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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.

Invasive alien crayfish threaten freshwater biodiversity and native crayfish fauna. However, the invasion success of an otherwise successful crayfish invader may be compromised by local environmental conditions or presence of local pathogens which can affect invader’s physiology, immune status, health, and fitness. Here we report the occurence of idiopathic necrotizing hepatopancreatitis and changes in the immune response in the signal caryfish, one of the most successful freshwater invaders, in a recently invaded Korana River in Croatia. We sampled and histologically analysed 73 signal crayfish individuals collected in three consecutive years (2018-2020) and used several standard immune parameters (encapsulation response, hemocyte count, phenoloxidaze activity and total prophenoloxidaze) to: i) compare immune response of the signal crayfish along its invasion range, ii) analyze effects of specific predictors (water temperature, crayfish abundance and body condition) on its immune response changes. Our results show very high prevalence of lesions in hepatopancreas of signal crayfish (>91%), with 14% of individuals displaying heavy and 51% moderate necroses and/or inflammation in the tubular and interstitial structures of the organ. Heavy histopathological changes in hepatopancreas were correlated with lower organosomatic condition. Immune response exhibited significant differences along the invasion range of the signal crayfish and was mostly affected by water temperature and population abundance. Obtained results offer a baseline for elucidating the role of immunocompetence in the signal crayfish invasion in the Korana River, while aetiology of necrotizing hepatopancreatitis needs to be further investigated to determine its potential impact on signal crayfish invasion success.

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.

Samo mali dio svih unesenih vrsta diljem svijeta postane invazivan. Međutim, ove vrste negativno utječu na funkcioniranje ekosustava i jedan su od vodećih uzroka gubitka bioraznolikosti, što čini identifikaciju i upravljanje uspješnim invazivnim vrstama prioritetom. Imunosni sustav invazivne vrste važna je komponenta invazivnog uspjeha jer omogućuje vrsti da se prilagodi uvjetima novog okoliša dok širi svoj areal. Usto, imunosni odgovor invazivne vrste može varirati duž invazivnog areala zbog raznih biotičkih i abiotičkih faktora. Ovdje smo analizirali koji faktori oblikuju imunosni odgovor jedne od najuspješnijih slatkovodnih invazivnih vrsta, signalnog raka, u rijeci Korani u Hrvatskoj, gdje je ova vrsta nedavno unesena. Koristili smo nekoliko standardnih imunosnih parametara (snaga reakcije inkapsulacije, broj hemocita, enzimska aktivnost fenoloksidaze i ukupna koncentracija profenoloksidaze) kako bi analizirali učinke specifičnih prediktora (temperatura vode, gustoća populacije i tjelesna kondicija) na imunosni odgovor rakova. Nadalje, usporedili smo imunosne odgovore invazivnog signalnog raka i zavičajnog uskoškarog raka, koji također nastanjuje rijeku Koranu. Imunosni odgovor najviše je oblikovan temperaturom vode i gustoćom populacije te se značajno razlikovao između dviju vrsta. Dobiveni rezultati upućuju na to da se imunosni sustav mijenja tijekom širenja areala te ukazuju na to da bi imunitet mogao imati ulogu u invazivnom uspjehu invazivnih vrsta beskralježnjaka.

