World of Crayfish™: a web platform towards real-time global mapping of freshwater crayfish and their pathogens.

Freshwater crayfish are amongst the largest macroinvertebrates and play a keystone role in the ecosystems they occupy. Understanding the global distribution of these animals is often hindered due to a paucity of distributional data. Additionally, non-native crayfish introductions are becoming more frequent, which can cause severe environmental and economic impacts. Management decisions related to crayfish and their habitats require accurate, up-to-date distribution data and mapping tools. Such data are currently patchily distributed with limited accessibility and are rarely up-to-date. To address these challenges, we developed a versatile e-portal to host distributional data of freshwater crayfish and their pathogens (using Aphanomyces astaci, the causative agent of the crayfish plague, as the most prominent example). Populated with expert data and operating in near real-time, World of Crayfish™ is a living, publicly available database providing worldwide distributional data sourced by experts in the field. The database offers open access to the data through specialized standard geospatial services (Web Map Service, Web Feature Service) enabling users to view, embed, and download customizable outputs for various applications. The platform is designed to support technical enhancements in the future, with the potential to eventually incorporate various additional features. This tool serves as a step forward towards a modern era of conservation planning and management of freshwater biodiversity.

Comprehensive analysis of the mRNA and miRNA transcriptome implicated in the immune response of Procambarus clarkii to Spiroplasma eriocheiris.

In recent years, the red swamp crayfish (Procambarus clarkii, P. clarkii) farming industry has suffered huge economic losses due to the pathogenic bacterium Spiroplasma eriocheiris (S. eriocheiris). To elucidate the immune response mechanism and identify hub immune genes as well as their associated microRNAs that regulate the host response of P. clarkii against S. eriocheiris infection, we conducted a comprehensive analysis on P. clarkii hemocyte mRNA and microRNA (miRNA) transcriptomes at different infection stages using third- and second-generation sequencing technologies. In full-length transcriptome functional annotation, 8155 unigenes were annotated, and 1168 potential new transcripts were predicted. In the mRNA transcriptome, a total of 3168 differentially expressed genes were identified at different infection stages, including 1492 upregulated and 1676 downregulated genes (duplicate genes excluded). Transcriptome analysis revealed 880 differentially expressed genes involved in multiple pathways and processes such as endocytosis, autophagy, lysosome, mTOR signaling, phagosome, and the Fanconi anemia pathway. Mfuzz analysis was employed to integrate and cluster the differential expression trends of genes across the three infection stages. In the miRNA transcriptome, 234 miRNAs and 966 predicted target genes were identified, with 86 differentially expressed miRNAs identified across the three time periods. A significant difference (P < 0.05) was observed for miRNAs including pcl-miR-146-3p, pcl-miR-74-3p, pcl-miR-225-5p, and pcl-miR-68-5p. These miRNAs are involved in multiple immune and autophagy-related pathways and have regulatory effects on immune genes including Vps26, lqf, and ERK-A. Based on the differentially expressed immune-related genes, we constructed a protein-protein interaction (PPI) network, which revealed the interactions among hub genes including Rac1, Akt1, Rho1, and Egfr. We also constructed a miRNA-gene interaction network in immune and autophagy-related processes, highlighting the potential regulatory effects of miRNAs including pcl-miR-183-5p, pcl-miR-146-3p, pcl-miR-176-5p, and pcl-miR-225-5p on proteins including LST8, SNAP29, Rab-7A, and ERK-A. To conclude, this study has identified hub immune genes and corresponding regulatory miRNAs in P. clarkii hemocytes in response to S. eriocheiris infection and explored the roles of these genes in selected pathways and processes. These findings are expected to provide further insights into the molecular mechanisms that confer resistance to S. eriocheiris infection in P. clarkii.

High variation of virulence in Aphanomyces astaci strains lacks association with pathogenic traits and mtDNA haplogroups.

