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Freshwater crayfish immunity has received great attention due to the need for urgent conservation. This concern has increased the understanding of the cellular and humoral defense systems, although the regulatory mechanisms involved in these processes need updating. There are, however, aspects of the immune response that require clarification and integration. The particular issues addressed in this review include an overall description of the oomycete Aphanomyces astaci , the causative agent of the pandemic plague disease, which affects freshwater crayfish, and an overview of crustaceans’ immunity with a focus on freshwater crayfish. It includes a classification system of hemocyte sub-types, the molecular factors involved in hematopoiesis and the differential role of the hemocyte subpopulations in cell-mediated responses, including hemocyte infiltration, inflammation, encapsulation and the link with the extracellular trap cell death pathway (ETosis). In addition, other topics discussed include the identity and functions of hyaline cells, the generation of neoplasia, and the emerging topic of the role of sessile hemocytes in peripheral immunity. Finally, attention is paid to the molecular execution of the immune response, from recognition by the pattern recognition receptors (PRRs), the role of the signaling network in propagating and maintaining the immune signals, to the effector elements such as the putative function of the Down syndrome adhesion molecules (Dscam) in innate immune memory.

Freshwater crayfish Pontastacus leptodactylus is an indigenous European species of considerable ecological and commercial significance. However, it is highly susceptible to the oomycete Aphanomyces astaci , the causative agent of crayfish plague. Although its importance is widely recognized, little information exists on how seasonality affects the physiology and welfare of this species. The present study was designed to (i) investigate seasonal effects on the physiological responses of P. leptodactylus in relation to abiotic factors and crayfish plague outbreak, and (ii) identify the most suitable period for restocking in temperate regions. To achieve this, we characterized for the first time the mRNA expression of hsp70, hsp90, hif-1α, ubiquitin, and gapdh (as endogenous control gene), alongside protein levels of Hsp70, Hsp90, hydroxylated Hif-1α, ubiquitin conjugates, cleaved caspases, Bax/Bcl-2 ratio, and interleukin-6 (IL-6). Our findings revealed that cold stress triggered both the heat shock response and apoptosis. Interestingly, despite the activation of apoptosis, which generally suppresses inflammation, IL-6 levels increased, suggesting a possible association with A. astaci infection. Moreover, elevated Hif-1α levels indicated hypoxic stress during warmer months, likely linked to reduced oxygen availability and turbidity. Overall results suggest that crayfish plague epidemics may intensify with rising temperatures, whereas winter appears to be the optimal period for restocking. Interestingly, in addition, freshwater crayfish in temperate latitudes are organisms that face both cold and heat stress seasonally in the same ecosystem.

Austropotamobius pallipes complex is an endangered freshwater crayfish species in Europe and the assessment of the health status of its wild populations is essential for conservation purposes. The two microsporidia Astathelohania contejeani and Nosema austropotamobii have been reported to cause in A. pallipes complex a chronic parasitic infection, known as ‘porcelain disease’, which reduces population fitness and leads the host to death. Due to the similar macroscopic signs produced, molecular biology analyses are required to unambiguously distinguish between these microsporidia. Focusing on A. pallipes complex populations located in Northern and Central Italy, the present study provides an evaluation of prevalence and distribution of A. contejeani and N. austropotamobii , and investigates the variables affecting the probability of detecting infected specimens during a survey (e.g. sex, crayfish density, longitude). Microsporidia were identified in 12 populations among the 42 monitored from 2011 to 2017, with an average prevalence of 3.12% for A. contejeani and 3.60% for N. austropotamobii , the latter being reported in a wider area than previously documented (from Lombardy to Liguria Regions). Notably, crayfish co-infected by both microsporidia were also detected in 4 populations. Moreover, it was observed that the probability of detecting a crayfish with a microsporidian infection significantly increased eastwards in the studied area, especially for N. austropotamobii . Our distribution map for microsporidiosis, combined with molecular screening, will be useful for planning breeding and translocation efforts for A. pallipes complex populations.

The crayfish plague pathogen Aphanomyces astaci (Oomycota: Saprolegniales) is native to North America but expanded with its crayfish hosts to other regions. In most of its invaded range, A. astaci haplotypes are associated with specific American crayfish, probably due to introduction bottlenecks, but haplotype diversity is higher and clear host-specific associations are lacking in its native range. However, little is known about the infection rate and load of this pathogen in North America. We investigated the distribution, prevalence and genetic variation of A. astaci in Pennsylvania (eastern USA), where multiple native and introduced crayfish species (family Cambaridae) occur. We used A. astaci -specific quantitative PCR to screen 533 individuals representing 8 crayfish species (2 Cambarus and 6 Faxonius ) from 49 sites. Faxonius limosus , an American species first introduced to Europe and carrier of A. astaci genotype group E, was of particular interest. We confirmed A. astaci infections in 76% of sites in all but 1 host taxon, with the pathogen infection rate and load comparable to established populations of North American crayfish studied in Europe and Japan. Despite the absence of highly infected hosts, we genotyped A. astaci from 14 sites. We only detected 2 mitochondrial haplotypes, but nuclear markers indicated the presence of at least 4 distinct pathogen genotypes, none documented from invaded areas in Europe or Asia. Genotype group E was not detected in F. limosus , possibly due to limited spatial distribution of the original strain. Our results highlight both benefits and limitations of combining multiple pathogen genotyping methods.