Invazivne strane vrste jedna su od najvećih prijetnji vrlo osjetljivim slatkovodnim ekosustavima, a predviđanje uspjeha invazije predstavlja globalni izazov. Invazivni uspjeh određen je, između ostaloga, i imunosnim sustavom, kojeg oblikuju patogeni i nepatogeni mikrobi domaćina te ekološki procesi koji proizlaze iz širenja areala. Ovo istraživanje ispituje promjene u sastavu mikrobnih zajednica i ulogu imunosnog odgovora tijekom širenja areala jedne od najuspješnijih invazivnih stranih vrsta slatkovodnih beskralježnjaka u Europi, signalnoga raka. Mikrobi u različitim tkivima jedinki duž invazivnog areala detektirani su analizom metagenoma (sekvenciranjem nove generacije), dok su razlike u imunosnom odgovoru jedinki ispitane analizom nekoliko standardnih imunosnih parametara. Navedene analize pokazale su da se mikrobne zajednice i imunosni odgovor signalnoga raka značajno mijenjaju tijekom širenja invazivnog areala te da su oblikovani, u različitoj mjeri, okolišnim čimbenicima i karakteristikama populacije. Ovo istraživanje također ukazuje na vrlo slabu istraženost patogena slatkovodnih deseteronožnih rakova te ističe potrebu za standardizacijom eksperimentalnih metoda prilikom ispitivanja patogenosti. Invasive alien species are among the largest threats to sensitive freshwater ecosystems, and predicting the success of an invasion represents a global challenge. Invasive success is determined, among other things, by the immune system, which is shaped by pathogenic and non-pathogenic host microbes and ecological processes arising from range expansion. This study examines changes in microbial communities and the role of immune response during range expansion of one of the most successful invasive alien species of freshwater invertebrates in Europe, the signal crayfish. Microbes in different tissues of individuals along the invasion range were detected by metagenome analysis (next generation sequencing), while differences in the immune response of individuals were examined by analysis of several standard immune parameters. These analyses have shown that microbial communities and the immune response of signal crayfish change significantly during range expansion and that they are shaped, to varying degrees, by environmental factors and population characteristics. This study also highlights a gap of knowledge regarding crayfish pathogens and underlines the need for standardization of experimental methods when testing pathogenicity.

Today we face an increased need to solve complex ecological challenges, such as synergistic effects of biological invasions and climate change. Thus, assessing effectiveness of invasive species management scenarios or conservation priorities for endangered native populations affected jointly by biological invasions and climate change, is crucial. While field and laboratory studies required for such assessments are costly and logistically challenging, mathematical modelling offers a good alternative. Bioenergetic mechanistic modelling, in particular, can integrate all relevant species-specific biological and ecological information, and can be applied to identify high-risk species in a changing environment and/or management scenario with the highest impact on invasive crayfish abundances. Here we demonstrate the application of Dynamic Energy Budget (DEB) models in: i) development of a conceptual population dynamics model for evaluation of effectiveness of different approaches to signal crayfish control and ii) comparison and assessment of individual growth and reproduction of two prominent crayfish invaders (signal crayfish and the marbled crayfish) and two native and endangered crayfish (noble crayfish and stone crayfish) under changing environmental conditions. Since DEB models capture dependence of metabolism, and therefore ontogeny, on environmental and population conditions, they can be used to investigate diverse biological and ecological questions. Based on the examples of the two studies presented here, we argue that mechanistic models in general, and bioenergetics models in particular, should be at the core of adaptive management and decision-making in nature conservation and ecosystem restoration.

Fungi and fungal-like organisms are among the most damaging pathogens for freshwater crayfish and may cause significant losses of both aquacultured and wild crayfish populations. Furthermore, the most devastating effects in wild populations are caused by fungal and fungal-like pathogens transmitted by invasive crayfish species (ICS) to native crayfish populations. Although research is often focused on pathogenic mycobiota, increasing evidence points out that mycobiota in general has an important role in biological invasions, since it can affect a number of interactions between the host and the environment. Also, the characteristics of the novel environment and the dispersal process may in turn affect the composition of invaders' mycobiota, and directly and indirectly affect their fitness and invasion success. Using ITS rRNA amplicon sequencing, we have analyzed the mycobiome of one of the most successful ICS in Europe – the signal crayfish – in the Korana River, Croatia. We explored the mycobiome of four types of crayfish samples (exoskeletal biofilm, hemolymph, hepatopancreas and intestine) and two types of environmental samples (water and sediment), and compared differences in their biodiversity and abundance between upstream and downstream segments of the signal crayfish' invasion range in Korana River. Since a very small number of mycobial features in hemolymph and hepatopancreas samples was recorded, they were excluded from further analyzes. Between the remaining sample groups, intestine showed significantly lower richness of mycobiota compared to exoskeletal biofilm, water and sediment. Beta diversity analyzes showed significant differences between mycobiomes of all four sample groups, confirming their uniqueness. Comparison between upstream and downstream river segments yielded opposite richness patterns of mycobiota for sediment (significantly higher richness downstream) and exoskeletal biofilm (significantly higher richness upstream). Furthermore, beta diversity analyzes also revealed significant differences in composition and abundance of sediment and exoskeletal mycobiota between upstream and downstream river segments, indicating that crayfish exoskeleton harbors a specific mycobiota, which is affected by parameters other than environment itself. Our findings represent the first insight into mycobiome of a successful ICS, and offer a baseline for assessing how mycobiome contributes to an invader’s overall health and its further invasion success.

