Immunity is an important component of invasion success since it enables invaders’ adaptation to conditions of the novel environment as they expand their range. Immune response of invaders may vary along the invasion range due to encountered parasites/microbial communities, conditions of the local environment, and ecological processes that arise during the range expansion. Here, we analyzed changes in the immune response along the invasion range of one of the most successful aquatic invaders, the signal crayfish, in the recently invaded Korana River, Croatia. We 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, and between species (comparison with co-occurring native narrow-clawed crayfish), and ii) analyze effects of specific predictors (water temperature, crayfish abundance, and body condition) on crayfish immune response changes. Immune response displayed species-specificity, differed significantly along the signal crayfish invasion range, and was mostly affected by water temperature and population abundance. Specific immune parameters showed density-dependent variation corresponding to increased investment in them during range expansion. Obtained results offer baseline insights for elucidating the role of immunocompetence in the invasion success of an invertebrate freshwater invader.

The stone crayfish, Austropotamobius torrentium, is a native European freshwater species sensitive to habitat alterations such as stream channelization and urban development, putting its populations at risk. This study aims to assess how habitat alteration and subsequent water quality changes affect the stone crayfish population in the Kustošak Stream (Croatia) through three selected sites under different levels of anthropogenic disturbance: (1) undisturbed; (2) recently modified, and (3) long-time modified sites. At each site, crayfish were captured, measured, and marked to estimate population size, structure, and crayfish condition. Additionally, we examined whether water quality (18 measured physicochemical parameters) affects relative crayfish abundance. We also used mitochondrial sequencing and microsatellite genotyping to assess species genetic diversity and population connectivity and to compare results among the sites. The results showed that habitat alteration caused an overall reduction in population abundance and changes in size structure; however, it had not yet resulted in detectable genetic differences. Partial least squares regression showed that crayfish abundance was affected by the physicochemical parameters of water, including, among others, oxygen, calcium ions, nitrates, pH, and water temperature. We discuss our findings in the context of the effects of anthropogenic disturbance on the viability and persistence of this EU priority species.

Transcription factors (TFs) have an important role in the regulation of the gene expression network. The role of TFs in the immune response of freshwater crayfish is poorly understood, but leveraging the regulatory mechanisms of immune response could augment the resistance against the invasive oomycete pathogen, Aphanomyces astaci. Previous studies indicated that the TFs CCAAT/enhancer-binding protein (C/EBP) and putative Krüppel homolog-1 protein (Kr-h1) might play a role in immune and stress response of the noble crayfish (Astacus astacus). Here, we aimed to further characterise these two gene products to gain a better understanding of their evolutionary origin, domain organisation and expression patterns across different crayfish tissues. Furthermore, we conducted an immune stimulation experiment to observe the potential changes in the gene expression of C/EBP and Kr-h1 under immune challenge in different crayfish tissues. Our results showed that both C/EBP and Kr-h1 are closely related to other C/EBPs and Kr-h1s in Malacostraca. Gene expression analysis revealed that both TFs are present in all analysed tissues, with higher expression of C/EBP in the gills and Kr-h1 in the abdominal muscle. Immune stimulation with laminarin (mimicking β-1-3-glucan in the oomycete cell wall) showed an activation of the crayfish immune system, with an overall increase in the total haemocyte count (THC) compared to untreated control and crayfish buffered saline (CBS) treatment. On the gene expression level, an up-regulation of the C/EBP gene was detected in the laminarin treated group in hepatopancreas and heart, while no changes were observed for the Kr-h1 gene. Our results indicate an early change in C/EBP expression in multiple tissues during immune stimulation and suggest its involvement in the immune response of the noble crayfish.

