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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.

The stone crayfish is a native European freshwater species with significant population declines caused by anthropogenic pressure onto its habitats, climate change and spreading of non-indigenous invasive crayfish and their pathogens (e.g., Aphanomyces astaci). We present the first record of an Austropotamobius torrentium population in a small stream from the southern lowlands of Romania, in an area without limestone or karst, known as a typical stone crayfish habitat in this country. No additional populations were found in the neighbouring streams. The mtDNA analysis based on sequencing a short fragment of the cytochrome c oxidase subunit I (COI) gene revealed that specimens from this newly discovered population belongs to the most widespread phylogroup of A. torrentium (CSE) and share the same haplotype as the specimens found in Croatia. Unfortunately, this crayfish population has disappeared most likely because of the crayfish plague since pathogen A. astaci was isolated from moribund crayfish. Our study highlights the vulnerability of isolated relic populations in the face of outside stressors.

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.

The stone crayfish Austropotamobius torrentium (Schrank, 1803), a freshwater crayfish species with intricate evolutionary history and high genetic diversity, is threatened due to anthropogenic pressure, climate change and spreading of invasive crayfish species and their pathogens. Maintenance of genetic diversity and the preservation of the integrity of a native species are one of pivotal conservation genetics goals. In order to develop appropriate conservation and management programs on the local and regional level, the aim of our study was to reveal the current population genetic structure of the endangered stone crayfish. We conducted a microsatellite analysis of 300 individuals from 15 sampling sites. Preliminary testing included 57 recently developed microsatellite markers. Ten out of 57 microsatellite loci were polymorphic and were used in further analyses. We also used seven additional, previously developed loci, cross- amplified on other Astacidae crayfish species. The obtained results enabled the identification of local genetic diversity hotspots, as well as definition of evolutionary significant and management units, which will be given priority in conservation programs.

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.

Microorganism Aphanomyces astaci (Schikora, 1906) causes the lethal disease crayfish plague, one of the reasons for decline of native decapod crayfish populationsin Europe. While A. astaci zoospores penetrate through the crayfish cuticle during the infection process, they encounter epibiontic bacterial communities on the cuticle surface. We have hypothesized that bacterial isolates originating from crayfish cuticle could inhibit the growth of A. astaci mycelium. To test this, we took cuticle swabs from five narrow-clawed crayfish, Astacus leptodactylus Eschscholtz, 1823 and five signal crayfish, Pacifastacus leniusculus (Dana, 1852) and inoculated them on PG1 medium. Next, we have isolated morphologically different individual colonies 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 33 isolates from narrow-clayed crayfish, 20exhibited great potential for A. astaci inhibition, while out of 25 isolates from signal crayfish, only 2 showed great inhibition potential. This result was surprising since narrow-clawed crayfish is known to be more sensitive to A. astaci than signal crayfish, but could be explained by a small sample size. Further, most of the potent inhibitors from both crayfish species belonged to genus Pseudomonas, for which the inhibition of fish pathogenic oomycete from genus Saprolegnia was previously shown. In conclusion, we have characterized cultivable symbiotic bacterial communities of crayfish and shown their potential for biocontrol of crayfish plague.

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.

Crayfish are the keystone species of freshwater ecosystems. However, in recent decades populations of indigenous European crayfish species (ICS) significantly declines due to numerous anthropogenic impacts, which include the spread of non-indigenous crayfish species (NICS) and climate change. Moreover, populations decline is increased since the migration of ICS to more favorable places in freshwater ecosystems is limited. NICS are carriers of the crayfish plague disease, whose causative agent is the microorganism Aphanomyces astaci (Schikora, 1906) (Oomycetes). This pathogen is main reason of ICS declines and increasing of NICS. The aim of this study was to investigate if increasing of water temperature (caused by global worming) would have an impact on virulence of pathogen A. astaci and the physiological condition of two native species of crayfish: the narrow-clawed crayfish (Astacus leptodactylus Eschscholtz, 1823) and the stone crayfish (Austropotamobius torrentium (Schrank, 1803)). Since we used new noninvasive method of molecular detection of pathogen from the crayfish cuticle we could select non-infected individuals for our experiments, as well as to identify the cause of death of crayfish during the experiment. Th both species were infected with 500 A. astaci strain B (PsI genotype) zoospores / mL at two water temperatures: 18 ° C (control temperature) and 22 ° C (elevated temperature – simulation of global warming). The mortality rate of both species was monitored for four weeks. Results show the reduction of virulence of A. astaci pathogen on elevated water temperature (22 °C), but it is also caused higher mortality range for endangered native species A. torrentium. In conclusion, global worming might decrease virulence of A. astaci (strain B, PsI genotype) but it could affect fitness of native crayfish species such as A. torrentium. This is the first study which present connection between virulence of pathogen A. astaci and the water temperature.