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.

Invazivne strane vrste predstavljaju prijetnju raznolikosti slatkovodnih ekosustava i populacijama zavičajnih vrsta slatkovodnih rakova, primarno zbog prijenosa bolesti račje kuge. No, kronična infekcija račjom kugom može i kod tolerantne invazivne strane vrste dovesti do kompromisa u raspodijeli energije između ulaganja u imunosni odgovor i rasta i reprodukcije, što u kombinaciji s drugim nepovoljnim okolišnim uvjetima može utjecati na njezin invazivni uspjeh. U ovom smo istraživanju ispitali da li imunosni odgovor na infekciju patogenom račje kuge negativno utječe na rast i preživljavanje juvenilnih jedinki invazivne strane vrste mramornog raka (Procambarus virginalis). U sklopu eksperimentalnih istraživanja, jedinke su izložene različitim koncentracijama patogena (2) i različitim režimima hranjenja (2). U svim pokusima, ponavljajuća infekcija rezultirala je smanjenim prirastom jedinki. Značajan porast mortaliteta inficiranih jedinki zabilježen je tek pri uvjetima značajne limitacije hrane, u grupi koja je bila vrlo blizu izgladnjivanja. S obzirom da su infekcije bile česte, a koncentracije patogena visoke, rezultati pokazuju kako je mramorni rak otporan na patogen Aphanomyces astaci i kako dodatni stresori (izgladnjivanje) marginalno povećavaju mortalitet inficiranih jedinki. Stoga, kronična infekcija račjom kugom vjerojatno neće imati značajan utjecaj na invazivni uspjeh mramornog raka, čak niti u nepovoljnim uvjetima.

Invazivne strane vrste jedna su od najvećih prijetnji vrlo osjetljivim slatkovodnim ekosustavima, a predviđanje uspjeha invazije predstavlja globalni izazov. Invazivni uspjeh određen je, između ostaloga, i imunosnim sustavom, kojeg oblikuju patogeni i nepatogeni mikrobi domaćina te ekološki procesi koji proizlaze iz širenja areala. Ovo istraživanje ispituje promjene u sastavu mikrobnih zajednica i ulogu imunosnog odgovora tijekom širenja areala jedne od najuspješnijih invazivnih stranih vrsta slatkovodnih beskralježnjaka u Europi, signalnoga raka. Mikrobi u različitim tkivima jedinki duž invazivnog areala detektirani su analizom metagenoma (sekvenciranjem nove generacije), dok su razlike u imunosnom odgovoru jedinki ispitane analizom nekoliko standardnih imunosnih parametara. Navedene analize pokazale su da se mikrobne zajednice i imunosni odgovor signalnoga raka značajno mijenjaju tijekom širenja invazivnog areala te da su oblikovani, u različitoj mjeri, okolišnim čimbenicima i karakteristikama populacije. Ovo istraživanje također ukazuje na vrlo slabu istraženost patogena slatkovodnih deseteronožnih rakova te ističe potrebu za standardizacijom eksperimentalnih metoda prilikom ispitivanja patogenosti.