Introduced into Europe from North America 150 years ago alongside its native crayfish hosts, the invasive pathogen Aphanomyces astaci is considered one of the main causes of European crayfish population decline. For the past two centuries, this oomycete pathogen has been extensively studied, with the more recent efforts focused on containing and monitoring its spread across the continent. However, after the recent introduction of new strains, the newly-discovered diversity of A. astaci in North America and several years of coevolution with its European host, a new assessment of the traits linked to the pathogen's virulence is much needed. To fill this gap, we investigated the presence of phenotypic patterns (i.e., in vitro growth and sporulation rates) possibly associated with the pathogen's virulence (i.e., induced mortality in crayfish) in a collection of 14 A. astaci strains isolated both in North America and in Europe. The results highlighted a high variability in virulence, growth rate and motile spore production among the different strains, while the total-sporulation rate was more similar across strains. Surprisingly, growth and sporulation rates were not significantly correlated with virulence. Furthermore, none of the analysed parameters, including virulence, was significantly different among the major A. astaci haplogroups. These results indicate that each strain is defined by a characteristic combination of pathogenic features, specifically assembled for the environment and host faced by each strain. Thus, canonical mitochondrial markers, often used to infer the pathogen's virulence, are not accurate tools to deduce the phenotype of A. astaci strains. As the diversity of A. astaci strains in Europe is bound to increase due to translocations of new carrier crayfish species from North America, there is an urgent need to deepen our understanding of A. astaci's virulence variability and its ability to adapt to new hosts and environments.

An old unknown: 40 years of crayfish plague monitoring in Switzerland, the water tower of Europe.

The oomycete Aphanomyces astaci is the causative agent of crayfish plague, a disease threatening susceptible freshwater crayfish species in Europe. To detect its spatiotemporal occurrence in Switzerland, we reviewed (1) the literature regarding occurrence of crayfish plague and North American crayfish carrier species and (2) the necropsy report archive of the Institute for Fish and Wildlife Health (FIWI) from 1968 to 2020. In the past, crayfish plague was diagnosed through several methods: conventional PCR, culture, and histology. When available, we re-evaluated archived Bouin's or formalin-fixed, paraffin-embedded samples collected during necropsies (1991-2020) with a recently published quantitative PCR. Literature research revealed putative reports of crayfish plague in Switzerland between the 1870s and 1910s and the first occurrence of three North American crayfish species between the late 1970s and 1990s. Finally, 54 (28.1%) cases were classified as positive and 9 (4.7%) cases as suspicious. The total number of positive cases increased by 14 (14.7%) after re-evaluation of samples. The earliest diagnosis of crayfish plague was performed in 1980 and the earliest biomolecular confirmation of A. astaci DNA dated 1991. Between 1980-1990, 1991-2000 and 2001-2010 crayfish plague spread from one to two and finally three catchment basins, respectively. Similar to other European countries, crayfish plague has occurred in Switzerland in two waves: the first at the end of the 19th and the second at the end of the 20th century in association with the first occurrence of North American crayfish species. The spread from one catchment basin to another suggests a human-mediated pathogen dispersal.

Prevalence of the crayfish plague pathogen in red swamp crayfish populations in western France: How serious is the risk for the native white-clawed crayfish?

