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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.

Croatia is situated in the south-east Europe on the western part of the Balkan Peninsula known as one of the major European refugial areas. Due to its position, Croatia encompasses four different biogeographical regions, and the past geological and climatic events created rich and diverse hydrology within the country. All that enabled continuous evolution and diversification of abundant freshwater fauna, including freshwater crayfish. Nowadays, four native European crayfish species, presented by divergent lineages, are distributed in Croatia. Three of them, namely the stone crayfish, white-clawed crayfish and noble crayfish, are endangered and protected by national and international legislation. The aim of this work is to show genetic diversity of the native crayfish species in the Croatian freshwaters. For that purpose, we overlapped diversity of different crayfish species haplotypes, obtained through DNA barcoding, with the map of Croatia, in the GIS programme package. Overall, 525 sequences were obtained: 118 for the stone crayfish (45 unique COI haplotypes), 274 for the noble crayfish (16 unique COI haplotypes), 96 for white-clawed crayfish (28 unique COI haplotypes) and 37 for narrow-clawed crayfish (23 unique COI haplotypes). The highest diversity for the stone crayfish was revealed in the Alpine biogeographical region, while the Continental biogeographical region hosts the highest diversity of the noble crayfish, and the Mediterranean region of the whiteclawed crayfish. Obtained results will serve, in combination with data from population genetics and species distribution modelling (useful for revealing the impact of ongoing climate change), as a basis for conservation of these endangered crayfish species.

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.

The stone crayfish is a native European freshwater species endangered due to anthropogenic pressure on its habitats, invasive alien crayfish species and climate change. Our aims were to assess its genetic diversity in Croatia using molecular phylogenetics and population genetics, and to reveal the impact of climate changes on its diversity using species distribution modelling (SDM). Phylogenetic reconstruction revealed the existence of high genetic diversity, maintained in seven geographically and genetically isolated mtDNA lineages which present distinct Evolutionary Significant Units (ESUs). Population genetic analyses based on microsatellites revealed moderate within-population genetic diversity and isolated populations with limited gene flow. Besides strong genetic structuring concordant with established ESUs, we discovered high level of inbreeding within the majority of populations. The SDM results predicted substantial decrease of suitable habitats for stone crayfish by 2070 ; 80% of its currently suitable habitats are predicted to be lost under high-warming climate change scenario. Moreover, 44% of investigated populations with high and/or unique genetic diversity are located in areas predicted to become unsuitable, thus having a high probability of extinction in the future. Considering ongoing climate change predictions, prompt actions including assisted migration and repopulation should take place in order to preserve the existing diversity of this endangered species.

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.

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.

The stone crayfish (Austropotamobius torrentium) is a native European species endangered by anthropogenically induced alteration of its habitats, climate change and invasive alien crayfish species. Diminishing populations’ trends across its distribution range highlight the importance of developing effective conservation that will ensure its long-term survival. Aiming to guide future conservation programs of the stone crayfish in Croatia, we combined population genetics and species distribution modelling (SDM) to reveal the impact of invasive signal crayfish (Pacifastacus leniusculus) and climate change on the stone crayfish diversity. Population genetic analyses based on microsatellites revealed moderate within-population genetic diversity and high differentiation among populations, reflecting isolated populations with limited gene flow. Alongside strong genetic structuring, we discovered high level of inbreeding coefficient indicating homozygote excess within the majority of populations. The SDM results predicted substantial reductions of suitable habitats for both species by 2080 under two representative concentration pathway (RCP) scenarios: RCP 4.5 and RCP 8.5, respectively describing low‐warming and high‐warming conditions. Obtained results indicated that many populations with high and/or unique genetic diversity are located in the areas predicted to become unsuitable in the future. Further, ecological niche analysis showed that stone crayfish and signal crayfish do not exploit equivalent niches in Croatia. Our study highlights the importance of conserving remnant populations and establishing new populations in areas likely to support stone crayfish in the future (climate change refugia or ark sites), by assisted migration and repopulation approaches that can help populations overcome the risks of inbreeding and maladaptation. The results of our research emphasizes once again the importance of multidisciplinary approach in the modern biodiversity conservation.

