The importance of population contextual data for large-scale biomonitoring using an apex predator: The Tawny Owl (Strix aluco).

Top predators are often used as sentinel species in contaminant monitoring due to their exposure and vulnerability to persistent, bioaccumulative and, in some cases, biomagnificable contaminants. Some of their ecological traits can vary in space and time, and are known to influence the contamination levels and therefore information on ecological traits should be used as contextual data for correct interpretation of large-scale contaminant spatial patterns. These traits can explain spatiotemporal variation in contaminant exposure (traits such as diet and dispersal distances) or contaminant impacts (traits such as population trend and clutch size). The aim of our research was to review the spatial variation in selected contextual parameters in the Tawny Owl (Strix aluco), a species identified by the COST Action European Raptor Biomonitoring Facility as one of the most suitable candidates for pan-European biomonitoring. A considerable variation in availability of published and unpublished contextual data across Europe was found, with diet being the most extensively studied trait. We demonstrate that the Tawny Owl is a suitable biomonitor at local scale but also that taking spatial variation of other contextual data (e.g. diet) into account is necessary. We found spatial gaps in knowledge about the species ecology and biology in Southern Europe, along with gaps in certain population parameters (e.g. population trends) in several countries. Based on our findings, we proposed a minimal recommended scheme for monitoring of population contextual data as one of the first steps towards a pan-European monitoring scheme using the Tawny Owl.

Disentangling phylogenetic relations and biogeographic history within the Cucujus haematodes species group (Coleoptera: Cucujidae).

Recent progress in the taxonomy of flat bark beetles (Cucujidae), specifically, in the genus Cucujus, has revealed great diversity in subtropical Asia, but the seemingly well-known temperate and boreal taxa need further attention because of their conservation status. Here, we used an integrative approach using morphology, DNA, and species distribution modelling to disentangle phylogenetic relations, verify the number of species, and understand the historical biogeography of Palearctic and Nearctic Cucujus beetles, particularly the C. haematodes species group. Species distinctiveness was supported for C. cinnaberinus, but present-day C. haematodes turned out to be a species complex made up of separate lineages in the western, middle and eastern parts of its Palearctic range. Cucujus muelleri was a member of that complex, being sister to Asian C. haematodes. Moreover, C. haematodes caucasicus was found to be phylogenetically closely related to Italian C. tulliae, and both to be sister to European C. haematodes. North American C. clavipes clavipes and C. c. puniceus resulted to be enough divergent to be considered different species. Interestingly, western American C. puniceus turned out to be closely related to the C. haematodes complex, whereas eastern American C. clavipes constituted a separate lineage, being distantly related to both C. puniceus and C. cinnaberinus. These patterns suggest former trans-continental connections among the ancestors of extant flat bark beetle species. Moreover, a divergent lineage of C. cinnaberinus was found in Calabria, which should be regarded at the very least as a subspecies. The ancestor of C. hameatodes group originated in mid-Miocene, and next, ca. 6.2 Mya, a line leading to C. cinnaberinus had split. Speciation of the American lineages occurred during Pliocene (4.4 Mya for C. clavipes and 3.3 Mya for C. puniceus). Species classified as C. haematodes, C. tulliae and C. muelleri, as well as distinct lineages within C. cinnaberinus split during mid Pleistocene (ca. 1.5 Mya). A comparison of species climatic requirements and their present distribution allowed to identify glacial refugia in south-eastern areas of North America (C. clavipes), south-western areas of North America (C. puniceus), and the Mediterranean and Caspian Sea Basins (European Cucujus species), or south-eastern areas of Asia and the foothills of the central Asian mountains (eastern C. haematodes). Subsequent climatic changes in the Holocene forced these beetles to move their ranges northwards along the coasts of the Pacific (C. puniceus) or Atlantic (C. clavipes), north-eastwards to central, northern, and eastern Europe (C. cinnaberinus and European C. haematodes) or Siberia (Asian C. haematodes). The combined use of molecular, morphological and climatic data allows a comprehensive understanding of the phylogenetic relations and past distributions of Cucujus beetles, highlighting the complexity of C. haematodes species group evolution.

