijeva 6, 10000 Zagreb 4Prirodoslovno-matematički fakultet, Biološki odsjek, Rooseveltov trg 6, 10000 Zagreb 5Institut za fiziku, Bijenička cesta 46, 10000 Zagreb U ovome projektu proučavan je učinak atmosferskih plazmenih mlazeva (atmospheric pressure plasma jet, APPJ) na obradu drvenih umjetnina u smislu njihove dezinfekcije. APPJ plazma bogata je hladnim reaktivnim elementima odgovornima za tretman materijala bez termalne štete [1, 2]. APPJ su stoga vrlo povoljne za obredu termo-osjetljivih materijala kao što je drvo. Ovo istraživanje uključuje primjenu APPJ za bezkontaktnu obradu površina drvenih umjetnina i njihove sterilizacije od mikroorganizama. Ova metoda komplementarna je standardnim metodama koje se koriste u konzervacijsko/restauratorskoj praksi a koje se temelje na uporabi kemikalija i kemijskih otapala, gamma-zraka i/ili mehaničke obrade. Metoda je ispitivana na djelovanje APPJ u svrhu dezinfekcije pozlaćenih drvenih test-pločica kontaminiranih gljivičnim micelijem Coniophora puteana (uzročnk truleži drva). Plinovi korišteni za rad APPJ bili su Ar, He i mješavina Ar/O2. Prilikom obrade drvenih pločica APPJ-om praćeni su procesi međudjleovanja mlaza i metarijala pomoću optičke emisijske spektroskopije [3, 4]. Dobivenio je da je plin Ar najučinkovitiji za sterilizaciju uzoraka jer gljivice nisu preživjele obradu što ovu metodu čini učinkovitom i usporedivu s standardnim metodama. No, prednost ove metode je da je površina pozlaćenih test-pločica ostala netaknuta, tj. bez vidljivih oštećenja, što često nije slučaj kod standardnih metoda. Detaljni rezultati ovog projekta objavljeni su u [5-7]. Zahvala: ovaj projekt financiran je od Zaklade Adris i manjim dijelom preko HrZZ projekta IP- 11-2013-2753. [1] M. Laroussi, T. Akan, Plasma Process. Polym. 2007, 4, 777 [2] R. Zaplotnik, M. Bišćan, N. Krstulović, D. Popović, and S. Milošević, Plasma Sources Sci. Technol. 24 (2015) 054004 [3] D. Vujošević, M. Mozetič, U. Cvelbar, N. Krstulović and S. Milošević, J. Appl. Phys. 101 (2007) 103305 [4] Nikša Krstulović, Irena Labazan, Slobodan Milošević, Uroš Cvelbar, Alenka Vesel and Miran Mozetič, J. Phys. D: Appl. Phys. 39 (2006) 3799–3804 [5] N. Krstulović, D. Mudronja, A. Bielen, I. Bošnjak, N. Krstulović, STERILIZATION OF WOODEN ARTIFACTS BY APPJ, 25th INTERNATIONAL SCIENTIFIC MEETING ON VACUUM SCIENCE AND TECHNIQUE, Book of Abstracts, 2018, Gozd Martuljek, Slovenia [6] N. Krstulović, D. Mudronja, N. Krstulović, Tretament of wooden artifacts with atmospheric pressure plasma jet, 24th International Scientific Meeting on Vacuum Science and Technique, Book of Abstracts, 2017, Zadar, Croatia [7] N. Krstulović, D. Mudronja, A. Bielen, I. Babić, N. Krstulović, THE PlasmaArt PROJECT – APPLICATION OF ATMOSPHERIC-PRESSURE PLASMA JETS IN CONSERVATION AND RESTORATION OF WOODEN ARTWORK, Portal 9 (2018) 145-158

