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.

Diversity and distribution of Aphanomyces astaci in a European hotspot of ornamental crayfish introductions.

Ornamental trade has become an important introduction pathway of non-native aquatic species worldwide. Correspondingly, there has been an alarming increase in the number of established crayfish of aquarium origin in Europe over the previous decade. The oomycete Aphanomyces astaci, the pathogen causing crayfish plague responsible for serious declines of European crayfish populations, is dispersed with introduced North American crayfish. The role of ornamental taxa in introducing and spreading different genotypes of this pathogen in open waters remains unclear. We investigated the distribution, prevalence, and diversity of A. astaci in Budapest, Hungary, which became a hotspot of aquarium crayfish introductions. Their establishment in this area was facilitated by locally abundant thermal waters. We screened for A. astaci in six host taxa from 18 sites sampled between 2018 and 2021: five cambarids (Cambarellus patzcuarensis, Faxonius limosus, Procambarus alleni, P. clarkii, P. virginalis) and one native astacid (Pontastacus leptodactylus). The pathogen was confirmed at five sampled sites in four host taxa: P. virginalis, P. clarkii, F. limosus, and for the first time in European open waters also in P. alleni. Genotyping was successful only in individuals from two different brooks where multiple host species coexisted but revealed unexpected patterns. Mitochondrial B-haplogroup of A. astaci, previously usually reported from Pacifastacus leniusculus or infected European species, was detected in P. virginalis at both sites, and in both F. limosus and P. virginalis sampled from a thermally stable tributary of Barát brook in 2018. In contrast, A-haplogroup of A. astaci was detected in coexisting F. limosus, P. virginalis and P. clarkii sampled in the same watercourse just a few hundred meters downstream in 2020. Additional genotyping methods indicated that a previously unknown A. astaci strain was associated with the latter haplogroup. One P. virginalis individual from 2020 was apparently co-infected by strains representing both mitochondrial haplogroups. The results indicated multiple sources of A. astaci in Budapest, likely directly associated with the introduction of ornamental species, interspecific transmission of this pathogen among ornamental hosts, and potential for a quick spatial or temporal turnover of dominant A. astaci strains at a certain locality. This highlights that in regions with high richness of potential A. astaci hosts, host taxon/pathogen genotype combinations become unpredictable, which might prevent reliable genotyping of pathogen sources in local crayfish mass mortalities.

Spatial insurance against a heatwave differs between trophic levels in experimental aquatic communities.

Climate change-related heatwaves are major threats to biodiversity and ecosystem functioning. However, our current understanding of the mechanisms governing community resistance to and recovery from extreme temperature events is still rudimentary. The spatial insurance hypothesis postulates that diverse regional species pools can buffer ecosystem functioning against local disturbances through the immigration of better-adapted taxa. Yet, experimental evidence for such predictions from multi-trophic communities and pulse-type disturbances, like heatwaves, is largely missing. We performed an experimental mesocosm study to test whether species dispersal from natural lakes prior to a simulated heatwave could increase the resistance and recovery of plankton communities. As the buffering effect of dispersal may differ among trophic groups, we independently manipulated the dispersal of organisms from lower (phytoplankton) and higher (zooplankton) trophic levels. The experimental heatwave suppressed total community biomass by having a strong negative effect on zooplankton biomass, probably due to a heat-induced increase in metabolic costs, resulting in weaker top-down control on phytoplankton. While zooplankton dispersal did not alleviate the negative heatwave effects on zooplankton biomass, phytoplankton dispersal enhanced biomass recovery at the level of primary producers, providing partial evidence for spatial insurance. The differential responses to dispersal may be linked to the much larger regional species pool of phytoplankton than of zooplankton. Our results suggest high recovery capacity of community biomass independent of dispersal. However, community composition and trophic structure remained altered due to the heatwave, implying longer-lasting changes in ecosystem functioning.

Crayfish plague in Czechia: Outbreaks from novel sources and testing for chronic infections.