Kopnene vode i njihova bioraznolikost predstavljaju iznimno vrijedan prirodni resurs i imaju važnu ulogu u gospodarstvu, kulturi, estetici, znanosti i obrazovanju. Predstavljaju dom mnogim organizmima, međutim smatra se da su kopnene vode (slatkovodni ekosustavi) među najugroženijim ekosustavima na Zemlji. Ugroženosti slatkovodnih ekosustava uvelike doprinosi antropogeni utjecaj. Biološke invazije jedan su od glavnih uzročnika brzog smanjenja biološke raznolikosti, a posljednjih desetljeća u značajnom su porastu. Invazivna vrsta je strana vrsta unesena na novo područje izvan svog prirodnog areala rasprostranjenosti, gdje se uspijeva nesmetano razmnožavati i širiti, a uzrokuje značajne ekološke i/ili ekonomske štete. Tematika invazivnih vrsta u zadnjih nekoliko desetljeća postaje sve više aktualna. U sprječavanju budućih unosa stranih vrsta bitno je usredotočiti se na edukaciju unutar akademske zajednice i među lokalnim stanovništvom, kroz koju bi se iznijeli detalji o posljedicama unosa stranih vrsta, koje se mogu negativno odraziti na bioraznolikost Hrvatske. U ovom radu kao primjer invazivnih beskralježnjaka Hrvatskih slatkovodnih ekosustava prikazani su sjevernoameričke vrste deseteronožnih rakova Pacifastacus leniusculus – signalni rak i Procambarus viriginalis – mramorni rak. S obzirom da su deseteronožni rakovi generalisti (s obzirom na njihove prehrambene sklonosti), imaju ključnu ulogu u vodenom staništu prenoseći energiju unutar hranidbene mreže između vodenih i kopnenih hranidbenih mreža, koristeći različite izvore hrane i služeći kao plijen za brojne kralježnjake, od riba do sisavaca. U Hrvatskoj je signalni rak prvi put zabilježen 2008. godine u rijeci Muri, dok je mramorni rak po prvi puta zabilježen u jezeru Šoderica 2014. godine. Tema obrađena u ovom radu može se uklopiti u koncepte koji se poučavaju u sklopu nastavnih predmeta Priroda i Biologija, gdje se organizmi prikazani u ovom radu mogu koristiti kao primjer za ostvarenje brojnih odgojno-obrazovnih ishoda. Jedan od tih ishoda je usporedba uspješnosti prilagodbi organizama na primjerima autohtonih, alohtonih i invazivnih stranih vrsta, pri ostvarenju kojega dolazi i do osvješćivanja nastavnika i učenika o negativnom utjecaju invazivnih vrsta beskralježnjaka na bioraznolikost, zdravlje ljudi i gospodarstvo i o ispravnom postupanju u slučaju rukovanja invazivnim vrstama.

The invasive oomycete pathogen Aphanomyces astaci significantly threatens native European crayfish populations, prompting investigations towards the effects of protective immunostimulation on the immune response of the vulnerable noble crayfish (Astacus astacus). Here, we evaluate the effect of three oomycete-derived immunostimulant treatments: laminarin (β-1,3-glucan found within the Ap. astaci cell wall), inactivated Ap. astaci spores and Ap. astaci hyphal homogenate. Our findings reveal immediate changes in the noble crayfish total haemocyte count (THC), differential haemocyte count (DHC), and gene expression. A short-term increase in the THC was observed in all treatments, with a gradual return to normal values 8 h post immunostimulation. Granular haemocytes seem to be involved in response to immunostimulation with inactivated Ap. astaci spores, while the number of semi-granular and hyaline haemocytes increased in response to laminarin and Ap. astaci hyphal homogenate. Analysis of the differentially expressed genes showed that the Prophenoloxidase pathway genes and Toll pathway genes are involved in the response to oomycete-derived immunostimulants. Prolonged effects of immunostimulation were reflected in the decreased C/EBP and Kr-h1 gene expression in the hyphal homogenate group as well as decreased Kr-h1 expression in the spore group. Taken together, our results indicate that immunostimulation causes a dynamic change in the noble crayfish immune system response, with similarities in the gene expression patterns between immunostimulated and Ap. astaci infected noble crayfish. As a future research focus, we highlight the importance of molecular characterisation of the genes involved in the anti-oomycete response which could provide valuable insights into pathogen resistance in freshwater crayfish. In the context of the Ap. astaci mediated downfall of the noble crayfish stocks across Europe, further exploration is needed regarding the benefits of the oomycete-derived immunostimulation that can potentially support conservation and aquacultural efforts.

Pharmaceuticals and personal care products (PPCPs) are now ubiquitous in freshwater ecosystems, entering water bodies through widespread use and incomplete removal during wastewater treatment. These compounds include psychoactive substances, antibiotics, anti-inflammatory drugs, hormones, chemotherapeutic drugs, fragrances, preservatives, UV filters, plasticizers and others. Although many studies report negative effects on survival and reproduction in non-target freshwater organisms, the overall magnitude and consistency of these impacts have remained unclear. We thus conducted the first systematic review and meta-analysis of PPCP effects at environmentally relevant concentrations across freshwater taxa. Our objectives were to quantify the overall effects of PPCP exposures on the fitness of freshwater fauna, and to test how the effects vary across taxa, and with developmental stage, sex, and experimental context. We synthesized data from 96 peer-reviewed experimental studies and open datasets spanning 28 species and 65 PPCP compounds. Our meta-analysis shows that exposure to environmental levels of PPCPs consistently reduced fitness components, with survival showing the strongest decline. Among compounds, pharmaceuticals—and particularly antibiotics—produced the largest effects. However, the responses were context dependent. Our synthesis also revealed a substantial gap in taxonomic and compound coverage. We found little statistical evidence of publication bias in our dataset and the results were robust to sensitivity analyses. This study provides the first quantitative, cross-taxon synthesis of PPCP impacts on freshwater animals. By integrating heterogeneous ecotoxicological evidence, our findings strengthen the basis for prioritizing high-risk compounds and improving environmental risk assessment and regulatory decision-making.