Disturbance in invasion? Idiopathic necrotizing hepatopancreatitis in the signal crayfish Pacifastacus leniusculus (Dana, 1852) in Croatia.

As the most successful crayfish invader and possible vector for infectious agents, signal crayfish Pacifastacus leniusculus is among the major drivers of the native crayfish species decline in Europe. We describe histopathological manifestation and frequency of newly detected idiopathic necrotizing hepatopancreatitis along the invasion range of the signal crayfish in the Korana River in Croatia. Our results show extremely high prevalence of necrotizing hepatopancreatitis (97.3%), with 58.9% of individuals displaying mild and 31.5% moderate histopathological changes in the hepatopancreas, also reflected in the lower hepatosomatic index of analysed animals. Recorded histopathological changes were more frequent in the invasion core where population density is higher. Our preliminary screening of co-occurring native narrow-clawed crayfish Pontastacus leptodactylus showed lower incidence (33.3%) and only mild hepatopancreatic lesions, but potentially highlighted the susceptibility of native crayfish populations to this disease. Pilot analyses of dissolved trace and macro elements in water, sediment fractions and crayfish hepatopancreas do not highlight alarming or unusually high concentrations of analysed elements. Hepatopancreas microbiome analysis, using 16S rRNA gene amplicon sequencing, identified taxonomic groups that should be further investigated, along with impacts of the disease on health and viability of both invasive and native crayfish populations.

New insights into the genetic diversity of the stone crayfish: taxonomic and conservation implications.

BACKGROUND: Austropotamobius torrentium is a freshwater crayfish species native to central and south-eastern Europe, with an intricate evolutionary history and the highest genetic diversity recorded in the northern-central Dinarides (NCD). Its populations are facing declines, both in number and size across its entire range. By extanding current knowledge on the genetic diversity of this species, we aim to assist conservation programmes. Multigene phylogenetic analyses were performed using different divergence time estimates based on mitochondrial and, for the first time, nuclear DNA markers on the largest data set analysed so far. In order to reassess taxonomic relationships within this species we applied several species delimitation methods and studied the meristic characters with the intention of finding features that would clearly separate stone crayfish belonging to different phylogroups. RESULTS: Our results confirmed the existence of high genetic diversity within A. torrentium, maintained in divergent phylogroups which have their own evolutionary dynamics. A new phylogroup in the Kordun region belonging to NCD has also been discovered. Due to the incongruence between implemented species delimitation approaches and the lack of any morphological characters conserved within lineages, we are of the opinion that phylogroups recovered on mitochondrial and nuclear DNA are cryptic subspecies and distinct evolutionary significant units. CONCLUSIONS: Geographically and genetically isolated phylogroups represent the evolutionary legacy of A. torrentium and are highly relevant for conservation due to their evolutionary distinctiveness and restricted distribution.

Non-destructive method for detecting Aphanomyces astaci, the causative agent of crayfish plague, on the individual level.

The pathogenic oomycete Aphanomyces astaci, transmitted mainly by invasive North American crayfish, causes the crayfish plague, a disease mostly lethal for native European crayfish. Due to its decimating effects on native crayfish populations in the last century, A. astaci has been listed among the 100 worst invasive species. Importantly, detecting the pathogen in endangered native crayfish populations before a disease outbreak would provide a starting point in the development of effective control measures. However, current A. astaci-detection protocols either rely on degradation-prone eDNA isolated from large volumes of water or, if focused on individual animals, include killing the crayfish. We developed a non-destructive method that detects A. astaci DNA in the microbial biofilm associated with the cuticle of individual crayfish, without the need for destructive sampling. Efficiency of the new method was confirmed by PCR and qPCR and the obtained results were congruent with the traditional destructive sampling method. Additionally, we demonstrated the applicability of the method for A. astaci monitoring in natural populations. We propose that the new method should be used in future monitoring of A. astaci presence in endangered European native crayfish individuals as an alternative to eDNA-based monitoring.

Insights into the molecular phylogeny and historical biogeography of the white-clawed crayfish (Decapoda, Astacidae).

In this study, the evolutionary history of the white-clawed crayfish (WCC) was evaluated using large-scale datasets comprising >1350 specimens from the entire distribution range. Using species delimitation methods on mitochondrial DNA (mtDNA) sequences, we propose four primary species hypotheses for WCC. Sequences for several nuclear regions were screened but none showed significant variation within WCC. This result favours a single secondary species hypothesis and indicates the existence of a mito-nuclear discordance in WCC. Therefore, mtDNA groups were considered only as genetic units that carry information about ancient divergences within WCC and not as taxonomic units. The reconstruction of ancestral ranges and divergence time estimates were used to link the current genetic structure with paleogeographic processes. These results showed that the emergence of mtDNA groups in WCC could be related to the Messinian Salinity Crisis, the climate cooling during the Pliocene and Pleistocene, and (paleo)shifting of the Adriatic Sea coastline in the Padanovenezian Plain. The most recent common ancestor of the mtDNA groups most likely originated from Dalmatia (eastern Adriatic coast) as indicated by the reconstruction of ancestral ranges. This ecoregion, along with the Gulf of Venice Drainages, harbours a high genetic diversity and should be emphasised as an area of the highest conservation priority.