Both strong immune system and fast growth rate are important determinants of invasion success of a species and their dispersal rate in a novel environment. Since effective immune function may be costly, increased investment in immune response would limit the individual's resources available for growth, leading to a trade-off between these traits in an invader. The aim of this study was to examine the energetic costs of mounting an immune response caused by infection by crayfish plague pathogen (Aphanomyces astaci strain Psl) on growth and mortality of individually kept juvenile marbled crayfish under laboratory conditions. Firstly, we conducted an infection trial using four different spore concentrations of A. astaci (7500, 15000, 20000, 30000 spores/ml) to determine the mortality of infected individuals during four weeks following the infection. We recorded mortality of 78% in individuals infected with concentration of 30000 spores/ml and no mortality at lower concentrations. Based on these results, we selected two concentrations (7500 and 15000 spores/ml) to test if repeated exposure to A. astaci leads to change in growth compared to non-infected individuals (control group). At least two repeated infections were conducted in four-week intervals using selected concentrations. Within these intervals we continuously recorded mortality of individuals and measured length and weight of control group and infected groups before succeeding infections. Significantly lower length and weight increments of repeatedly infected individuals compared to control group were observed. We discuss our findings in the context of potential long-term effects of trade-offs between chronic and acute activation of the immune system and growth on the invasive success of non-indigenous crayfish species in novel environment. This research was funded by Croatian Science Foundation Installation Grant (HRZZ UIP-2017-05- 1720).

Uvod: Posljednjih se godina intenzivno istražuju nove metode izolacije eteričnih ulja, s ciljem povećanja njihovog prinosa. Ciljevi: Cilj ovog istraživanja bio je istražiti da li različiti predtretmani vodene destilacije mogu povećati prinos eteričnog ulja kadulje, ružmarina i lovora te utvrditi eventualne promjene u kemijskom sastavu eteričnih ulja. Metode: Vodenoj destilaciji predhodili su različiti predtretmani: klasična ekstrakcija refluksiranjem, ekstrakcija refluksiranjem uz dodatak enzima pektinaze, celulaze i ksilanaze te ultrazvučna ekstrakcija. Kemijski sastav dobivenih ulja analiziran je GC- MS i NMR analizom. Rezultati: Svi predtretmani povećali su prinos eteričnih ulja u usporedbi s negativnom kontrolom, pri čemu je kvaliteta ulja ostala nepromijenjena. No, predtretman ekstrakcijom refluksiranjem s dodatkom enzima nije povećao prinos ulja u odnosu na sami predtretman ekstrakcijom refluksiranjem (kontrola bez enzima). Ovo upućuje na to da je povećanje prinosa ulja uzrokovala ekstrakcija refluksiranjem, a ne enzimska aktivnost, i suprotno je navodima mnogih ranijih istraživanja. Međutim, u tim istraživanjima prinos ulja nakon predtretmana enzimima uspoređen s negativnom kontrolom (bez predtretmana), umjesto s kontrolom bez enzima. Zaključno, rezultati ovog istraživanja pokazali su da predtretmani ultrazvukom i ekstrakcijom refluksiranjem mogu biti korisni u proizvodnji eteričnih ulja navedenih biljaka, ali da primjena enzima u ovu svrhu treba biti dodatno istražena.

U ovom su radu određivani fizikalno-kemijski pokazatelji kakvoće tekućica i stajaćica u Hrvatskoj mjerenjem pHvrijednosti, električne provodnosti, otopljenog kisika, sadržaja iona, hranjivih tvari (dušikovi spojevi, ukupni fosfor i fosfati), režima kisika (kemijska i biokemijska potrošnja kisika) i ukupnog organskog ugljika. Iz dobivenih je rezultata vidljivo da površinskim vodama panonske regije prijeti onečišćenje nitratima, amonijem i organskim tvarima, čije je podrijetlo najvjerojatnije iz neobrađenih otpadnih voda. Također, nekim površinskim vodama prijeti eutrofikacija jer su u dva uzorka prisutne koncentracije fosfora iznad 0, 35 mg P/L, a u dva su uzorka koncentracije nitrata veće od 2, 5 mg N/L. Analize su, također, pokazale da jedan od četiri testirana ribnjaka nije pogodan za uzgoj riba jer je koncentracija amonija bila veća od 0, 2 mg NH4 /L, a biokemijska potrošnja kisika veća od 5 mg O2/L.