The crayfish plague pathogen Aphanomyces astaci, which is among the most studied pathogens of aquatic invertebrates, co-evolved with North American crayfish species but threatens crayfish on other continents. The pathogen causes mass mortalities, particularly in Europe. In this study we document 12 crayfish plague outbreaks that occurred from 2014 to 2019 in Czechia and, by using available molecular techniques (microsatellite and mtDNA markers), we reveal the A. astaci genotypes involved. Our results provide the first evidence of strains from genotype group D, originally associated with the host Procambarus clarkii, causing Astacus astacus and Austropotamobius torrentium mass mortalities in Czechia. Moreover, mtDNA sequencing confirmed two distinct haplotypes of the D haplogroup, indicating two independent sources of infection, presumably originating from ornamental crayfish in the pet trade or spreading from crayfish established in neighbouring countries. Genotype group A was recorded in two As. astacus mortalities, and genotype group E, associated with Faxonius limosus, in two Au. torrentium and three As. astacus mortalities. Microsatellite genotyping also reidentified the unusual genotype SSR-Up in two As. astacus outbreaks, ten years after its first documented occurrence. In addition, we tested healthy-appearing indigenous crayfish from 25 localities for potential chronic infections. No traces of A. astaci DNA were detected; chronic infections in European crayfish species thus do not seem a pervasive phenomenon in Czechia. However, their role as A. astaci latent reservoirs, especially in Pontastacus leptodactylus populations introduced to the country since the late 19th century, cannot be excluded.

Experimental evaluation of the potential for crayfish plague transmission through the digestive system of warm-blooded predators.

The crayfish plague pathogen (Aphanomyces astaci) can be transmitted through the digestive system of fish, but its dispersal through mammalian and bird digestive tracts has been considered unlikely, and direct experimental evidence remains scarce. We present a small-scale transmission experiment with European otter and American mink fed with infected crayfish, and experiments testing survival of cultures of five A. astaci strains at temperatures corresponding to those inside mammal and bird bodies. The pathogen was neither isolated from predator excrements nor transmitted to susceptible crayfish exposed to excrements. In agar-based artificial media, it occasionally survived for 15 min at 40.5°C and for 45 min at 37.5°C, but not so when incubated at those temperatures for 45 min and 75 min, respectively. The five tested strains differed in resistance to high temperatures, two (of genotype groups E and D) being more susceptible than other three (of groups A, B and D). Their survival to some extent varied when exposed to the same temperature after several weeks or months, suggesting that some yet-unknown factors may influence A. astaci resistance to temperature stress. Overall, we support the notion that passage through the digestive tract of warm-blooded predators makes A. astaci transmission unlikely.

Lineage diversity and reproductive modes of the Daphnia pulex group in Chinese lakes and reservoirs.

Recent studies of the distribution and diversity of freshwater zooplankton have indicated that the previously understudied Eastern Palearctic region is an important biogeographic hotspot. Here, we explored the lineage diversity and reproductive modes of the Daphnia pulex species group across China. Members of this group are often keystone species of standing water bodies and are frequently used as a model system for ecological, evolutionary and, more recently, genomic studies. We found members of the D. pulex group in seven of seventy-six Chinese water bodies examined. We analyzed their phylogenetic position using mitochondrial markers, and explored the genetic structure of six populations using microsatellite markers. Mitochondrial DNA analysis suggested the presence of two distinct species complexes in China: the D. pulex complex that has a global distribution, and an apparently endemic Eastern Palearctic D. mitsukuri complex. Microsatellite analyses of six populations suggested that three of these reproduced by cyclical parthenogenesis, as evidenced by high clonal diversity and the absence of deviations from the Hardy-Weinberg equilibrium. In contrast, three other populations showed remarkably low diversity of multilocus genotypes. This suggests an obligate parthenogenetic reproductive mode, which was confirmed in one of the populations by comparison of genotypes of Daphnia adults and dormant embryos. All presumably obligate parthenogenetic clones were heterozygous at the majority of microsatellite loci, suggesting their hybrid origin. This was further supported by analyses of a small GTPase nuclear gene (rab4), as two alleles within single individuals belonged to different clades. Interestingly, one putatively obligate parthenogenetic clone carried three distinct alleles suggesting higher ploidy and potential gene flow between the D. pulicaria and D. mitsukuri complexes. Our data show that the expansion of the D. pulex complex in the Eastern Palearctic was associated with widespread hybridization.

Establishment of a new microsporidian genus and species, Pseudoberwaldia daphniae (Microsporidia, Opisthosporidia), a common parasite of the Daphnia longispina complex in Europe.