The crayfish plague pathogen Aphanomyces astaci has been implicated in a number of mass mortalities and irreversible population declines of native crayfish across Europe. At present, the reservoirs of the pathogen in Europe are mainly populations of invasive North American crayfish species. In southwestern Europe, including France, a particularly widespread invader is the red swamp crayfish Procambarus clarkii. Recent distribution data confirm that P. clarkii is present in at least 75 French departments, i.e. more than 78% of those in metropolitan France. We analysed the prevalence and pathogen load of A. astaci in 42 populations of this species in western France (Nouvelle Aquitaine region), where the species is most densely distributed, particularly in a wide range of environments around the Gironde estuary. The pathogen was detected by two different quantitative PCR assays in more than three quarters of the populations studied (34 out of 42); 163 out of 480 analysed crayfish individuals tested positive for the presence of A. astaci. In most cases, individual infection levels were very low, detectable with quantitative PCR but not sufficient for pathogen genotyping. In seven P. clarkii individuals from four populations, however, we were able to assess A. astaci variation by microsatellite markers and sequencing of mitochondrial markers. All these host specimens carried A. astaci genotype group D, haplotype d1, which has caused the majority of crayfish plague outbreaks in neighbouring Spain. In contrast, the French outbreaks genotyped to date (including eight newly analysed in this study) were mostly caused by strains of genotype group B, specific to the signal crayfish Pacifastacus leniusculus. Haplotype d1 found in P. clarkii was involved in one of the newly characterised outbreaks. Our study confirms that P. clarkii is a potentially important reservoir of the crayfish plague pathogen in France, but not the main source of the pathogen in mass mortalities of A. pallipes, probably due to different ecological requirements of the different invasive host crayfish. However, as P. clarkii continues to spread, the threat posed by this species to native crayfish is likely to increase.

Gradual effects of gradient concentrations of perfluorooctane sulfonate on the antioxidant ability and gut microbiota of red claw crayfish (Cherax quadricarinatus).

Perfluorooctane sulfonate (PFOS) is a typical persistent organic pollutant that is characterized by environmental persistence, bioaccumulation, and toxicity. In this study, we investigated the gut microbial response of the red claw crayfish Cherax quadricarinatus after 28 days of exposure to 0 ng/L, 1 ng/L, 10 µg/L, or 10 mg/L of PFOS as a stressor. We measured oxidative stress-related enzyme activities and expression of molecules related to detoxification mechanisms to evaluate the toxic effects of PFOS. We found that PFOS disturbed microbial homeostasis in the gut of C. quadricarinatus, resulting in increased abundance of the pathogen Shewanella and decreased abundance of the beneficial bacterium Lactobacillus. The latter especially disturbed amino acid transport and carbohydrate transport. We also found that the activities of glutathione S-transferase and glutathione peroxidase were positively correlated with the expression levels of cytochrome P450 genes (GST1-1, GSTP, GSTK1, HPGDS, UGT5), which are products of PFOS-induced oxidative stress and play an antioxidant role in the body. The results of this study provided valuable ecotoxicological data to better understand the biological fate and effects of PFOS in C. quadricarinatus.

Effects of Dietary Melatonin on Antioxidant Capacity, Immune Defense, and Intestinal Microbiota in Red Swamp Crayfish (Procambarus clarkii).

The aim of this study was to investigate the effects of melatonin (MT) feed supplementation on the antioxidant capacity, immune defense, and intestinal flora in Procambarus clarkii (P. clarkii). Six groups of P. clarkii were fed test feeds containing different levels of MT: 0 mg/kg (control), 22.5, 41.2, 82.7, 165.1, and 329.2 mg/kg for a duration of 2 months. The specific growth rate, hepatosomatic index, and condition factor were recorded highest in the test group of shrimp fed an MT concentration of 165.1 mg/kg. Compared to the control group, the rate of apoptosis was lower in hepatopancreas cells of P. clarkii supplemented with high concentrations of MT. Analyses of antioxidant capacity and immune-response-related enzymes in the hepatopancreas indicated that dietary supplementation of MT significantly augmented both the antioxidant system and immune responses. Dietary MT supplementation significantly increased the expression levels of antioxidant-immunity-related genes and decreased the expression levels of genes linked to apoptosis. Dietary MT was associated with an elevation in the abundance of the Firmicutes and a reduction in the abundance of the Proteobacteria in the intestines; besides, resulting in an increase in the abundance of beneficial bacteria, such as Lactobacilli. The broken-line model indicated that the suitable MT concentration was 154.09-157.09 mg/kg. MT supplementation enhanced the growth performance of P. clarkii, exerting a positive influence on the intestinal microbiota, and bolstered both immune response and disease resistance. Thus, this study offered novel perspectives regarding the application of dietary MT supplementation within the aquaculture field.

