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.

Feminising Wolbachia disrupt Armadillidium vulgare insulin-like signalling pathway.

The endosymbiont Wolbachia feminises male isopods by making them refractory to the insulin-like masculinising hormone, which shunts the autocrine development of the androgenic glands. It was, therefore, proposed that Wolbachia silences the IR receptors, either by preventing their expression or by inactivating them. We describe here the two IR paralogs of Armadillidium vulgare. They displayed a conventional structure and belonged to a family widespread among isopods. Av-IR1 displayed an ubiquist expression, whereas the expression of Av-IR2 was restricted to the gonads. Both were constitutively expressed in males and females and throughout development. However, upon silencing, altered gland physiology and gene expression therein suggested antagonistic roles for Av-IR1 (androinhibiting) and Av-IR2 (androstimulating). They may function in tandem with regulating neurohormones, as a conditional platform that conveys insulin signalling. Wolbachia infection did not alter their expression patterns: leaving the IRs unscathed, the bacteria would suppress the secretion of the neurohormones, thus inducing body-wide IR deactivation and feminisation. Adult males injected with Wolbachia acquired an intersexed physiology. Their phenotypes and gene expressions mirrored the silencing of Av-IR1 only, suggesting that imperfect feminisation stems from a flawed invasion of the androstimulating centre, whereas in fully feminised males invasion would be complete in early juveniles. TAKE AWAY: Two antagonistic Insulin Receptors were characterised in Armadillidium vulgare. The IRs were involved in androstimulating and androinhibiting functions. Wolbachia-induced feminisation did not prevent the expression of the IRs. Imperfectly feminised intersexes phenocopied the silencing of Av-IR1 only. Wolbachia would deactivate the IRs by suppressing neurosecretory co-factors.

The shutting down of the insulin pathway: a developmental window for Wolbachia load and feminization.

Using the isopod Armadillidium vulgare as a case study, we review the significance of the "bacterial dosage model", which connects the expression of the extended phenotype to the rise of the Wolbachia load. In isopods, the Insulin-like Androgenic Gland hormone (IAG) induces male differentiation: Wolbachia feminizes males through insulin resistance, presumably through defunct insulin receptors. This should prevent an autocrine development of the androgenic glands so that females differentiate instead: feminization should translate as IAG silencing and increased Wolbachia load in the same developmental window. In line with the autocrine model, uninfected males expressed IAG from the first larval stage on, long before the androgenic gland primordia begin to differentiate, and exponentially throughout development. In contrast in infected males, expression fully stopped at stage 4 (juvenile), when male differentiation begins. This co-occurred with the only significant rise in the Wolbachia load throughout the life-stages. Concurrently, the raw expression of the bacterial Secretion Systems co-increased, but they were not over-expressed relative to the number of bacteria. The isopod model leads to formulate the "bacterial dosage model" throughout extended phenotypes as the conjunction between bacterial load as the mode of action, timing of multiplication (pre/post-zygotic), and site of action (soma vs. germen).

Shedding Light on the Antimicrobial Peptide Arsenal of Terrestrial Isopods: Focus on Armadillidins, a New Crustacean AMP Family.

In crustaceans, antimicrobial peptides (AMPs) are clustered into four major groups according to their amino acid composition and structure: (1) single-domain peptides containing cysteine residues such as anti-lipopolysaccharide-factor (ALF), (2) multi-domain or chimeric AMPs such as crustins, (3) non-conventional AMPs, and (4) linear single-domain AMPs. The majority of AMPs has been described in commercially exploited crustaceans, particularly decapods living in aquatic environments (crab, shrimp, lobster, and crayfish). Here, we aimed at establishing the AMPs repertoire of terrestrial isopods (Oniscidea), an original suborder of crustaceans adapted to life outside of the aquatic environment. Using transcriptomic data from 21 species, we identified 110 ALF and 73 crustin sequences. We also characterized the full-length sequence of armadillidins from 17 species, similar to the AMP previously described in the terrestrial isopod Armadillidium vulgare. Furthermore, we tested the antimicrobial activity of three armadillidin peptides characterized from three distantly related species. This analysis revealed similar activity spectra against pathogens, despite extensive structural variation among the tested peptides. In addition to conventional crustacean AMPs, our work highlights armadillidins as a new and independent family of AMPs specific to the Oniscidea, thus opening new perspectives concerning the study of the immune system of terrestrial isopods.