Microsporidia are among the most common microparasites of cladocerans and have potentially significant impact on host populations. However, many of these pathogens are known only from molecular-based studies. We provide ultrastructural data supported by molecular phylogeny for a common microsporidium infecting the Daphnia longispina complex, important planktonic filter-feeders in reservoirs and ponds in the temperate Holarctic region. This parasite, previously characterized only by molecular means, infects adipose cells around the Daphnia midgut and eventually fills the centre of the host body with ovoid-shaped spores. A new microsporidian genus and species belonging to the Agglomeratidae superclade is described as Pseudoberwaldia daphniae gen. et sp. nov. Molecular data indicate its widespread presence in Central European reservoirs (reported as isolate "MIC1") but also in Swedish coastal rockpools ("Ängskärs-klubben"). The most closely related lineage was reported from a caddisfly larva; we thus speculate that this taxon may have an insect secondary host in its life cycle. Morphological characterization and differential diagnosis of most commonly encountered microsporidian taxa infecting hosts in the D. longispina complex in Europe opens new possibilities for studies of their ecological and evolutionary interactions.

The signal crayfish (Pacifastacus leniusculus) in Lake Tahoe (USA) hosts multiple Aphanomyces species.

The genus Aphanomyces (Oomycetes) comprises approximately 50 known species of water molds in three lineages. One of the most notorious is Aphanomyces astaci, the causative agent of crayfish plague. In this study, fresh isolates of Aphanomyces were collected from 20 live specimens of the signal crayfish Pacifastacus leniusculus (Dana, 1852) from Lake Tahoe, California, providing 35 axenic cultures of A. astaci as well as two apparently undescribed Aphanomyces spp. isolates. Based on the results of ITS-, chitinase-, mitochondrial rnnS- and rnnL-sequences and microsatellite markers combined, the Lake Tahoe A. astaci isolates were identical to isolates of A. astaci B-haplogroup commonly detected in Europe, and infection experiments confirmed their high virulence towards noble crayfish. One of the two undescribed Aphanomyces spp. isolates was highly similar to an Aphanomyces lineage detected previously in crustacean zooplankton (Daphnia) in Central Europe, while the other was distinct and most closely related (ITS sequence similarity of 93%) to either A. astaci or to Aphanomyces fennicus isolated recently from Astacus astacus in Finland. Neither of the two Aphanomyces spp. isolates caused crayfish mortality under experimental conditions. Our results indicate that the populations of North American signal crayfish can act as carriers of both pathogenic and non-pathogenic Aphanomyces at the same time. Furthermore, considering that a limited number of crayfish individuals from a single location yielded multiple distinct Aphanomyces isolates, our results suggest that substantial species diversity within this genus remains undescribed.

Cytonuclear diversity and shared mitochondrial haplotypes among Daphnia galeata populations separated by seven thousand kilometres.

BACKGROUND: The zooplanktonic cladocerans Daphnia, present in a wide range of water bodies, are an important component of freshwater ecosystems. In contrast to their high dispersal capacity through diapausing eggs carried by waterfowl, Daphnia often exhibit strong population genetic differentiation. Here, to test for common patterns in the population genetic structure of a widespread Holarctic species, D. galeata, we genotyped two sets of populations collected from geographically distant areas: across 13 lakes in Eastern China and 14 lakes in Central Europe. The majority of these populations were genotyped at two types of markers: a mitochondrial gene (for 12S rRNA) and 15 nuclear microsatellite loci. RESULTS: Mitochondrial DNA demonstrated relatively shallow divergence within D. galeata, with distinct haplotype compositions in the two study regions but one widely distributed haplotype shared between several of the Chinese as well as European populations. At microsatellite markers, clear separation was observed at both large (between China and Europe) and small (within Europe) geographical scales, as demonstrated by Factorial Correspondence Analyses, Bayesian assignment and a clustering method based on genetic distances. Genetic diversity was comparable between the sets of Chinese and European D. galeata populations for both types of markers. Interestingly, we observed a significant association between genetic distance and geographical distance for D. galeata populations in China but not in Europe. CONCLUSIONS: Our results indicate relatively recent spread of D. galeata across wide expanses of the Palaearctic, with one mtDNA lineage of D. galeata successfully establishing over large distances. Despite a clear differentiation of Chinese and European D. galeata at a nuclear level, the pattern of genetic variation is nevertheless similar between both regions. Overall, our findings provide insights into the genetic population structure of a cladoceran species with extremely wide geographical range.