Transmission of crayfish plague by invasive crayfish is one of the main drivers of native European crayfish populations declines. The causative agent of crayfish plague Aphanomyces astaci (Schikora, 1906) is listed among the 100 worst invasive species in the world, and it has been spreading throughout Europe since its introduction in the late 19th century. The future of the interplay between this pathogen and its hosts is hard to predict, but it has been shown for other pathogen-host pairs that elevated water temperature caused by climate change favours spreading of pathogens. Therefore, the aim of this study was to investigate whether a 4 °C increase in water temperature will also increase the virulence of A. astaci (strain B, PsI genotype) towards two indigenous crayfish species: the stone crayfish Austropotamobius torrentium (Schrank, 1803) and the narrow-clawed crayfish Astacus leptodactylus Eschscholtz, 1823. Before the experiment, we have tested all animals for A. astaci presence and only A. astaci-free animals were included into the experiment. After acclimatization, we have infected the crayfish with 500 zoospores per mL at water temperature of 18⁰C (control conditions) and 22⁰C (increased water temperature - simulation of climate change). After four weeks the results have shown that the mortality of both crayfish species was significantly lower at increased water temperature (22⁰C). This suggests that the increase of water temperatures caused by climate change will probably not result in the increased spread of A. astaci strain B in stone crayfish and narrow-clawed crayfish populations. Results will be discussed in adaptation and evolutionary perspective as well as in the context of A. astaci strains that have different water temperature preference. In conclusion, this study presents the first experimental data on the balance between A. astaci and indigenous crayfish in the context of climate change.

Anthropogenically induced climate change has multiple negative effects on ecosystems. Many freshwater organisms are especially vulnerable to climate change-related habitat alterations since animals have limited possibilities to migrate to areas with more favourable conditions. Thus, crayfish as keystone species and ecosystem engineers of many freshwater ecosystems could be negatively affected. Furthermore, it has been shown for multiple pathogen-host pairs that elevated water temperature caused by climate change favours spreading of pathogens. The aim of this study was to investigate whether a 4 °C increase in water temperature (from 18 to 22 °C) will also increase the virulence of Aphanomyces astaci (Schikora, 1906), the causative agent of crayfish plague. Transmission of crayfish plague by invasive crayfish species is one of the main drivers of native European crayfish populations declines. We used a highly virulent A. astaci strain B (PsI genotype, transmitted by the most successful invasive crayfish) and two indigenous crayfish species: the stone crayfish Austropotamobius torrentium (Schrank, 1803) and the narrow-clawed crayfish Astacus leptodactylus Eschscholtz, 1823. In their natural habitat in Croatia, the selected species are either in direct contact (A. leptodactylus) or in the close proximity (A. torrentium) to invasive crayfish and the presence of A. astaci was already detected in some populations. Before the experiment, we have tested all animals for A. astaci presence, using a non-invasive PCR method. Only A. astaci-free animals were included into the experiment. After acclimatization, we have infected the crayfish with 500 zoospores per mL at water temperature of 18⁰C (control conditions) and 22⁰C (increased water temperature - simulation of climate change). After four weeks the results have shown that the mortality of both crayfish species was significantly lower at increased water temperature (22⁰C). This suggests that the increase of water temperatures caused by climate change will probably not result in the increased spread of A. astaci strain B in stone crayfish and narrow-clawed crayfish populations. Results will be discussed in adaptation and evolutionary perspective as well as in the context of A. astaci strains that have different water temperature preference. In conclusion, this study presents the first experimental data on the balance between A. astaci and indigenous crayfish in the context of climate change. As such, it can serve as a starting point for new research in this area and the development of adjusted conservation programs.

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.