The stone crayfish (Austropotamobius torrentium) and noble crayfish (Astacus astacus) are native European freshwater species with significant population declines caused by anthropogenic pressure on their habitats, invasive crayfish species, and climate change. In order to develop effective conservation for these threatened species, prerequisite is, among others, a sound knowledge of their genetic diversity patterns. The aim of this research was to assess genetic diversity of both native species in the freshwater habitats of Croatia using DNA barcoding based on sequencing a short fragment of the mtDNA cytochrome c oxidase subunit I (COI) gene. In addition, species distribution modelling was used to reveal the impact of ongoing climate change on their genetic diversity. Overall, 392 sequences were obtained for these two species from the study area (118 for the stone crayfish and 274 for the noble crayfish). Phylogenetic reconstruction revealed the existence of high genetic diversity within stone crayfish, maintained in seven distinct mtDNA phylogroups (containing 45 unique COI Croatian haplotypes). Since those geographically and genetically isolated phylogroups are highly relevant for conservation due to their evolutionary distinctiveness and restricted distribution, we declared them as distinct evolutionary significant units. Results for the noble crayfish showed that all Croatian haplotypes belong to two previously discovered genetic lineages, with the indication of a novel lineage (containing 16 unique COI Croatian haplotypes). Overlapping genetic data with potential future distribution of these two keystone species revealed their vulnerability ; most of the populations with high and/or unique genetic diversity are located in the areas predicted to become unsuitable, and consequently have a high probability of being lost in the future. DNA barcoding proved to be a useful tool for studying species genetic diversity and guiding their conservation. Considering ongoing climate extremes and predictions, prompt actions should take place in order to preserve the existing diversity.

The Kustošak Stream is partially located within the Medvednica Nature Park, which is known for abundant stone crayfish (Austropotamobius torrentium) populations. This species is sensitive to changes in habitat so any anthropogenic impact puts stone crayfish populations at risk. This study aimed to assess how modifications of the natural features of a freshwater habitat and subsequent water quality changes affect populations of the stone crayfish in the Kustošak Stream in Zagreb, Croatia. For the study we selected three spatially close locations but under differing levels of anthropogenic disturbance: (1) no disturbance, (2) recently modified part of the stream during construction and (3) long time modified part of the stream through channelization and artificial modification of streambed. During a week-long period in September 2021, at each site, we measured physicochemical parameters of the water and collected crayfish with baited handmade traps. Caught individuals were used to estimate population size by mark-recapture method, and to analyse population structure by recording sex ratio, animal size and condition (Fulton’s condition factor). We then compared all the measured population and water parameters between sites. Results showed that population density of the site without disturbance (1) was significantly higher in comparison to the two sites that are anthropogenically impacted. Also, the crayfish size structure of this site differed significantly as opposed to the two hydromorphologically altered sites. Furthermore, PLS analyses has shown that crayfish abundance (expressed as CPUE) is strongly affected by physicochemical parameters of water, including among others: oxygen, calcium ions, nitrates, nitrites, COD, pH and water temperature. We discuss our findings in the context of effects of anthropogenic disturbance on viability and persistence of this EU priority species.

Climate change imperils the persistence of numerous keystone species, including European freshwater crayfish. The stone crayfish (Austropotamobius torrentium) is a native European freshwater species endangered due to anthropogenic pressure on its habitats, invasive alien crayfish species and climate change. In order to guide the conservation of the stone crayfish within the hotspot of its genetic diversity in Europe, we combined population genetics and species distribution modelling (SDM) to reveal the impact of climate change and invasive signal crayfish (Pacifastacus leniusculus) on the stone crayfish diversity. Population genetic analyses based on newly developed microsatellites revealed moderate within-population genetic diversity and high differentiation among populations, reflecting isolated populations with limited gene flow. Alongside strong genetic structuring concordant with previously established Evolutionary Significant Units using mtDNA data, we discovered high level of inbreeding, indicating homozygote excess within the majority of populations. The SDM results predicted substantial decreases of suitable habitats for stone crayfish by 2070 ; 80% of its currently suitable habitat is predicted to be lost under high‐warming climate change scenario (RCP8.5). Overlapping genetic data with future habitat suitability revealed that 44% of populations with high and/or unique genetic diversity, including three highly divergent and geographically restricted evolutionary lineages, are located in the areas predicted to become unsuitable in the future. Further, SDMs revealed substantial decrease of future habitat suitability for invasive signal crayfish, suggesting that climate change represents greater threat to stone crayfish. Our study highlights the importance of conserving remnant populations through assisted migration and repopulation approaches that could enable species long-term preservation. The results of our research emphasize the importance of multidisciplinary approach in the modern biodiversity conservation.