Comparison of exoskeleton microbial communities of co-occurring native and invasive crayfish species.

Host-associated microbial communities are an important determinant of individual fitness and have recently been highlighted as one of the factors influencing the success of invasive species. Invasive hosts introduce their microbes into the new environment, and then both the host and its associated microbes enter into a series of interactions with the native macroscopic and microscopic biota. As these processes are largely unexplored, we aimed to compare the exoskeletal microbial communities of co-occurring and phylogenetically related crayfish: the native narrow-clawed crayfish Pontastacus leptodactylus and the invasive signal crayfish Pacifastacus leniusculus from the recently invaded Korana River, Croatia. The results of high-throughput 16S rRNA sequencing showed that the exoskeletal microbiome of both species is very diverse, significantly influenced by the local environment and dominated by low abundance bacterial families from the phylum Proteobacteria. Furthermore, the exoskeletal microbiomes of the crayfish species differed significantly in the composition and abundance of Amplicon Sequence Variants (ASVs), suggesting that they are to some extent shaped by species-specific intrinsic factors, despite sharing a common habitat. However, over 95% of the bacterial genera associated with the exoskeleton were detected in the exoskeleton samples of both native and invasive crayfish. We paid particular attention to two known crayfish pathogens, Aphanomyces astaci and Saprolegnia parasitica, and find that both species carry low amounts of both pathogens. On the side, we find that a non-standard ddPCR protocol outperforms standard qPCR test for A. astaci under low concentration conditions. Taken together, our results indicate the possibility of bidirectional mixing and homogenisation of exoskeleton microbiome. As such, they can serve as a baseline in future detangling of the processes that act together to shape the microbiomes of co-occuring native and invasive congeners during biological invasions.

Intestinal microbiome in crayfish: Its role upon growth and disease presentation.

The intestine-associated microbiota in crustaceans are considered a key element for maintaining homeostasis and health within the organisms. Recently, efforts have been made to characterize bacterial communities of freshwater crustaceans, including crayfish, and their interplay with the host's physiology and the aquatic environments. As a result, it has become evident that crayfish intestinal microbial communities display high plasticity, which is strongly influenced by both the diet, especially in aquaculture, and the environment. Moreover, studies regarding the characterization and distribution of the microbiota along the gut portions led to the discovery of bacteria with probiotic potential. The addition of these microorganisms to their food has shown a limited positive correlation with the growth and development of crayfish freshwater species. Finally, there is evidence that infections, particularly those from viral etiology, lead to low diversity and abundance of the intestinal microbial communities. In the present article, we have reviewed data on the crayfish' intestinal microbiota, highlighting the most frequently observed taxa and emphasizing the dominance of phylum within this community. In addition, we have also searched for evidence of microbiome manipulation and its potential impact on productive parameters, and discussed the role of the microbiome in the regulation of diseases presentation, and environmental perturbations.

PcEiger links the Imd/Relish pathway to ROS production in the intestine of the red swamp crayfish.

In the arthropod gut, commensal microbiota maintain the immune deficiency (Imd)/Relish pathway for expression of antimicrobial peptides, whereas pathogenic bacteria induce dual oxidase 2 (Duox2) for production of extracellular microbicidal reactive oxygen species (ROS). The Imd/Relish pathway and the Duox2/ROS system are regarded as independent systems. Here, we report that these two systems are bridged by the tumor necrosis factor (TNF) ortholog PcEiger in the red swamp crayfish Procambarus clarkii. PcEiger expression is induced by commensal bacteria or the Imd/Relish pathway. PcEiger knockdown alters bacterial abundance and community composition due to variations in the oxidative status of the intestine. PcEiger induces Duox2 expression and ROS production by regulating the activity of the transcription factor Atf2. Moreover, PcEiger mediates regulation of the Duox2/ROS system by commensal bacteria and the Imd/Relish pathway. Our findings suggest that the Imd/Relish pathway regulates the Duox2/ROS system via PcEiger in P. clarkii, and they provide insights into the crosstalk between these two important mechanisms for arthropod intestinal immunity.