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.

Integrating population genetics and species distribution modelling to guide conservation of the noble crayfish, Astacus astacus, in Croatia.

The noble crayfish, Astacus astacus, is an indigenous European freshwater species. Its populations show significant declines caused by anthropogenic pressure on its habitats, climate change and the spread of invasive species. Diminishing populations' trends and loss of genetic diversity highlight the need for effective conservation that will ensure their long-term survival. We combined population genetics and species distribution modelling (SDM) to reveal the impact of climate change and invasive species on the noble crayfish, and to guide future conservation programs of current populations. Our study showed that Croatian populations of A. astacus harbour an important part of species genetic diversity and represent significant genetic reservoir at the European level. The SDM results predicted substantial reductions of suitable habitats for A. astacus by the 2070; only 13% of its current potential distribution is projected to remain stable under pessimistic Representative Concentration Pathway (RCP 8.5) emission scenario. Moreover, most of the populations with high genetic diversity are located in the areas predicted to become unsuitable, and consequently have a high probability of being lost in the future. Further, SDM results also indicated considerable decrease of future habitat suitability for invasive crayfish species in Croatia, suggesting that climate change poses a major threat to already endangered A. astacus. The obtained results help in the identification of populations and areas with the highest conservation value which should be given the highest priority for protection. In order to preserve present diversity in areas that are predicted as suitable, we propose assisted migration and repopulation approaches, for enhancing populations' size and saving maximum genetic variability. The result of our research emphasizes once again the benefits of multidisciplinary approach in the modern biodiversity conservation.

Immune Response in Crayfish Is Species-Specific and Exhibits Changes along Invasion Range of a Successful Invader.

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

Genetic diversity and structure of the noble crayfish populations in the Balkan Peninsula revealed by mitochondrial and microsatellite DNA markers.

BACKGROUND: The noble crayfish (Astacus astacus) is a native European species in decline, with a contracting range and diminishing populations and abundance. Previous studies revealed this species significant genetic diversity in the south-eastern Europe, with populations from the western and the southern part of the Balkan Peninsula being the most divergent. However, sampling of populations from the western part of the Balkans was limited and insufficient for investigating genetic diversity and population divergence for the purpose of conservation planning and management. Thus, the major aim of this study was to fill in this knowledge gap by studying mitochondrial and microsatellite DNA diversity, using 413 noble crayfish from 18 populations from waterbodies in the western part of the Balkan Peninsula. METHODS: Phylogenetic analysis of studied populations and their mitochondrial diversity were studied using COI and 16S sequences and population genetic structure was described using 15 microsatellite loci. RESULTS: Phylogeographic analysis revealed new divergent mitochondrial haplotypes for the populations in the westernmost part of the Balkan Peninsula in the tributaries of the Sava and Drava rivers. Microsatellite data indicated that these populations harbour an important component of genetic diversity within A. astacus. The results suggest that the western part of the Balkans played an important role as microrefugia during the Pleistocene climate fluctuations, allowing the long term persistence of A. astacus populations in this region. These results will also be important to supporting conservation decision making and planning.

The Pontastacus leptodactylus (Astacidae) Repeatome Provides Insight Into Genome Evolution and Reveals Remarkable Diversity of Satellite DNA.

Pontastacus leptodactylus is a native European crayfish species found in both freshwater and brackish environments. It has commercial importance for fisheries and aquaculture industries. Up till now, most studies concerning P. leptodactylus have focused onto gaining knowledge about its phylogeny and population genetics. However, little is known about the chromosomal evolution and genome organization of this species. Therefore, we performed clustering analysis of a low coverage genomic dataset to identify and characterize repetitive DNA in the P. leptodactylus genome. In addition, the karyogram of P. leptodactylus (2n = 180) is presented here for the first time consisting of 75 metacentric, 14 submetacentric, and a submetacentric/metacentric heteromorphic chromosome pair. We determined the genome size to be at ~18.7 gigabase pairs. Repetitive DNA represents about 54.85% of the genome. Satellite DNA repeats are the most abundant type of repetitive DNA, making up to ~28% of the total amount of repetitive elements, followed by the Ty3/Gypsy retroelements (~15%). Our study established a surprisingly high diversity of satellite repeats in P. leptodactylus. The genome of P. leptodactylus is by far the most satellite-rich genome discovered to date with 258 satellite families described. Of the five mapped satellite DNA families on chromosomes, PlSAT3-411 co-localizes with the AT-rich DAPI positive probable (peri)centromeric heterochromatin on all chromosomes, while PlSAT14-79 co-localizes with the AT-rich DAPI positive (peri)centromeric heterochromatin on one chromosome and is also located subterminally and intercalary on some chromosomes. PlSAT1-21 is located intercalary in the vicinity of the (peri)centromeric heterochromatin on some chromosomes, while PlSAT6-70 and PlSAT7-134 are located intercalary on some P. leptodactylus chromosomes. The FISH results reveal amplification of interstitial telomeric repeats (ITRs) in P. leptodactylus. The prevalence of repetitive elements, especially the satellite DNA repeats, may have provided a driving force for the evolution of the P. leptodactylus genome.