Oomycetes (water moulds) are a group of eukaryotic microorganisms that include many pathogens of terrestrial and aquatic species. Among freshwater oomycetes, microorganism Aphanomyces astaci (Schikora, 1906) is a causative agent of lethal disease crayfish plague and a reason for decline of native decapod crayfish populations all over Europe. During the infection, germinating A. astaci zoospores penetrate through the crayfish cuticle, followed by growth of hyphae through the host tissue. However, upon the arrival of zoospores on the crayfish cuticle, they encounter host epibiontic biofilm communities. While the protective role of commensal microbes has been shown in other models, the interaction of A. astaci and host biofilm is completely unexplored. Therefore, the aim of this work was to explore the potential of bacterial isolates originating from crayfish cuticle to inhibit A. astaci growth. We took swabs of cuticle biofilm from five specimens of narrow-clawed crayfish, Astacus leptodactylus Eschscholtz, 1823 and five specimens of signal crayfish, Pacifastacus leniusculus (Dana, 1852) and inoculated them on PG1 medium. Next, we have isolated morphologically different individual colonies from mixed cultures and taxonomically identified each isolate by MALDI–TOF mass spectrometry and 16S rRNA gene sequencing. Dominant bacterial genera among isolates were Pseudomonas and Acinetobacter. Furthermore, we tested the potential of isolates to inhibit the growth of A. astaci mycelium by plate inhibition assay. Out of 46 isolates from narrow-clayed crayfish, 13 exhibited great potential for A. astaci inhibition (28 %), while out of 44 isolates from signal crayfish, only 7 showed great inhibition potential (16 %). Most of the potent inhibitors belonged to genus Pseudomonas, for which inhibition of fish pathogenic oomycete from genus Saprolegnia was previously shown. In conclusion, we have characterized cultivable crayfish-associated biofilm communities and shown their potential as a source of anti- oomycete compounds.

Waterbodies of the Plitvice Lakes National Park are inhabited by two indigenous crayfish species, noble and stone crayfish. Native European crayfish populations are in decline, amongst others due to the lethal disease crayfish plague caused by pathogen Aphanomyces astaci. Presence of this pathogen was previously recorded in the Plitvice Lakes, but those analyses included small sample size. Thus, our goal was to analyse the presence of A. astaci on both crayfish species during two years monitoring period, using larger sample size, at multiple locations within the Park. For A. astaci detection we used IAA Gotland 201920non-invasive sampling method based on collecting swabs of mixed epibiotic microbial communities from crayfish carapace. DNA was isolated from swab samples and PCR was used to detect the presence of the pathogen. Positive samples were classified into semi-quantitative categories of pathogen load by qPCR, and microsatellite genotyping identified presence of strain A (As-genotype). Obtained results will be used in future management plans aiming to protect vulnerable native species in NP, with focus on prevention of unintentional spread of the pathogen.

Background: Oomycetes are globally important pathogens of plants and animals, causing significant economic losses in agriculture and aquaculture. Since current methods for their treatment include chemicals harmful to humans and the environment, new environmentally friendly methods for their control should be developed. Objectives: The aim of this study was to examine anti-oomycete effects of propolis, a natural substance with known antimicrobial activity but poorly investigated anti-oomycete potential. Methods: Two propolis formulations were used: P1 and P2, and their chemical composition was determined spectrophotometrically. Their anti- oomycete activity was determined using an in vitro plate assay and a range of pathogenic oomycetes that parasitize on animals (Aphanomyces astaci, crayfish pathogen, and Saprolegnia parasitica, parasite of salmonid fish) and plants (Phytophora cactorum and P. plurivora). Results: Both propolis samples contained high, mg / mL concentrations of bioactive compounds: flavonoids, total phenols, hydroxyacetic acids and flavanols. Accordingly, high inhibitory activity of propolis was demonstrated against all tested Oomycetes in vitro: mycelium growth was slowed down from 30 % to 75 % in the presence of propolis formulations and the degree of inhibition was positively correlated with the concentration of flavonoids in the samples. In conclusion, this is one of the first studies that demonstrates strong inhibitory effect of propolis towards pathogenic Oomycetes. In the future, the effect of propolis should be tested in vivo, e.g. as food additive in freshwater aquaculture facilities.