The immune response is among the major drivers of an individual’s health since it is responsible for protecting organisms against pathogens. Consequently, it is one of the drivers of the invasion success of a species. This study aimed to compare the immune response of the resistant native species, the narrowclawed crayfish (Pontastacus leptodactylus) and the successful invasive alien species, the signal crayfish (Pacifastacus leniusculus) from the Korana River, at three sites where these species co-occur in mixed populations. We have performed between- species comparisons of specimens caught with LiNi traps in September 2020. The standard immune response parameters, frequently reported in crayfish, were compared: total hemocyte count, the strength of the encapsulation response and enzyme activity of phenoloxidase (PO) and total prophenoloxidase (proPO). Also, since the immune response is considered an important fitness component, we have compared it to the body condition parameters (hepatosomatic index and Fulton’s condition factor) of each species. Compared to native narrow-clawed crayfish, invasive signal crayfish had significantly higher values of two immune response parameters (encapsulation response and total prophenoloxidase) and exhibited an overall better immune response. We discuss our findings in the context of the potential contribution of these differences in the immune response to the observed displacement of the narrow-clawed crayfish by the signal crayfish from mixed populations in the Korana River.

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).

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.

Investment in immunocompetence can have direct impacts on the invasion success. During range expansion, individuals enter the novel environment where they can benefit from both the increase or decrease in immune investment. Increased immune investment may be required during contact with novel pathogens, but it is also energetically costly for the dispersing individuals. Thus, if investment in immunocompetence drives range expansion, we would expect to observe differences in immune response along the invasion range of an invader. In this pilot study, we compared the immune response of one of the most successful crayfish invaders in Europe, the signal crayfish, along its invasive range in the Korana River, Croatia. Comparisons were made between individuals from the invasion core and invasion front sites, which were subjected to the same treatment in the laboratory. We measured the immune response parameters (total hemocyte count, encapsulation response, total and specific phenoloxidase and prophenoloxidase activity), morphological parameters and crayfish condition parameters (Fulton's condition factor, total plasma protein concentration). Immune response parameters were analysed separately, as well as their combined effect in order to measure the overall immune status. No significant differences were found in overall immune status between individuals from invasion core and invasion front. However, some parameters, i.e. the specific and total prophenoloxidase activity, exhibited significant differences – they were significantly higher in individuals on the invasion front compared to those in the invasion core, which potentially indicates higher immune investment in the individuals on the invasion front. We discuss our findings in the context of the theories regarding immune investment during the range expansion of the invasive species. This research was funded by Croatian Science Installation Grant (HRZZ UIP-2017-05-1720).

Background: Invasive non-indigenous crayfish species (NICS) negatively impact ecosystems and native crayfish populations by, among others, introducing pathogens into the new environment. Literature suggests that hemolymph of apparently healthy crayfish is unsterile and can, in stressful conditions, contain a higher bacterial load which may be correlated with higher bacterial load in hepatopancreas. Understanding how bacterial load affects crayfish immunity and fitness would help us assess whether the healthiest individuals are the first to disperse to new environment. Objectives: To determine whether bacterial load in hemolymph could be used as crayfish fitness proxy and to establish whether there is a link between bacterial load in hemolymph and hepatopancreas of the most successful crayfish invader in Europe – the signal crayfish (Pacifastacus leniusuclus), in the Korana River, Croatia. Methods: We collected over 110 individuals along the Korana River and sampled cuticule swabs, and hepatopancreas, gut and hemolymph tissues and recorded any visible signs of decreased fitness, such as injuries, potential diseases (melanisations) or presence of parasites in examined tissues. We compared bacterial presence, load and diversity in hemolymph and hepatopancreas of 48 individuals by using 16S ribosomal DNA PCR. Results: Our results show that 18.75% of individuals had bacteria present in the hepatopancreas, and 10.42% individuals in the hemolymph. Out of them, only 2 (4.2 %) individuals had bacteria present in both tissues. Individuals with bacteria recorded in the examined tissues had higher occurence of morphological signs of decreased fitness (75%) than individuals with no bacterial load in the tissues (55%).