Detection of Herpesviruses in Wild Bird Casualties in Slovenia.

The complete host range of avian herpesviruses in wild birds is unknown, and information about nucleotide sequences is available only in limited cases. The aim of this study was to detect the presence of herpesviruses in wild birds and to gain more information about their phylogenetic relationship. Oropharyngeal and cloacal swabs from 447 wild birds from 15 different orders presented as wildlife casualties were examined for herpesvirus presence with PCR targeting a fragment of the DNA polymerase gene. Herpesviruses were detected in oropharyngeal and/or cloacal swabs in 34 (7.5%) birds belonging to 11 species from six different avian orders: Accipitriformes, Charadriiformes, Columbiformes, Falconiformes, Passeriformes, and Strigiformes. The results of phylogenetic analysis showed that various herpesviruses sequences are present in the wild bird population. Some herpesviruses are host species-specific, whereas in some cases very similar sequences were detected through different avian orders, which confirms findings that herpesviruses are not always restricted to bird species. It seems that herpesvirus transmission could occur by predation from avian prey, and even by superpredation-for example, large owls, such as the Eurasian eagle owl (Bubo bubo) or Ural owl (Strix uralensis), preying on smaller raptors. This can lead to greater infection exposure and is in line with the fact that raptors were the most infected species group. Nevertheless, the individual or simultaneous detection of herpesviruses in oropharyngeal and cloacal swabs shows that both swab samples should be used for herpesvirus detection in wild birds.

Herpesvirus Infection in a Breeding Population of Two Coexisting Strix Owls.

Birds are a frequent host of a large variety of herpesviruses, and infections in them may go unnoticed or may result in fatal disease. In wild breeding populations of owls, there is very limited information about the presence, impact, and potential transmission of herpesvirus. The herpesvirus partial DNA polymerase gene was detected using polymerase chain reaction in oropharyngeal swabs of 16 out of 170 owls examined that were captured in or near nest boxes. Herpesvirus was detected in Ural owls (Strix uralensis), in both adults and young, but not in tawny owls (Strix aluco). In yellow-necked mice (Apodemus flavicollis), as the main prey of tawny owls and Ural owls in the area, herpesvirus was detected in the organs of 2 out of 40 mice captured at the same locations as the owls. Phylogenetic analysis showed that the herpesvirus sequences detected in the Ural owls differed from the herpesvirus sequences detected in the yellow-necked mice. The results indicate that herpesvirus infection exists in the breeding wild Ural owl population. However, herpesvirus-infected owls did not show any clinical or productivity deviances and, based on a phylogenetic comparison of detected herpesvirus sequences and sequences obtained from Genbank database, it seems that mice and other rodents are not the source of owl infections. The most probable transmission pathway is intraspecific, especially from adults to their chicks, but the origin of herpesvirus in owls remains to be investigated.

The European Stag Beetle (Lucanus cervus) Monitoring Network: International Citizen Science Cooperation Reveals Regional Differences in Phenology and Temperature Response.

To address the decline in biodiversity, international cooperation in monitoring of threatened species is needed. Citizen science can play a crucial role in achieving this challenging goal, but most citizen science projects have been established at national or regional scales. Here we report on the establishment and initial findings of the European Stag Beetle Monitoring Network (ESBMN), an international network of stag beetle (Lucanus cervus) monitoring schemes using the same protocol. The network, started in 2016, currently includes 14 countries (see results) but with a strong variation in output regarding the number of transects (148 successful transects in total) and transect walks (1735). We found differences across European regions in the number of stag beetles recorded, related to phenology and temperature, but not for time of transect start. Furthermore, the initial experiences of the ESBMN regarding international cooperation, citizen science approach, and drop-out of volunteers is discussed. An international standardised protocol that allows some local variation is essential for international collaboration and data management, and analysis is best performed at the international level, whereas recruiting, training, and maintaining volunteers is best organised locally. In conclusion, we appeal for more joint international citizen science-based monitoring initiatives assisting international red-listing and conservation actions.

A review of constraints and solutions for collecting raptor samples and contextual data for a European Raptor Biomonitoring Facility.