IGFBP-rP1, a strongly conserved member of the androgenic hormone signalling pathway in Isopoda.

The first protein which has been described to interact with the malacostracan Androgenic Gland Hormone (AGH) is a binding protein called IGFBP-rP1. It has been identified and studied in several species of decapods, in which its interaction with the masculinizing hormone and its expression patterns have been established in several ways. However, this protein remains uncharacterised to date in the other malacostracan orders, like Amphipoda and Isopoda, although they were historically the first ones in which the androgenic gland and the corresponding hormone were respectively described. In this article, we identified the IGFBP-rP1 of isopods and established its implication in the pathway of the AGH with a silencing approach in the model species Armadillidium vulgare. We also showed that this gene is expressed in all the tissues of males and females, with a similar pattern in animals infected with Wolbachia, a feminizing endosymbiont of several isopod species. The expression pattern did not differ during the development of uninfected and infected animals either. We finally studied the evolution of the IGFBP-rP1 in 68 isopod species, looking for conserved motifs and evidence of natural selection. Altogether, our results showed that this gene is constitutively expressed and strongly conserved in isopods, in which it likely constitutes a key element of the insulin/IGF signalling pathway. However, we also illustrated that IGFBP-rP1 is not sufficient on its own to explain the different developmental paths taken by the males and the females or feminized genetic males.

Prevalence of the pathogen Aphanomyces astaci in freshwater crayfish populations in Croatia.

The Oomycete Aphanomyces astaci is an obligate crayfish parasite that co-evolved with American crayfish species, and they therefore generally live in a balanced relationship. On the contrary, European native crayfish are highly susceptible to A. astaci, and infestation with it causes development of the lethal disease termed crayfish plague. Until now, 5 A. astaci strains have been described from the freshwater crayfish present in Europe. In this study we aimed to investigate the occurrence of the pathogen A. astaci in Croatian native and non-native crayfish populations, as well as to genotype established strains using microsatellite markers and obtain information on the pathogen's epidemiology. Our results showed that the pathogen is widespread in both native and non-native crayfish populations. Agent level, when positive, in non-native crayfish was generally low; in native species it was higher. Genotyping from microsatellites proved the presence of the B (Ps) strain in non-native species (Pacifastacus leniusculus), while the A (As) strain was detected from viable native species (Astacus astacus and Austropotamobius torrentium) that are distributed in areas lacking non-native crayfish. The genotype from A. torrentium differed from a typical A (As) by 1 allele. Strain B (Ps) was identified in native Astacus leptodactylus from the population that co-occurs with P. leniuscuslus. Interestingly, in 1 A. leptodactylus population both A (As) and B (Ps) strains were present.

Effect of experimental exposure to differently virulent Aphanomyces astaci strains on the immune response of the noble crayfish Astacus astacus.

European crayfish are sensitive to the crayfish plague pathogen, Aphanomyces astaci, carried by North American crayfish species due to their less effective immune defence mechanisms against this disease. During a controlled infection experiment with a susceptible crayfish species Astacus astacus using three A. astaci strains (representing genotype groups A, B, and E), we investigated variation in their virulence and in crayfish immune defence indicators (haemocyte density, phenoloxidase activity, and production of reactive oxygen species). Experimental crayfish were exposed to two dosages of A. astaci spores (1 and 10 spores mL(-1)). The intensity and timing of the immune response differed between the strains as well as between the spore concentrations. Stronger and faster change in each immune parameter was observed in crayfish infected with two more virulent strains, indicating a relationship between crayfish immune response and A. astaci virulence. Similarly, the immune response was stronger and was observed earlier for the higher spore concentration. For the first time, the virulence of a strain of the genotype group E (isolated from Orconectes limosus) was experimentally tested. Total mortality was reached after 10 days for the two higher spore dosages (10 and 100 spores mL(-1)), and after 16 days for the lowest (1 spore mL(-1)), revealing equally high and rapid mortality as caused by the genotype group B (from Pacifastacus leniusculus). No mortality occurred after infection with genotype group A during 60 days of the experimental trial.