Pathogenic oomycete Aphanomyces astaci is transmitted mainly by invasive North American crayfish, and causes the crayfish plague, a disease mostly lethal for native European crayfish. Detecting the pathogen in endangered native crayfish populations before the disease outbreak would provide a starting point in the development of effective control measures. However, current A. astaci-detection protocols either rely on eDNA isolated from large volumes of water or, if focused on individual animals, include crayfish killing. Thus, we have developed a non-invasive method that detects A. astaci DNA in microbial biofilm associated with the cuticle of individual crayfish, bypassing the unnecessary killing step. Efficiency of the new method was confirmed by PCR and qPCR and the obtained results were congruent with the traditional invasive method. We propose that the new method should be used in future monitoring of A. astaci presence in endangered European native crayfish individuals.

The signal crayfish (Pacifastacus leniusculus) is the most successful crayfish invader in Europe. In Croatia it was firstly recorded in the Mura-Drava river basin in 2008, as a result of natural dispersal by Slovenian populations. In 2012 it was recorded in the Korana River in the continental part of Croatia, where it was illegally introduced. In both rivers, a high dispersal rate was observed. The aim of this study was to explore the range expansion of the signal crayfish 10 years after its first record in Croatia. We collected literature data and conducted field studies in summer 2018 along the Korana and Drava rivers. In the Korana River, we observed an increase in the range of P. leniusculus by 5 km in 2 years. The invasion range now stretches along a total of 30 km. Dispersal rates were higher in the upstream direction, potentially due to differences in competitive pressure from native crayfish. Abundance of the native narrow clawed crayfish (Astacus leptodactylus) was six times lower at the upstream invasion front than downstream. In the centre of the P. leniusculus invasion range in the Korana river no native crayfish were captured, suggesting a displacement of the narrow clawed crayfish by the invasive signal crayfish. In the Drava River, the extent of the newly recorded invasion front corresponds to projections from an earlier study that estimated signal crayfish dispersal over 10 years. Our new records suggest that signal crayfish dispersal rates in this river have remained among the highest in Europe.

Brljansko i Visovačko jezero nalaze se u zaštićenom dijelu ekosustava rijeke Krke. Jezerski dijelovi Krke posebno su izloženi riziku onečišćenja zbog sporije izmjene i duljeg vremenskog zadržavanja vode. U radu su proučavani sadržaji 30 metala/metaloida u vodi i probavnoj žlijezdi školjkaša Anodonta spp. iz Brljanskog i Visovačkog jezera, kao bioindikatorskih organizama za praćenje onečišćenja u jezerskim sustavima. Cilj je bio vidjeti postoji li razlika u izloženosti metalima između ovih jezera. Uzorci vode i školjkaša uzeti su na dubinama između 1 i 4 m u svibnju 2016. godine. Koncentracije metala određene su pomoću HR ICP-MS-a. Analiza uzoraka jezerske vode ukazala je na relativno niske razine otopljenih metala u vodi obaju jezera. Koncentracije Cd, Co, U, Mo, Mn, Li, Fe, Sr, Mg i Na bile su značajno više na Brljanu, a Se, Rb, Cr i V na Visovcu. Školjkaši iz Brljana akumulirali su više Mo, Sb, U, V, Al i Na, dok su Rb, Ag, Cd, Cr, Mn, Co, Ni, Cu, Zn, Sr, Ba, Tl i Ca bili značajno viši u školjkaša iz Visovca. Uočena je slaba povezanost sadržaja metala u probavnoj žlijezdi s otopljenim metalima u vodi. Dobiveni rezultati mogu poslužiti kao polazište za usporedbu u budućim studijama praćenja onečišćenja vodenog okoliša metalima.