Aim Climate change affects freshwater organisms and their interactions, i.e. the pathogens are mostly predicted to increase their distribution. We have studied the complex relationship between crayfish host, its epibiontic communities and its pathogen Aphanomyces astaci at different water temperatures. Elevated water temperature caused by climate change simultaneously affects crayfish fitness, composition of cuticle- associated communities and the virulence of its pathogens. Methods We have infected two crayfish species, Austropotamobius torrentium and Astacus leptodactylus, with A. astaci zoospores at increased water temperature (22 ⁰C, simulation of climate change) and control temperature (18 ⁰) and monitored crayfish mortality. We took cuticle swabs before the experiment and immediately after death to test for A. astaci presence. Thus, we could determine the cause of death of each animal (A. astaci-induced or other causes). Also, we analyzed the epibiontic microbiome composition by Illumina Miseq. Results The composition of cuticle-associated microbial communities was mostly determined by temperature. Further, at increased temperature crayfish mortality was reduced and mostly could not be ascribed to A. astaci infection. The observed higher diversity of epibiontic communities at elevated temperature could have contributed to host resistance to A. astaci. Conclusions We present the first data on cuticle-associated microbiota of A. torrentium and A. leptodactylus, as well as their correlation with temperature and A. astaci. This can help to predict the spreading of crayfish plague, as one of the most important causes of decline of indigenous European crayfish, in the future climate change scenarios.

Competitive exclusion is one of the mechanisms by which invasive crayfish displace native crayfish species. In this study, we examined competitive interactions between one of the most successful crayfish invaders of European freshwater ecosystems, the signal crayfish (Pacifastacus leniusculus), and the native narrow-clawed crayfish (Astacus laptodactylus). Both species already co-occur in the same river system in Croatia (the Korana River), where the invasive signal crayfish is gradually displacing the native narrow-clawed crayfish. In order to test the ability of one species to dominate the other, agonistic interactions between pairs of conspecifics (approximately 22 pairs per species) were compared to the characteristics and relative success in heterospecific dyads (mixed pairs, 5 in total). Comparisons between intraspecific pairs showed that context dynamics did not differ significantly between the species, except in fight number and duration. The signal crayfish dyads had engaged in fewer fights than the native narrow-clawed, but their duration was significantly longer. In mixed pair trials, P. leniusculus clearly dominated A. leptodactylus, being more likely to win heterospecific agonistic encounters. Furthermore, signal crayfish initiated a higher number of interactions and physical contacts than the native A. leptodactylus. This indicates that the success of P. leniusculus in agonistic encounters with the native counterpart might stem from its readiness to engage in and escalate a fight, and also through its readiness to withstand a fight for a longer period of time.

The marble crayfish (Procambarus fallax f. virginalis) is a relatively new non-indigenous crayfish species that has established populations in Europe. Until today, it is the only known decapod species with parthenogenetic reproduction. Established population of marble crayfish was recently discovered in a gravel pit in Northwest Croatia. Apart from parthenogenetic reproduction, the invasion success of this species stems from its early maturation, and higher fecundity compared to other crayfish species. Therefore, the aim of this study was to examine the year cycle of the marble crayfish in Croatia, and its potential (number of ovarian eggs) and realized fecundity (number of pleopodal eggs and juveniles). Our results show that all 140 examined individuals were reproductively active during the whole sampling period. Peaks of reproduction activity were recorded from September to November, when both individuals with pleopodal eggs or juveniles as well as individuals with ripe ovarian eggs were recorded. The smallest reproductively active female was 40.81 mm TL, while reproductive output increased with size (TL). The average number of ovarian eggs was 297. The average number of pleopodal eggs was 15% lower than the number of ovarian eggs, while number of juveniles attached to pleopods was reduced by 50% from ovarian egg count. This represents a lower brood loss compared to other crayfish species. Due to almost constant reproduction, early maturation and relatively high potential and realized fecundity in an established population in Croatia, the marble crayfish represents a major threat to the surrounding waterbodies and their native crayfish fauna.

Until recently Croatian freshwater ecosystems harboured only indigenous European crayfish species (ICS). The first appearance of the non- indigenous crayfish species (NICS), the invasive spiny-cheek crayfish Orconectes limosus, was recorded in Kopački rit in 2003 where it has spread naturally through the Danube River from Hungary. In 2008, another invasive species, the signal crayfish Pacifastacus leniusculus, was recorded in the Mura River, because of natural spread from Slovenia. Since that time, we are witnessing continuous NICS spread via natural or human- mediated ways throughout Croatian freshwaters. Due to their biological and ecological characteristics, they have severe adverse impacts on ICS. Moreover, they are latent carriers of the pathogen Aphanomyces astaci, causative agent of the disease crayfish plague that is lethal for ICS. The aim of this presentation is to give an overview on the NICS invasion in Croatia and their impact on ICS. Today, NICS comprise 43% of the total number of crayfish species present in Croatia. Since 2003, NICS have occupied 30 out of 327 10x10km grid cells of Continental Croatia. At least in two localities, they have displaced ICS in a short period of time (1-3 years), and their dispersal speed ranges between 24 km/year (downstream) and 2.5 km/year (upstream). In contact areas between ICS and NICS, transmission of A. astaci was recorded.