Is subterranean lifestyle reversible? Independent and recent large-scale dispersal into surface waters by two species of the groundwater amphipod genus Niphargus.

Groundwater is an extreme environment due to its absence of light, resource scarcity and highly fragmentary nature. Successful groundwater colonizers underwent major evolutionary changes and exhibit eye and pigment loss (troglomorphies). Consequently, their chances of dispersal and survival in the well-connected surface waters are greatly decreased, resulting in significant endemism. The West Palaearctic subterranean amphipod genus Niphargus comprises hundreds of narrowly endemic and troglomorphic species. Nevertheless, a few are known to occur in surface waters, two of which, N. hrabei and N. valachicus, have extremely large ranges that even exceed those of many surface-water amphipods. We tested if this pattern results from a secondary colonization of the relatively well-connected epigean environment, and whether this ecological shift promoted the large-scale dispersal of these species. Results showed that despite their ecological and zoogeographic similarities, N. hrabei and N. valachicus are not closely related and independently colonized surface waters. Their phylogeographic patterns indicate Middle to Late Pleistocene dispersal episodes throughout the Danube lowlands, and relatively modest yet significant genetic differentiation among populations. Clustering based on morphology revealed that the two species are phenotypically closer to each other than they are to most other epigean congeners. We presume that the ecological shift to surface environments was facilitated by their ability to thrive in hypoxic waters where rheophilic competitors from the family Gammaridae cannot survive. In conclusion, our results indicate that adaptation to groundwater is not a one-way evolutionary path and that troglomorphic species can occasionally recolonize and widely disperse in surface waters.

Multiple origins and strong phenotypic convergence in fish-cleaning palaemonid shrimp lineages.

Several species of palaemonid shrimps are known to act as fish-cleaning symbionts, with cleaning interactions ranging from dedicated (obligate) to facultative. We confirmed five evolutionarily independent origins of fish cleaning symbioses within the family Palaemonidae based on a phylogenetic analysis and the ancestral state reconstruction of 68 species, including 13 fish-cleaners from the genera Ancylomenes, Brachycarpus, Palaemon, Periclimenes, and Urocaridella. We focus in particular on two distantly related lineages of fish cleaning shrimps with allopatric distributions, the Indo-West Pacific Ancylomenes and the western Atlantic monophyletic Ancylomenes/Periclimenes group, which exhibit striking similarities in morphology, colouration and complex behaviour. Specifically, representatives of both lineages are similar in: (1) the general body shape and colour pattern; (2) the utilization of sea anemones as conspicuous cleaning stations; and (3) the use of sideways body swaying to visually promote their bright colour spots in order to attract fish clients. Such morphological, ecological and ethological convergences are apparently due to adaptations to fish cleaning linked to the establishment of similar modes of communication with fish clients in these species.

Molecular and structural assessment of microsporidia infecting daphnids: The "obtusa-like" microsporidia, a branch of the monophyletic Agglomeratidae clade, with the establishment of a new genus Conglomerata.

Microsporidia (Opisthosporidia, Microsporidia) are frequent parasites of planktonic cladocerans, including Daphnia (Crustacea, Branchiopoda). Analysis of available molecular data (ITS region and partial ssu and lsu rDNA) of these parasites indicates that many microsporidia infecting daphnids have a common ancestor and represent a large clade, which splits during evolution into a number of well supported subclades. These subclades are cytologically different but may be most conveniently characterised by their specific ITS barcode. We have analysed one of these subclades and we describe a new microsporidian genus and species combination, and assemble a large group of structurally indistinguishable microsporidian parasites that infect adipose cells of their hosts and form pyriform spores of a certain type ("obtuse spores"). Obtuse spores are non-infectious by feeding to their crustacean hosts and it is plausible that microsporidia forming them actually are parasites of insects with aquatic larval stages, with an obligate two-host life cycle, analogous to the Amblyospora life cycle involving copepods and mosquitoes.

Aphanomyces astaci genotypes involved in recent crayfish plague outbreaks in central Italy.