Toxic effects of metals depend not only on their total cellular concentrations, but also on their distribution within the cell(1). The aim of this study was to compare the intracellular distribution of nine selected metals and metalloids (essential: Cu, Zn, Fe, Mo and Se ; nonessential: Cd, Ag, Pb and As) in the digestive gland (DG) of freshwater bivalves Anodonta exulcerata from the Visovac Lake and Anodonta anatina from the Brljan Lake, both located in the Krka Nacional Park, protected area of the karst Krka River in Croatia. Upstream from this protected area there is a potential source of anthropogenic contamination via municipal and industrial wastewater effluents from the town of Knin. The freshwater bivalves are often applied as bioindicators of environmental contamination in the lacustrine ecosystems. The digestive gland of bivalves is a main organ for metal storage and detoxification, and therefore widely used as a target organ in biomonitoring studies(2). Metal/metalloid contents in the cytosolic tissue fraction as well as the metal/metalloid distribution among cytosolic biomolecules were assessed. Cytosolic metal/metalloid concentrations were determined by high resolution inductively coupled plasma mass spectrometry (HR ICP-MS). The separation of cytosolic biomolecules was performed by size exclusion high performance liquid chromatography (SEC-HPLC) on Superdex 200 10/300 GL column, and subsequent metal/metalloid analyses were performed off- line by HR ICP-MS. Cytosolic concentrations of Cu, Fe, Mo, Ag and Pb were higher in DG of bivalve A. anatina from the Brljan Lake, while cytosolic Cd was higher in DG of A. exulcerata from the Visovac Lake. Analysis of the metal distribution among cytosolic biomolecules allowed the definition of molecular masses of biomolecules involved in binding of particular metals. Resulting metal/metalloid profiles were the same in both studied bivalve species for all studied elements at different levels of metal accumulation. Exception was chromatographic profile of Pb with one additional distinctive peak for DG of A. anatina with increased cytosolic Pb from the Brljan Lake. The results of this study represent the first data on cytosolic metal/metalloid concentrations and their intracellular distribution in the DG of bivalves from genus Anodonta in the Visovac and Brljan Lakes and as such can serve as a starting point for comparison in future studies of metal contamination and conservation of these species. REFERENCES 1. Naimo T.J., Ecotoxicology, 1995, 4, 341-362. 2. Farris J.L. and Van Hassel J.H., Freshwater Bivalve Ecotoxicology, 2007, CRC Press.

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.

Thallium is nonessential and highly toxic metal. It has high water solubility and shows the tendency of accumulation in living organisms. As a priority pollutant, it should be monitored both in water and in the organisms. So far, there are relatively few studies of Tl bioaccumulation in freshwater bivalves, especially in karst waters. We have studied Tl accumulation in the digestive gland of freshwater bivalves Anodonta spp. from two karst lakes in Croatia, Brljan and Visovac. The lakes are integral part of the Krka River ecosystem. Brljan Lake is situated upstream from Visovac Lake and is consequently more affected by the pollution of wastewaters from the Town of Knin. In Visovac Lake, organisms were sampled in shallow (1-3 m, VS) and deep (~13 m, VD) part of the lake, and at Brljan Lake only in shallow part (2-4 m, BR). In addition to Tl, we also analyzed the content of 23 trace elements and 4 macro elements in digestive gland in order to detect possible coaccumulation of Tl with other elements. Metals were determined by HR ICP-MS. Concentrations of dissolved Tl in water were very low in both lakes (5-6 ng/L). Whole digestive gland tissue of bivalves from VS location had about ten times higher Tl concentrations (0.039 μg/g) compared to the other two locations (0.004 and 0.005 μg/g at VD and BR location, respectively). Thallium concentration ranges in the cytosolic fraction of digestive gland were as follows: BR: 0.0008-0.004 μg/g, VS: 0.0002-0.0009 μg/g and VD 0.0004-0.0008 μg/g. Correlation analysis confirmed specific coaccumulation patterns at each of the investigated locations. Obtained results indicate low Tl exposure by water and suggest the need for further sediment research as a potential source of Tl for these bivalves.