Aphanomyces astaci, the crayfish plague pathogen, first appeared in Europe in the mid-19(th) century and is still responsible for mass mortalities of native European crayfish. The spread of this parasite across the continent is especially facilitated by invasive North American crayfish species that serve as its reservoir. In France, multiple cases of native crayfish mortalities have been suggested to be connected with the presence of the signal crayfish Pacifastacus leniusculus, which is highly abundant in the country. It shares similar habitats as the native white-clawed crayfish Austropotamobius pallipes and, when infected, the signal crayfish might therefore easily transmit the pathogen to the native species. We investigated the prevalence of A. astaci in French signal crayfish populations to evaluate the danger they represent to local populations of native crayfish. Over 500 individuals of Pacifastacus leniusculus from 45 French populations were analysed, plus several additional individuals of other non-indigenous crayfish species Orconectes limosus, O. immunis and Procambarus clarkii. Altogether, 20% of analysed signal crayfish tested positive for Aphanomyces astaci, and the pathogen was detected in more than half of the studied populations. Local prevalence varied significantly, ranging from 0% up to 80%, but wide confidence intervals suggest that the number of populations infected by A. astaci may be even higher than our results show. Analysis of several individuals of other introduced species revealed infections among two of these, O. immunis and P. clarkii. Our results confirm that the widespread signal crayfish serves as a key reservoir of Aphanomyces astaci in France and therefore represents a serious danger to native crayfish species, especially the white-clawed crayfish. The prevalence in other non-indigenous crayfish should also be investigated as they likely contribute to pathogen transmission in the country.

The oomycete Aphanomyces astaci, the causative agent of crayfish plague, is listed as one of the 100 worst invasive species in the world, destroying the native crayfish populations throughout Eurasia. The aim of this study was to examine the potential of selected mitochondrial (mt) genes to track the diversity of the crayfish plague pathogen A. astaci. Two sets of primers were developed to amplify the mtDNA of ribosomal rnnS and rnnL subunits. We confirmed two main lineages, with four different haplogroups and five haplotypes among 27 studied A. astaci strains. The haplogroups detected were (1) the A-haplogroup with the a-haplotype strains originating from Orconectes sp., Pacifastacus leniusculus and Astacus astacus; (2) the B-haplogroup with the b-haplotype strains originating from the P. leniusculus; (3) the D-haplogroup with the d1 and d2-haplotypes strains originating from Procambarus clarkii; and (4) the E-haplogroup with the e-haplotype strains originating from the Orconectes limosus. The described markers are stable and reliable and the results are easily repeatable in different laboratories. The present method has high applicability as it allows the detection and characterization of the A. astaci haplotype in acute disease outbreaks in the wild, directly from the infected crayfish tissue samples.

Ecological theory and observational evidence suggest that symbiotic interactions such as cleaning symbioses can shift from mutualism to parasitism. However, field experimental evidence documenting these shifts has never been reported for a cleaning symbiosis. Here, we demonstrate shifts in a freshwater cleaning symbiosis in a system involving crayfish and branchiobdellid annelids. Branchiobdellids have been shown to benefit their hosts under some conditions by cleaning material from host crayfish's gill filaments. The system is uniquely suited as an experimental model for symbiosis due to ease of manipulation and ubiquity of the organisms. In three field experiments, we manipulated densities of worms on host crayfish and measured host growth in field enclosures. In all cases, the experiments revealed shifts from mutualism to parasitism: host crayfish growth was highest at intermediate densities of branchiobdellid symbionts, while high symbiont densities led to growth that was lower or not significantly different from 0-worm controls. Growth responses were consistent even though the three experiments involved different crayfish and worm species and were performed at different locations. Results also closely conformed to a previous laboratory experiment using the same system. The mechanism for these shifts appears to be that branchiobdellids switched from cleaning host gills at intermediate densities of worms to consuming host gill tissue at high densities. These outcomes clearly demonstrate shifts along a symbiosis continuum with the maximum benefits to the host at intermediate symbiont densities. At high symbiont densities, benefits to the host disappear, and there is some evidence for a weak parasitism. These are the first field experimental results to demonstrate such shifts in a cleaning symbiosis.