Characterization of fibroblast growth factor receptor 4 (FGFR4) from the red swamp crayfish Procambarus clarkii and its role in antiviral and antimicrobial immune responses.

FGFRs involved multiple physiological processes, such as endocrine homeostasis, wound repair, and cellular behaviors including proliferation, differentiation and survival. In the present study, the homologs of fibroblast growth factor receptor 4 (FGFR4) were identified and characterized from the red swamp crayfish Procambarus clarkii for the first time. The full-length cDNAs of pcFGFR4 were 2878 bp with 2451 bp open reading frame (ORF), respectively. The deduced pcFGFR4 protein contained an immunoglobulin, two immunoglobulin C-2 Type, a transmembrane region and a catalytic domain. Real-time PCR analysis showed that pcFGFR4 were highly expressed in muscle and hemocyte. Moreover, the expression levels of pcFGFR4 in the hepatopancreas and hemocyte were positively stimulated after challenge with Aeromonas hydrophila and WSSV, implying the involvement of pcFGFR4 against bacterial and viral infections in innate immune responses. While pcFGFR4 were silenced in vivo, the expression levels of antimicrobial peptide (AMP) genes (pcALF1-5,8 and pcCrustin1-2) and NF-κB signaling components (pcDrosal and pcRelish) were significantly reduced. Additionally, NF-κB signaling could be markedly activated by overexpression of pcFGFR4 in HEK293T cells. Finally, our results indicated that pcFGFR4 regulated crayfish's innate immunity by modulating NF-κB signaling. These findings may provide new insights into pcFGFR4-mediated signaling cascades in crustaceans and provide a better understanding of crustacean innate immune system.

Detection of Zoonotic Bacteria and Paragonimus kellicotti in Red Swamp Crayfish (Procambarus clarkii) and the Assessment of Traditional Crayfish Boils.

ABSTRACT: Studies of red swamp crayfish (Procambarus clarkii) outside of the United States confirm the presence of a variety of zoonotic pathogens, but it is unknown whether these same pathogens occur in P. clarkii in the United States. The U.S. commercial crayfish industry generates $200 million yearly, underscoring the need to evaluate this consumer commodity. The study objectives were to evaluate specific zoonotic pathogens present on P. clarkii from Alabama and Louisiana, states in the southeastern United States, and to determine the effectiveness of traditional food preparation methods to reduce pathogens. Experiment A evaluated the presence of Escherichia coli, Salmonella, Staphylococcus aureus, and Vibrio spp. in crayfish and environmental samples over a 2-month collection period (May to June 2021). Crayfish sampling consisted of swabbing the cephalothorax region; 15 samples were tested for E. coli, Salmonella, and S. aureus, and an additional 15 samples for Vibrio spp. Additionally, crayfish shipping materials were sampled. In experiment B, 92 crayfish were evaluated for Paragonimus kellicotti. Experiment C compared live and boiled crayfish for the presence of Vibrio spp. In experiments A and B, all 60 (100%) crayfish samples and 13 (81.25%) of 16 environmental samples showed growth characteristic of Vibrio spp. Three (5%) of 60 samples showed E. coli growth, with no statistical difference (P = 0.5536) between farms. P. kellicotti, Salmonella, and S. aureus were not recovered from any samples. In experiment C, all 10 (100%) of the live preboiled crayfish samples showed characteristic growth, whereas 1 (10%) of 10 samples of crayfish boiled in unseasoned water showed Vibrio growth (P < 0.0001). These results confirm that Vibrio spp. and E. coli may be present on U.S. commercial crayfish and that care should be taken when handling any materials that come into contact with live crayfish because they can potentially be contaminated.

Sources of protein diet differentially stimulate the gut and water microbiota under freshwater crayfish, marron (Cherax cainii, Austin 2002) culture.