The oomycete Aphanomyces astaci is the causative agent of crayfish plague in native European freshwater crayfish. Molecular analyses showed that several distinct genotype groups of this pathogen, apparently associated with different original host taxa, are present in Europe. Tracking their distribution may contribute to understanding the introduction pathways of A. astaci. We used microsatellite markers to genotype the pathogen strains involved in 7 mass mortalities of the endangered indigenous crayfish Austropotamobius pallipes that occurred between 2009 and 2016 in the Abruzzi and Molise regions, central Italy. Three A. astaci genotype groups (A, B, and D, with the latter represented by 2 distinct multilocus genotypes) were identified, suggesting the existence of multiple infection sources even in a relatively small area. Most crayfish plague episodes were due to genotype groups associated with the North American host species Pacifastacus leniusculus and Procambarus clarkii, although these crayfish are not widespread in the study area. A. astaci genotype group A was detected not only in crayfish plague outbreaks but also in apparently healthy Astacus leptodactylus imported for human consumption from Armenia and kept alive in an aquaculture facility. Imports of chronically infected A. leptodactylus from Armenia, Turkey, and possibly Eastern Europe are an underestimated introduction pathway for A. astaci. Although we cannot exclude the presence of latently infected native populations of A. pallipes in the region, A. astaci infections in legally imported crayfish species considered vulnerable to crayfish plague may represent further reservoirs of A. astaci; this should be reflected in the policies regulating the trade of live crayfish.

Divergent clades or cryptic species? Mito-nuclear discordance in a Daphnia species complex.

BACKGROUND: Genetically divergent cryptic species are frequently detected by molecular methods. These discoveries are often a byproduct of molecular barcoding studies in which fragments of a selected marker are used for species identification. Highly divergent mitochondrial lineages and putative cryptic species are even detected in intensively studied animal taxa, such as the crustacean genus Daphnia. Recently, eleven such lineages, exhibiting genetic distances comparable to levels observed among well-defined species, were recorded in the D. longispina species complex, a group that contains several key taxa of freshwater ecosystems. We tested if three of those lineages represent indeed distinct species, by analyzing patterns of variation of ten nuclear microsatellite markers in six populations. RESULTS: We observed a discordant pattern between mitochondrial and nuclear DNA, as all individuals carrying one of the divergent mitochondrial lineages grouped at the nuclear level with widespread, well-recognized species coexisting at the same localities (Daphnia galeata, D. longispina, and D. cucullata). CONCLUSIONS: A likely explanation for this pattern is the introgression of the mitochondrial genome of undescribed taxa into the common species, either in the distant past or after long-distance dispersal. The occurrence of highly divergent but rare mtDNA lineages in the gene pool of widespread species would suggest that hybridization and introgression in the D. longispina species complex is frequent even across large phylogenetic distances, and that discoveries of such distinct clades must be interpreted with caution. However, maintenance of ancient polymorphisms through selection is another plausible alternative that may cause the observed discordance and cannot be entirely excluded.

Epigean gammarids survived millions of years of severe climatic fluctuations in high latitude refugia throughout the Western Carpathians.

Isolated glacial refugia have been documented in Central Europe for a number of taxa, but conclusive evidence for epigean aquatic species has remained elusive. Using molecular data (mitochondrial and nuclear markers), we compared the spatial patterns of lineage diversity of the widely distributed Gammarus fossarum species complex between two adjacent biogeographically and geomorphologically distinct Central European regions: the Bohemian Massif and the Western Carpathians. We investigated if the observed patterns of spatial diversity are more likely to stem from historical or present-day factors. Phylogenetic and phylogeographic analyses revealed eight phylogenetically diverse lineages: two exhibiting local signatures of recent demographic expansion inhabit both regions, while the other six display a relict distributional pattern and are found only in the Western Carpathians. Molecular dating indicates that these lineages are old and probably diverged throughout the Miocene (7-18Ma). Furthermore, their distribution does not seem to be constrained by the present boundaries of river catchments or topography. The contrasting spatial patterns of diversity observed between the two regions thus more likely result from historical rather than contemporaneous or recent factors. Our results indicate that despite the high latitude and proximity to the Pleistocene ice sheets, the Western Carpathians functioned as long-term glacial refugia for permanent freshwater fauna, allowing the uninterrupted survival of ancient lineages through millions of years of drastic climatic fluctuations.

Recent acute crayfish mortality reveals Aphanomyces astaci presence in Bosnia and Herzegovina.

Although the introduction of the crayfish plague pathogen Aphanomyces astaci to Europe is responsible for substantial declines in native crayfish populations throughout the whole continent, its presence has never been officially confirmed in many European regions, including most of the Balkan Peninsula. We demonstrate that the recent crayfish mortality observed in Bosnia and Herzegovina (Mostarsko blato karst field, Neretva river drainage) was caused by A. astaci. The causative strain is known only from European crayfish, indicating that A. astaci poses a threat to native species in this region, even in the absence of its main vectors, the North American crayfish.