Symbiont community assembly is driven by host–symbiont and symbiont–symbiont interactions. The effects that symbionts exert on their hosts are often context‐dependent, and existing theoretical frameworks of symbiont community assembly do not consider the implications of variable outcomes to assembly processes. We hypothesized that symbiont–symbiont interactions become increasingly important along a parasitism/mutualism continuum because; (i) negative outcomes favour host resistance which in turn reduces symbiont colonization and subsequently reduce symbiont–symbiont interactions, whereas (ii) positive host outcomes favour tolerance and consequently higher symbiont colonization rates, leading to stronger interactions among symbionts. We found support for this hypothesis in the cleaning symbiosis between crayfish and ectosymbiotic branchiobdellidan worms. The symbiosis between crayfish and their worms can shift from parasitism/commensalism to mutualism as crayfish age. Here, field surveys identified changes in worm density, diversity and composition that were concomitant to changing symbiosis outcomes. We conducted several laboratory experiments and behavioural assays to relate patterns from the field to their likely causal processes. Young crayfish typically hosted only two relatively small worm species. Older crayfish hosted two additional larger species. In laboratory experiments, young crayfish exhibited a directed grooming response to all worm species, but were unable to remove small species. Conversely, adult crayfish did not exhibit grooming responses to any worm species. Relaxed grooming allowed the colonization of large worm species and initiated symbiont–symbiont intraguild predation that reduced the abundance and altered the behaviour of small worm species. Thus, the dominant processes of symbiont community assembly shifted from host resistance to symbiont–symbiont interactions through host ontogeny and a concomitant transition towards mutualism. This work shows that host resistance can have a prevailing influence over symbiont community assembly when symbiosis is disadvantageous to the host. However, when symbiosis is advantageous and resistance is relaxed, symbiont colonization rate and consequently abundance and diversity increases and interactions among symbionts become increasingly important to symbiont community assembly.

Australian spiny mountain crayfish (Euastacus, Parastacidae) and their ecotosymbiotic temnocephalan flatworms (Temnocephalida, Platyhelminthes) may have co-occurred and interacted through deep time, during a period of major environmental change. Therefore, reconstructing the history of their association is of evolutionary, ecological, and conservation significance. Here, time-calibrated Bayesian phylogenies of Euastacus species and their temnocephalans (Temnohaswellia and Temnosewellia) indicate near-synchronous diversifications from the Cretaceous. Statistically significant cophylogeny correlations between associated clades suggest linked evolutionary histories. However, there is a stronger signal of codivergence and greater host specificity in Temnosewellia, which co-occurs with Euastacus across its range. Phylogeography and analyses of evolutionary distinctiveness (ED) suggest that regional differences in the impact of climate warming and drying had major effects both on crayfish and associated temnocephalans. In particular, Euastacus and Temnosewellia show strong latitudinal gradients in ED and, conversely, in geographical range size, with the most distinctive, northern lineages facing the greatest risk of extinction. Therefore, environmental change has, in some cases, strengthened ecological and evolutionary associations, leaving host-specific temnocephalans vulnerable to coextinction with endangered hosts. Consequently, the extinction of all Euastacus species currently endangered (75%) predicts coextinction of approximately 60% of the studied temnocephalans, with greatest loss of the most evolutionarily distinctive lineages.

A new genus and species within the family Polymorphidae Meyer, 1931 were erected to accommodate cystacanths recovered from the mesentery of individuals from a non-native population of the red swamp crayfish Procambarus clarkii (Girard), collected from South Carolina (USA). Morphological characteristics of the specimens collected included in both sexes a spindle-shaped body with a slender hindtrunk, two fields of markedly different sized spines on the foretrunk, the presence of a middle row of smaller scythe-shaped hooks on a proboscis armed with 19–20 longitudinal rows of 14–16 hooks; and in males, six cement glands, absence of genital spines, and a digitiform and spinose bursa (observed inverted). Sequencing portions of both the mitochondrial cytochrome c oxidase I (COI) and large subunit ribosomal RNA genes was completed, followed by phylogenetic analysis of a concatenated alignment. Sequences from our specimens appeared in a clade with those of Hexaglandula corynosoma (Travassos, 1915) and Ibirhynchus dimorpha (Schmidt, 1973) but were 27% divergent from both using the COI marker. The genetic divergence of this parasite from other polymorphid genera, along with unique morphological features, justified erecting a new genus and new species. Herein we describe Heterospinus mccordi n. gen. n. sp. bringing the total number of genera within the family Polymorphidae to 16, and we emend and update the latest key that was provided for the genera within this family. This is the first record of polymorphids infecting P. clarkii outside of its native range. The definitive host remains unknown.