Invasion biology in non-free-living species: interactions between abiotic (climatic) and biotic (host availability) factors in geographical space in crayfish commensals (Ostracoda, Entocytheridae).

In invasion processes, both abiotic and biotic factors are considered essential, but the latter are usually disregarded when modeling the potential spread of exotic species. In the framework of set theory, interactions between biotic (B), abiotic (A), and movement-related (M) factors in the geographical space can be hypothesized with BAM diagrams and tested using ecological niche models (ENMs) to estimate A and B areas. The main aim of our survey was to evaluate the interactions between abiotic (climatic) and biotic (host availability) factors in geographical space for exotic symbionts (i.e., non-free-living species), using ENM techniques combined with a BAM framework and using exotic Entocytheridae (Ostracoda) found in Europe as model organisms. We carried out an extensive survey to evaluate the distribution of entocytherids hosted by crayfish in Europe by checking 94 European localities and 12 crayfish species. Both exotic entocytherid species found, Ankylocythere sinuosa and Uncinocythere occidentalis, were widely distributed in W Europe living on the exotic crayfish species Procambarus clarkii and Pacifastacus leniusculus, respectively. No entocytherids were observed in the remaining crayfish species. The suitable area for A. sinuosa was mainly restricted by its own limitations to minimum temperatures in W and N Europe and precipitation seasonality in circum-Mediterranean areas. Uncinocythere occidentalis was mostly restricted by host availability in circum-Mediterranean regions due to limitations of P. leniusculus to higher precipitation seasonality and maximum temperatures. The combination of ENMs with set theory allows studying the invasive biology of symbionts and provides clues about biogeographic barriers due to abiotic or biotic factors limiting the expansion of the symbiont in different regions of the invasive range. The relative importance of abiotic and biotic factors on geographical space can then be assessed and applied in conservation plans. This approach can also be implemented in other systems where the target species is closely interacting with other taxa.

Genotoxicity monitoring of freshwater environments using caged crayfish (Astacus leptodactylus).

Genotoxicity of freshwater pollution was assessed by measuring DNA damage in haemocytes of caged freshwater crayfish Astacus leptodactylus by the means of Comet assay and micronucleus test, integrated with the measurements of physiological (total protein concentration) and immunological (total haemocyte count) haemolymph parameters as biomarkers of undergone stress. Crayfish were collected at the reference site (River Mreznica) and exposed in cages for 1 week at three polluted sites along the Sava River (Zagreb, Sisak, Krapje). The long term pollution status of these locations was confirmed by chemical analyses of sediments. Statistically significant increase in DNA damage measured by the Comet assay was observed at all three polluted sites comparing to the crayfish from reference site. In addition, native crayfish from the mildly polluted site (Krapje) cage-exposed on another polluted site (Zagreb) showed lower DNA damage than crayfish from the reference site exposed at the same location indicating adaptation and acclimatisation of crayfish to lower levels of pollution. Micronuclei induction showed similar gradient of DNA damage as Comet assay, but did not reach the statistical significance. Observed increase in total haemocyte count and total protein content in crayfish from polluted environments in the Sava River also confirmed stress caused by exposure to pollution. The results of this study have proved the applicability of caging exposure of freshwater crayfish A. leptodactylus in environmental genotoxicity monitoring using Comet assay and micronucleus test.

Genotoxic, physiological and immunological effects caused by temperature increase, air exposure or food deprivation in freshwater crayfish Astacus leptodactylus.

The aim of this research was to investigate influence of different environmental stressors, such as temperature increase, air exposure and food deprivation on DNA integrity of a bioindicator species, freshwater crayfish Astacus leptodactylus. DNA damage was measured in crayfish haemocytes using Comet assay and micronucleus test. Crayfish haemolymph was subsequentially sampled during their 7 days of exposure to increased temperatures (25 and 30 degrees C) and during 24 h of air exposure. Both groups were also monitored through the following 7 days of recovery period. Food deprived crayfish were monitored over a period of 2 weeks. Alterations of measured physiological and immunological haemolymph parameters (THC, lactate, glucose and protein concentration) indicated stress response in exposed crayfish. However, only the stress induced by increased temperature significantly increased DNA damage in freshwater crayfish while food deprivation or air exposure did not cause a significant genotoxic effect.