To reduce the reliance on fishmeal (FM), other protein sources have been evaluated on cultured animals. In a 60-days feeding trial, marrons (Cherax cainii) were fed a FM diet and five test diets containing 100% of plant-based protein sources such as soybean, lupin and valorised animal-based proteins such as poultry-by-product, black soldier fly and tuna hydrolysate. At the end of the trial, DNA samples from marron gut and rearing water were investigated through DNA-based 16S rRNA gene sequencing. Plant-based diets increased abundance for Aeromonas, Flavobacterium and Vogesella, whereas animal and insect proteins influenced diverse bacterial groups in the gut linked to various metabolic activities. Insect meal in the water favoured the growth of Firmicutes and lactic acid bacteria, beneficial for the marron health. Aeromonas richness in the gut and reared water signified the ubiquitous nature of the genus in the environment. The higher bacterial diversity in the gut and water with PBP and BSF was further supported by qPCR quantification of the bacterial single-copy gene, rpoB. The overall results suggested that PBP and BSF can exhibit positive and influential effects on the gut and water microbial communities, hence can be used as sustainable ingredients for the crayfish aquaculture.

An outbreak of crayfish rickettsiosis caused by Coxiella cheraxi in redclaw crayfish (Cherax quadricarinatus) imported to Israel from Australia.

The redclaw crayfish (Cherax quadricarinatus) is a freshwater decapod crustacean, cultured in numerous countries worldwide for both food and ornamental purposes. Redclaw crayfish has become an important aquaculture species due to its physical and biological traits, relatively easy breeding, and a short growing-out period to reach commercial size. Bacterial infections are the second-most studied pathogens of freshwater crayfish. However, redclaw crayfish rickettsiosis, caused by Coxiella cheraxi, was reported in only a few scientific papers in the early 2000s, in Australia and Ecuador. Coxiella cheraxi is a rod-shaped intracellular bacterium that can cause mortality of 22%-80% in naturally infected crayfish. In experimental infections, mortality rates may be even higher (40%-90%). Coxiella cheraxi is closely related to Coxiella burnetii, the agent of Q-fever, which affects ruminants (goats, sheep, and cattle) and occasionally may cause zoonotic infections. According to the scientific knowledge available, C. cheraxi is a species-specific pathogen because it has been only detected in Cherax quadricarinatus and thus far, there is no evidence of a zoonotic potential. In this study, we describe an outbreak of rickettsiosis in a batch of redclaw crayfish imported to Israel from an Australian hatchery, observed 2 months after introduction in a quarantine facility. Initial mortality was evaluated through histopathology, revealing infection by rickettsia-like organisms (RLO) that were subsequently investigated by molecular analysis and transmission electron microscopy examination. Phylogenetic analysis revealed that the detected RLO were closely related to C. cheraxi from a single source (Australian strain TO98), available in free publicly accessible databases. After 5 months in quarantine, almost 99% of the crayfish population had died. Our findings raise valuable questions related to aquatic animal trade and the importance of mitigation measures, such as quarantine and routine diagnostic procedures, to limit the spread of infectious diseases.

Gut microbiome alterations in the crustacean Pacifastacus leniusculus exposed to environmental concentrations of antibiotics and effects on susceptibility to bacteria challenges.

Gut-associated microbiota in crustaceans are recognized as a key element for maintaining homeostasis and health in the animal. Since the richness of these microbial communities is strongly influenced by the local environment, especially in aquatic organisms, it is important to address to what extent environmental variations can affect these communities. In the present study, we used high-throughput 16S rRNA sequencing technology to study the composition of gut-associated microbiota of the crayfish Pacifastacus leniusculus after exposure to environmentally-relevant concentrations of an antibiotic, namely sulfamethoxazole. Also, we examined if alterations of microbiota caused by environmentally-relevant concentrations of this antibiotic affected the host susceptibility to bacterial diseases, including Vibrio species. As a result, we found high individual variability of bacterial abundance and composition in the intestinal microbiome of crayfish, in both antibiotic-exposed and antibiotic-free crayfish. However, an increase of chitinolytic bacteria including Vibrio spp. was detected in some animals exposed to the antibiotic. Moreover, when crayfish susceptibility to bacterial infections was tested, the antibiotic-exposed crayfish survived longer than the control crayfish group. This study represents the first approach for investigating the interplay between crayfish and intestinal bacteria during antibiotic-pollution scenarios. Results herein should be considered by scientists before planning experiments under laboratory conditions, especially to study environmental effects on aquatic animals' intestinal health and immune status.