Invasive non-indigenous crayfish species (NICS) impact native crayfish populations through competition and hybridization, but more importantly, through transmission of pathogens which can spread rapidly and have a detrimental effect on existing native species/populations. The impact of pathogens on freshwater crayfish has been studied extensively, especially in species important for aquaculture. However, for many alleged pathogens, the pathogenicity has never been experimentally confirmed since the infection trials have never been conducted. Detection of a potential pathogen in moribund or dead crayfish has often led to the assumption that the organism is able to cause disease. Therefore, the aim of this study was to analyze the available literature to determine which microbes (bacteria, viruses and fungi) can be classified as ‘proven’ pathogens for freshwater crayfish, and which of them are classified inadvertently or incorrectly. We analyzed over 70 scientific publications and classified the alleged crayfish pathogens into the following categories: a) pathogen – pathogenicity confirmed by infection trials, b) potential pathogen – although classified as pathogen in literature, no infection trials have been recorded, c) non-pathogen – infection trials failed to prove pathogenicity for crayfish and d) indirect pathogen/opportunist – infection trials indicate possible crayfish mortality under stress conditions. We recorded 39 microbial species belonging to these categories, with the majority of them (41 %) classified as pathogens, including bacterial, viral and fungal representatives. We discuss our results in the context of microbial potential effects on both invasive non-indigenous and native crayfish populations during the range expansion of the invasive NICS to the novel environment.

U krškim područjima Hrvatske prirodne koncentracije metala su uglavnom niske, zbog čega su vodeni sustavi tih područja vrlo osjetljivi na povišene koncentracije metala. Visovačko jezero dio je ekosustava u zaštićenom dijelu rijeke Krke. Dosadašnja istraživanja ukazala su na povišenje razine nekih ekotoksičnih metala u sedimentima ovog jezera kao posljedicu antropogenog djelovanja, no za bolju procjenu rizika onečišćenja metalima potrebno je istražiti njihov utjecaj na organizme. Školjkaš Anodonta exulcerata korišten je kao bioindikator u ovom radu. Analiziran je sadržaj metala u jezerskoj vodi i u probavnoj žlijezdi školjkaša s plićeg i dubljeg lokaliteta. Određeni su molekularni biomarkeri izloženosti i učinaka zagađivala: metalotioneini, ukupni glutation, ukupni proteini i malondialdehid. Analiza uzoraka jezerske vode ukazala je na relativno niske razine otopljenih metala u vodi Visovačkog jezera. Uočene su značajne razlike u bioraspoloživosti pojedinih metala u plićem i dubljem dijelu Visovačkog jezera. Analiza raspodjele metala između topljive i netopljive frakcije tkiva probavne žlijezde ukazala je na specifične obrasce raspodjeljivanja pojedinih elemenata između ovih dviju frakcija pri povećanoj akumulaciji. Koncentracije metalotioneina ukazale su na značajno veću izloženost školjkaša s dubljeg lokaliteta. Koncentracija glutationa bila je značajno povišena u školjkaša s plićeg lokaliteta. In the karst areas of Croatia natural concentrations of metals are generally low, which is why water systems in these areas are very sensitive to elevated metal concentrations. Visovac Lake is a part of the ecosystem in the protected part of the Krka River. Previous research has shown an increase in some ecotoxic metals’ levels in sediments of this lake as a result of anthropogenic activity but in order to better assess the risk of metal contamination, it is necessary to estimate their influence on the organisms. Bivalve Anodonta exulcerata was used as a bioindicator in this paper. The content of metal was analyzed in the lake water and in the digestive gland of the bivalves from the shallower and deeper site. Molecular biomarkers of exposure and effect were measured: metallothionein, total glutathione, total protein and malondialdehyde. Analysis of lake water samples showed relatively low levels of dissolved metals in Visovac Lake water. Significant differences in the bioavailability of certain metals were observed in the shallower and deeper parts of the Lake Visovac. The analysis of the distribution of metal between the soluble and insoluble fraction of digestive gland showed specific patterns of distribution of some elements between these two fractions at increased accumulation. Concentrations of metallothionein showed a significantly greater exposure of bivalves from a deeper locality. The glutathione concentration was significantly elevated in bivalves from the shallower site.