A naturally heterogeneous landscape can effectively slow down the dispersal of aquatic microcrustaceans.

Several studies have suggested that aquatic microcrustaceans are relatively efficient dispersers in a variety of landscapes, whereas others have indicated dispersal limitation at large spatial scales or under specific circumstances. Based on a survey of a set of recently created ponds in an area of approximately 18 × 25 km, we found multiple indications of dispersal limitation affecting the community assembly of microcrustacean communities. Spatial patterns in the community composition were better explained by the geomorphological structure of the landscape than by mere geographic distances. This suggests that ridges separating the network of valleys act as dispersal barriers, and as such may channel the dispersal routes of the studied taxa and, likely, also of their animal vectors. Dispersal limitation was further supported by a strong positive relationship between species richness and the abundance of neighboring water bodies, suggesting that isolation affects colonization rates. Finally, the apparent dispersal limitation of microcrustaceans is further corroborated by the observation of low colonization rates in newly dug experimental ponds in the study area.

The southwestern Carpathians as an ancient centre of diversity of freshwater gammarid amphipods: insights from the Gammarus fossarum species complex.

Gammarus fossarum is a diverse species complex of epigean freshwater amphipods throughout Europe. Due to their poor dispersal capabilities and ubiquity, these crustaceans may serve as a model for investigating the influence of historical factors on the contemporary distribution and diversity patterns of freshwater macrozoobenthos. Here, we investigate the fine-scale phylogeographic structure of this complex across its range in the southwestern Carpathian Mountains, which comprises two areas that are geographically isolated from its main European distribution area as well as from each other. Given the Tertiary age of many freshwater Gammarus species, we hypothesize that the southwestern Carpathian populations reflect a relict distribution pattern. We used two mitochondrial and three nuclear markers from 32 localities to reconstruct phylogenetic relationships and estimate the timings of divergence among southwestern Carpathian and non-Carpathian lineages. Cryptic diversity was evaluated from mitochondrial markers by employing phylogenetic and distance-based methods. We distinguished at least 16 cryptic microendemic taxa, some of them coexisting, distributed in the southwestern Carpathians in a mosaic-like pattern. These lineages form a monophyletic group together with several lineages from southeastern Europe. Estimated divergence times indicate a Middle Miocene origin of this clade, with many deep splits dating back to more than 10 Ma. This time frame corresponds with a period of intense geological subsidence in the region that gave birth to the Pannonian Basin. We conclude that subsidence could have been an important driver of diversification in freshwater Gammarus and that the southwestern Carpathians represent an ancient centre of diversity for these crustaceans.

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.

Population structure of a microparasite infecting Daphnia: spatio-temporal dynamics.

BACKGROUND: Detailed knowledge of spatial and temporal variation in the genetic population structure of hosts and parasites is required for understanding of host - parasite coevolution. As hot-spots of contemporary coevolution in natural systems are difficult to detect and long-term studies are restricted to few systems, additional population genetic data from various host - parasite systems may provide important insights into the topic. This is particularly true for parasites, as these players have been under-investigated so far due to the lower availability of suitable molecular markers. Here, we traced genetic variation (based on sequence variants in the internal transcribed spacer region, ITS) among seven geographically isolated populations of the ichthyosporean Caullerya mesnili, a common microparasite of the cladoceran Daphnia (here, the D. longispina hybrid complex). At three sites, we also studied parasite genetic variation over time (three to four sampling points) and tested for associations between parasite genotypes and host species. RESULTS: Parasite (and host) populations were significantly structured across space, indicating limited dispersal. Moreover, the frequency of parasite genotypes varied significantly over time, suggesting rapid evolutionary change in Caullerya. However, the distribution of parasite genotypes was similar across different host species, which might in turn have important consequences for parasite epidemiology. CONCLUSIONS: The approach proposed here can be applied to track spatial and temporal changes in the population structure of other microparasite species for which sequence variation in the ITS or other highly variable genome regions has been documented but other types of polymorphic markers are lacking. Screening of parasite sequence variants allows for reliable detection of cross-species infections and, using advanced sequencing techniques in the near future, for detailed studies of parasite evolution in natural host - parasite systems.