Microbiome of the Successful Freshwater Invader, the Signal Crayfish, and Its Changes along the Invasion Range.

Increasing evidence denotes the role of the microbiome in biological invasions, since it is known that microbes can affect the fitness of the host. Here, we demonstrate differences in the composition of an invader's microbiome along the invasion range, suggesting that its microbial communities may affect and be affected by range expansion. Using a 16S rRNA gene amplicon sequencing approach, we (i) analyzed the microbiomes of different tissues (exoskeleton, hemolymph, hepatopancreas, and intestine) of a successful freshwater invader, the signal crayfish, (ii) compared them to the surrounding water and sediment, and (iii) explored their changes along the invasion range. Exoskeletal, hepatopancreatic, and intestinal microbiomes varied between invasion core and invasion front populations. This indicates 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]), including the differences in invasion core and front population structure. Hemolymph had the most distinct microbiome compared to other tissues and differed between upstream (rural) and downstream (urban) river sections, indicating that its microbiome is potentially more driven by the effects of the abiotic environment. 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. IMPORTANCE 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, while 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, and explored its changes along the signal crayfish invasion range in the Korana River, Croatia. Our study sets the perspective for future research required to assess the contribution of these changes to an individual's overall health status and resilience of dispersing populations and their impact on invasion success.

Lytic Polysaccharide Monooxygenases as Chitin-Specific Virulence Factors in Crayfish Plague.

The oomycete pathogen Aphanomyces astaci, also known as "crayfish plague", is an obligate fungal-like parasite of freshwater crustaceans and is considered responsible for the ongoing decline of native European crayfish populations. A. astaci is thought to secrete a wide array of effectors and enzymes that facilitate infection, however their molecular mechanisms have been poorly characterized. Here, we report the identification of AA15 lytic polysaccharide monooxygenases (LPMOs) as a new group of secreted virulence factors in A. astaci. We show that this enzyme family has greatly expanded in A. astaci compared to all other oomycetes, and that it may facilitate infection through oxidative degradation of crystalline chitin, the most abundant polysaccharide found in the crustacean exoskeleton. These findings reveal new roles for LPMOs in animal-pathogen interactions, and could help inform future strategies for the protection of farmed and endangered species.

Etiological characteristics of "tail blister disease" of Australian redclaw crayfish (Cherax quadricarinatus).

In November 2019, an acute disease outbreak in Australian redclaw crayfish (Cherax quadricarinatus) occurred in a farm in Hubei, China, with a cumulative mortality rate of over 80%. One of the characteristic symptoms of the disease was blisters on the tail. This symptom is also common in diseased Procambarus clarkii every year in this country, but the causative agent has not been determined. This study analyzed the etiological characteristics of this disease. Bacterial isolation and identification combined with high-throughput sequencing analysis were conducted to obtain the microbiota characteristics in the hemolymph, hepatopancreas, and intestines. Results showed that this outbreak was caused by infection from Aeromonas hydrophila and Aeromonas veronii. The underlying cause was stress imposed on crayfish during transferring from outdoor pond to indoor pond because of temperature drops. Aeromonas infection caused remarkable changes in the structure of the microbial composition in the hemolymph, hepatopancreas, and intestines of the crayfish. The abundance of Aeromonas in the hemolymph of the sick crayfish was as high as 99.33%. In particular, KEGG metabolic pathway analysis showed that some antibiotic synthesis, enterobactin biosynthesis, and myo-inositol degradation pathways were abundant in healthy crayfish hemolymphs, which may be the mechanism of maintaining crayfish health. Conversely, inhibition of these pathways led to the disorder of microbiota structure, finally leading to the occurrence of diseases. To the knowledge of the authors, this study was the first to use high-throughput amplicon sequencing targeting the 16S rRNA gene to find the causative bacteria in aquatic animals. This protocol can provide more comprehensive and reliable evidence for pathogen identification, even if the pathogenic bacteria are anaerobes or other hard-to-culture bacteria.