Microsatellite markers for direct genotyping of the crayfish plague pathogen Aphanomyces astaci (Oomycetes) from infected host tissues.

Aphanomyces astaci is an invasive pathogenic oomycete responsible for the crayfish plague, a disease that has devastated European freshwater crayfish. So far, five genotype groups of this pathogen have been identified by applying random amplified polymorphic DNA analysis on axenic cultures. To allow genotyping of A. astaci in host tissue samples, we have developed co-dominant microsatellite markers for this pathogen, tested them on pure cultures of all genotype groups, and subsequently evaluated their use on tissues of (1) natural A. astaci carriers, i.e., North American crayfish species, and (2) A. astaci-infected indigenous European species from crayfish plague outbreaks. Out of over 200 potential loci containing simple sequence repeat (SSR) motifs identified by 454 pyrosequencing of SSR-enriched library, we tested 25 loci with highest number of repeats, and finally selected nine that allow unambiguous separation of all known RAPD-defined genotype groups of A. astaci from axenic cultures. Using these markers, we were able to characterize A. astaci strains from DNA isolates from infected crayfish tissues when crayfish had a moderate to high agent level according to quantitative PCR analyses. The results support the hypothesis that different North American crayfish hosts carry different genotype groups of the pathogen, and confirm that multiple genotype groups, including the one originally introduced to Europe in the 19th century, cause crayfish plague outbreaks in Central Europe. So far undocumented A. astaci genotype seems to have caused one of the analysed outbreaks from the Czech Republic. The newly developed culture-independent approach allowing direct genotyping of this pathogen in both axenic cultures and mixed genome samples opens new possibilities in studies of crayfish plague pathogen distribution, diversity and epidemiology.

Prevalence of the crayfish plague pathogen Aphanomyces astaci in populations of the signal crayfish Pacifastacus leniusculus in France: evaluating the threat to native crayfish.

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.

Intense transpositional activity of insertion sequences in an ancient obligate endosymbiont.

The streamlined genomes of ancient obligate endosymbionts generally lack transposable elements, such as insertion sequences (IS). Yet, the genome of Wolbachia, one of the most abundant bacterial endosymbionts on Earth, is littered with IS. Such a paradox raises the question as to why there are so many ISs in the genome of this ancient endosymbiont. To address this question, we investigated IS transpositional activity in the unculturable Wolbachia by tracking the evolutionary dynamics and history of ISWpi1 elements. We show that 1) ISWpi1 is widespread in Wolbachia, being present in at least 55% of the 40 sampled strains, 2) ISWpi1 copies exhibit virtually identical nucleotide sequences both within and among Wolbachia genomes and possess an intact transposase gene, 3) individual ISWpi1 copies are differentially inserted among Wolbachia genomes, and 4) ISWpi1 occurs at variable copy numbers among Wolbachia genomes. Collectively, our results provide compelling evidence for intense ISWpi1 transpositional activity and frequent ISWpi1 horizontal transmission among strains during recent Wolbachia evolution. Thus, the genomes of ancient obligate endosymbionts can carry high loads of functional and transpositionally active transposable elements. Our results also indicate that Wolbachia genomes have experienced multiple and temporally distinct ISWpi1 invasions during their evolutionary history. Such recurrent exposition to new IS invasions may explain, at least partly, the unusually high density of transposable elements found in the genomes of Wolbachia endosymbionts.