Brljansko i Visovačko jezero su sastavni dio ekosustava zaštićenog dijela rijeke Krke. Dosadašnja istraživanja ukazala su na povećanje razine nekih ekotoksičnih metala u sedimentima ovih jezera kao posljedicu antropogenog djelovanja. Cilj rada bio je istražiti postoji li razlika u izloženosti metalima između Brljanskog i Visovačkog jezera. S obzirom na smještaj, Brljansko jezero se nalazi uzvodnije i stoga je pod jačim utjecajem onečišćenja otpadnih voda grada Knina. Dodatni cilj je bio istražiti postoji li razlika u izloženosti metalima između školjkaša uzorkovanih u plićim (2 m) i dubljim (13 m) dijelovima Visovačkog jezera. Procjenu izloženosti proveli smo na osnovu analize sadržaja metala u jezerskoj vodi, sadržaja metala u probavnoj žlijezdi školjkaša Anodonta spp. kao bioindikatorskog organizma te određivanjem sadržaja metalotioneina (MT) i ukupnog glutationa (tGSH) kao biomarkera izloženosti metalima. Razine metala u vodi bile su niske i nisu se značajno razlikovale među istraživanim lokacijama. Međutim, za većinu analiziranih metala ustanovili smo značajne razlike između lokaliteta s obzirom na sadržaj metala u probavnoj žlijezdi školjkaša. Koncentracije MT u školjkašima uzorkovanim u dubljim dijelovima istraživanih jezera nisu se razlikovale značajno, dok su razine tGSH bile značajno više na lokalitetu Brljan. Također smo utvrdili statistički značajne razlike u sadržaju MT i tGSH u školjkaša prikupljenih na različitim dubinama Visovačkog jezera. Dobiveni rezultati potvrdili su razlike u akumulaciji metala u školjkaša Anodonta spp. iz Brljanskog i Visovačkog jezera. Daljnja istraživanja, koja bi uz određivanje metala u sedimentu, uključivala i određivanje biomakera toksičnog djelovanja metala su neophodna za konačnu procjenu rizika izloženosti metalima u ovim jezerima.

Slatkovodni rakovi vrlo su pogodni modeli za istraživanje agonističkog ponašanja. Agonističko ponašanje je ponašanje koje uključuje agresivnost, obranu i izbjegavanje borbe. Slatkovodni rakovi agresivne su životinje, a njihove česte borbe igraju važnu ulogu u formiranju njihove društvene strukture. Rakovi se međusobno bore za resurse kao što su hrana, sklonište i partneri, koji su im potrebni za uspješan rast, preživljavanje i reprodukciju. U ovom radu ukratko je opisano agonističko ponašanje slatkovodnih rakova te faktori koji na njega utječu. U određivanju ishoda borbe ulogu imaju razni vanjski i unutarnji faktori te iskustva raka u prošlim borbama iz kojih izlaze kao pobjednici (efekt pobjednika) ili gubitnici (efekt gubitnika). Borbe mogu biti različitih intenziteta, a svaka rezultira pobjednikom (dominantni rak) i gubitnikom (podređeni rak). Nizom borbi formira se linearna hijerarhija dominacije, na čijem je vrhu alfa rak. Freshwater crayfish are very suitable models in studying agonistic behaviour. Agonistic behaviour is a type of behaviour which involves aggression, defence and avoiding fights. Crayfish are aggressive animals and their frequent fights play an important role in formation of their social structure. Crayfish fight each other to gain resources such as food, shelter and mates, which they need for successful growth, survival and reproduction. In this work, agonistic behaviour has been shortly described, along with the factors that affect it. Various intrinsic and extrinsic factors define the battle outcome as well as experience gained in previous fights (known as the winner effect and the loser effect). Fights are characterised by different intensities and each of them generates the dominant crayfish and its subordinate. The alpha crayfish is on the top of the linear dominance hierarchy, created by a series of battles.