A new insight to characterize immunomodulation based on hepatopancreatic transcriptome and humoral immune factor analysis of the Cherax quadricarinatus infected with Aeromonas veronii.

Cherax quadricarinatus is a type of large freshwater crayfish that is characterized by rapid growth and formidable adaptability. It has also been widely cultured and studied as a model organism. Aeromonas veronii is the dominant pathogen in aquatic environments and the primary threat to aquaculture's economic stability. To better understand the interactions between C. quadricarinatus and A. veronii, high-throughput RNA sequencing of the C. quadricarinatus hepatopancreas was carried out on a control group, susceptible group (6 h after infection), and resistant group (48 h after infection). A total of 65,850,929 genes were obtained. Compared with the control group, 2616 genes were up-regulated and 1551 genes were down-regulated in the susceptible group; while 1488 genes were up-regulated and 1712 genes were down-regulated in the resistant group. GO and KEGG analysis showed that these differentially expressed genes (DEGs) were associated with multiple immune pathways, including Toll-like receptors (TLRs), antigen processing and presentation, NOD-like receptor signaling pathway, phagosome, lysosome, JAK-STAT signaling pathway. qRT-PCR showed that infection by A. veronii changed the expression pattern of the serine proteinase inhibitor (SPI), crustacean hyperglycemic hormone (CHH), anti-lipopolysaccharide factor (ALF), and extracellular copper/zinc superoxide dismutase (SOD1), all of which were significantly higher than in the control group up to 48 h after infection. In addition, detection of superoxide dismutase (SOD), catalase (CAT), lysozyme (LZM), and phenoloxidase (PO) activity, as well as ceruloplasmin (CP) concentration at different times after infection showed diverse trends. Furthermore, pathological sections obtained 24 h after infection show lesions on the hepatopancreas and intestinal tissues caused by A. veronii. The results of this study provide a foundation for analyzing the immune mechanism of C. quadricarinatus infected with A. veronii at the transcriptional level and a theoretical basis for screening disease-resistant individuals to ensure healthy economic development of the aquatic industry.

Patterns of infection in a native and an invasive crayfish across the UK.

Invasive crayfish and the introduction of non-native diseases pose a significant risk for the conservation of endangered, white-clawed crayfish (Austropotamobius pallipes). Continued pollution of waterways is also of concern for native species and may be linked with crayfish disease dynamics. We explore whether crayfish species or environmental quality are predictors of infection presence and prevalence in native A. pallipes and invasive signal crayfish (Pacifastacus leniusculus). We use a seven-year dataset of histology records, and a field survey comparing the presence and prevalence of infectious agents in three isolated A. pallipes populations; three isolated P. leniusculus populations, and three populations where the two species had overlapped in the past. We note a lower diversity of parasites (Simpson's Index) in P. leniusculus ('Pacifastacus leniusculus Bacilliform Virus' - PlBV) (n = 1 parasite) relative to native A. pallipes (n = 4 parasites), which host Thelohania contejeani, 'Austropotamobius pallipes bacilliform virus' (ApBV), Psorospermium haeckeli and Branchiobdella astaci, at the sites studied. The infectious group present in both species was an intranuclear bacilliform virus of the hepatopancreas. The prevalence of A. astaci in A. pallipes populations was higher in more polluted water bodies, which may reflect an effect of water quality, or may be due to increased chance of transmission from nearby P. leniusculus, a species commonly found in poor quality habitats.