The crayfish plague pathogen Aphanomyces astaci (Oomycetes) is native to North America, but has expanded through Europe via invasive North American crayfish. Alien crayfish, such as Orconectes limosus, Pacifastacus leniusculus and Procambarus fallax f. virginalis, co- evolved with the pathogen and are generally resistant to the disease, acting as A. astaci carriers. On the contrary, native crayfish species are highly susceptible and infestation with A. astaci causes their death. Crayfish plague has caused major declines of native European freshwater crayfish, and A. astaci is therefore listed among the world's 100 worst invasive alien species according to the Invasive Species Specialist Group. Monitoring of A. astaci in Europe is of the upmost importance for the conservation of native crayfish populations. However, current methods for detection of A. astaci on crayfish are invasive. Namely, animals are sacrificed, DNA is extracted from pieces of cuticle and A. astaci presence is confirmed by PCR based methods. To avoid sacrificing endangered native crayfish, the main goal of this research was to develop a noninvasive method of A. astaci detection. We have firstly optimized the procedure of DNA isolation from crayfish mixed epibiotic microbial communities, including A. astaci. Then, we have improved the sensitivity of the PCR-based A. astaci detection and tested the protocol on individuals, which were infected under controlled laboratory conditions. In perspective, this protocol will allow to detect native individuals in the early phases of the infection, remove them from the natural habitat, and develop methods for their treatment.

Signal crayfish (Pacifastacus leniusculus) is a native to North America, and it was introduced from there into both Europe and Japan where it rapidly spread as an invasive species. In Croatia, it is recorded in the rivers Mura and Drava, where it had spread downstream from Slovenia, and also in the Korana River, where it had been recently illegally introduced. In these invaded areas signal crayfish outcompetes native crayfish species. Moreover, it is also a spreading vector of the pathogen Aphanomyces astaci, causative agent of crayfish plague, a disease lethal for native crayfish species. Since the knowledge on the genetic diversity of this species is limited, microsatellite markers and mitochondrial gene for 16S rRNA were analysed to explore the phylogenetic relations between the two Croatian populations (Mura and Korana) as well as their relation to other already studied populations. Morphometric characteristics were also studied to determine if there are any significant differences between the studied populations and if these are concordant with the genetic analyses results. Results showed that the genetic diversity of Pacifastacus leniusculus is slightly higher within the Mura population in comparison to the Korana population, but no clear separation between these two populations was obtained. Genetic diversity of the signal crayfish within Croatia is slightly lower in comparison to those already investigated Scandinavian and Western European populations. This study contributes to the knowledge of the signal crayfish, its characteristics, genetic diversity and also offers a possible estimation of its invasive success.

The marble crayfish (Procambarus fallax f. virginalis) is a relatively new non-indigenous crayfish species (NICS) that has established populations in Europe. Established population of marble crayfish was recently discovered in a gravel pit in Northwest Croatia, making it the third NICS present in Croatian freshwaters. In this study we looked into marble crayfish distribution in this region of Croatian territory, and examined some basic population characteristics such as year cycle characteristics and size structure. Fieldwork showed that the marble crayfish is only present in the gravel pit and its canals, while in neighboring waterbody (only 0.45 km away) – the Drava River, the signal crayfish (Pacifastacus leniusculus) presence was recorded. Marble crayfish in the gravel pit were reproductively active during the whole sampling period (from May to November 2015). Peaks of reproduction activity were recorded from September to November, when both individuals with pleopodal eggs or juveniles as well as individuals with ripe ovarian eggs were recorded. The smallest reproductively active female was 40.81 mm TL. Gonadosomatic index was the highest in May and September, indicating the most pronounced egg production in that period. Size structure of caught crayfish also varied during the sampling period, with significantly increased proportion of smaller-sized individuals in the catch during the peak reproduction activity period. Collected information regarding distribution, size structure and fecundity of the marble crayfish in Croatia represents baseline data for the development of management strategies aimed at its control.

White-clawed crayfish is a native European species distributed naturally within water bodies of Southern and Western Europe. Due to anthropogenic influence onto their habitats and spread of crayfish plague many populations disappeared and/or were dramatically reduced in their sizes. Hence white-clawed crayfish is treated as endangered and is protected by both international and local legislation. Currently, based on molecular research, white-clawed crayfish is recognised as a complex including two distinct species: Austropotamobius pallipes and Austropotamobius italicus, with four subspecies in the latter. So far, results of white-clawed morphological traits research have not yielded unambiguous and clear separations. Therefore, the aim of this study was to use large morphological and meristical data sets originating from white- clawed crayfish caught in rivers of the Adriatic Sea drainage, and analyse them using the multivariate statistical method. In total 249 crayfish from 9 populations belonging to different subspecies were studied (analysed). Results showed that males and females differ significantly between populations in measured meristical and morphometrical traits. Meristical traits were significantly correlated to the crayfish body size, therefore not reliable for distinguishing populations. From measured morphometrical traits, the most discriminant ones for separating both males and females were those describing rostrum, carapace and claw shape. Overall, percentage of correctly classified crayfish was high (98% for males, 96% for females). Additionally, grouping and separation of studied populations obtained by canonical analyses was in accordance with their taxonomical classification (i.e. subspecies), previously achieved with a molecular approach.

Signal crayfish (Pacifastacus leniusculus) is one of the most successful crayfish invaders of European freshwater ecosystems. Until 2012, its distribution in Croatia was limited to the Drava river basin where it exhibits extremely high rates of downstream dispersal. In 2012 its presence was recorded in the karstic river of the Sava river Basin, the Korana River, where it has been deliberately introduced. This area hosts numerous populations of native crayfish (Astacus leptodactylus, A. astacus and Austropotamobius torrentium). In order to estimate the extent of this recent invasion and its potential threat to existing populations of native crayfish, we examined signal crayfish distribution in the Korana River and analyzed size and structure of recorded populations. Preliminary research showed that the current signal crayfish distribution in the Korana River is localized within 20 kilometers of its lower reach. The downstream end of its distribution range is less than 20 kilometers upstream from the confluence with the Mrežnica River. Thus, recorded signal crayfish invasion has a high potential to spread on a vast majority of surrounding water bodies. Recorded populations differed in relative population abundance, with the downstream populations exhibiting a higher catch per unit effort when compared to the populations at the upstream end of the distribution. Based on obtained data, we identified and prioritized native crayfish populations which are under immediate threat from signal crayfish spread. All obtained results are discussed in the context of potential management activities aimed at conservation of invaded karstic rivers and their native crayfish fauna.

Our research on a population of Austropotamobius pallipes italicus (Faxon, 1914) in the protected karstic limnocrenic spring Modro oko (Dalmatia, southern Croatia) was carried out between February and October 1995. Data were collected on the density of crayfish population, the depth at which specimens were caught, water salinity, water temperature, alkalinity, concentration of nitrite, ammonia and phosphate and the presence of chlorophyll a. We also examined submersed vegetation as a potential microhabitat for this species. The aim of our research was to discover a possible correlation between some of these parameters and the number of crayfish. Small negative correlation was found between population density and water salinity and population density and concentration of phosphate. A positive correlation was found between the number of crayfish and the water temperature and the number of crayfish and the depth at which they were caught. The water temperature was relatively stable through the year.

The crayfish plague pathogen, the oomycete Aphanomyces astaci, has been decimating populations of European crayfish species for more than 150 years, and is therefore considered one of the 100 worst world's invasive species. A. astaci is highly specialised for a parasitic life, but it can be isolated from moribund crayfish and grown on synthetic media, as it is the case also for several other oomycetes (chapter 7). The life of A. astaci includes three basic forms: mycelium in host's tissues, and the infective units occurring in water, zoospores and cysts. All North American crayfish species tested so far have shown some resistance to A. astaci, i.e., they could carry the infection for long, serving as vectors of the pathogen. Massive sporulation from infected North American crayfish starts when the host is moulting, stressed, or dying (chapter 4). However, I could show in my experiments that some sporulation occurs even from apparently healthy and non-moulting American crayfish hosting A. astaci, so infected North American crayfish must be considered a permanent source of the infection (chapter 4). Five genotype groups of A. astaci have already been distinguished. Strains from a particular genotype group probably share the same original host crayfish species of North American origin. Nevertheless, they can...