The COST Action 'European Raptor Biomonitoring Facility' (ERBFacility) aims to develop pan-European raptor biomonitoring in support of better chemicals management in Europe, using raptors as sentinel species. This presents a significant challenge involving a range of constraints that must be identified and addressed. The aims of this study were to: (1) carry out a comprehensive review of the constraints that may limit the gathering in the field of raptor samples and contextual data, and assess their relative importance across Europe; and (2) identify and discuss possible solutions to the key constraints that were identified. We applied a participatory approach to identify constraints and to discuss feasible solutions. Thirty-one constraints were identified, which were divided into four categories: legal, methodological, spatial coverage, and skills constraints. To assess the importance of the constraints and their possible solutions, we collected information through scientific workshops and by distributing a questionnaire to stakeholders in all the countries involved in ERBFacility. We obtained 74 answers to the questionnaire, from 24 of the 39 COST participating countries. The most important constraints identified were related to the collection of complex contextual data about sources of contamination, and the low number of existing raptor population national/regional monitoring schemes and ecological studies that could provide raptor samples. Legal constraints, such as permits to allow the collection of invasive samples, and skills constraints, such as the lack of expertise to practice necropsies, were also highlighted. Here, we present solutions for all the constraints identified, thus suggesting the feasibility of establishing a long-term European Raptor Sampling Programme as a key element of the planned European Raptor Biomonitoring Facility.

DETECTION OF HERPESVIRUSES IN PASSERINE BIRDS CAPTURED DURING AUTUMN MIGRATION IN SLOVENIA.

Herpesviruses (HVs) were detected by PCR in the cloacal swabs of 0.76% (4/525) clinically healthy free-living passerine birds from 32 different species captured in mist nets in Slovenia during the 2014 and 2017 autumn migrations. Herpesviruses were detected in the Eurasian Blackcap (Sylvia atricapilla), the Common Blackbird (Turdus merula), and the Eurasian Blue Tit (Cyanistes caeruleus). Phylogenetic analysis of partial DNA polymerase gene nucleotide sequences of the HV strains showed a distant relationship with other alphaherpesviruses of birds. In the phylogenetic tree, the HVs detected were clustered together with HV detected in Sulphur-crested Cockatoo and Neotropic Cormorants, as well as with known HVs such as gallid HV1, psittacid HV1 and HV2, and passerine HV1. Different sequences of HVs with relatively low identity were detected in our study, suggesting that different HVs were circulating in passerines sampled during the autumn migration in Slovenia.

A schematic sampling protocol for contaminant monitoring in raptors.

Birds of prey, owls and falcons are widely used as sentinel species in raptor biomonitoring programmes. A major current challenge is to facilitate large-scale biomonitoring by coordinating contaminant monitoring activities and by building capacity across countries. This requires sharing, dissemination and adoption of best practices addressed by the Networking Programme Research and Monitoring for and with Raptors in Europe (EURAPMON) and now being advanced by the ongoing international COST Action European Raptor Biomonitoring Facility. The present perspective introduces a schematic sampling protocol for contaminant monitoring in raptors. We provide guidance on sample collection with a view to increasing sampling capacity across countries, ensuring appropriate quality of samples and facilitating harmonization of procedures to maximize the reliability, comparability and interoperability of data. The here presented protocol can be used by professionals and volunteers as a standard guide to ensure harmonised sampling methods for contaminant monitoring in raptors.

Supporting dataset and methods for body sizes and concentrations of chemical elements measured in elytra and abdomens of Stag Beetles Lucanus cervus.

The dataset presented in this data paper supports "Breaking down insect stoichiometry into chitin-based and internal elemental traits: Patterns and correlates of continent-wide intraspecific variation in the largest European saproxylic beetle" (Orlowski et al. 2020). Here we present the supplementary data and description of methods on the following: (1) mass of elytra and abdomens across 28 local Stag Beetle Lucanus cervus populations in Europe. (2) Population origin and coverage of six major land-cover types, including transport infrastructure, measured in three radii (500 m, 1000 m and 5000 m) around the sampling sites of these populations. (3) The relationship between the mass and concentrations of elements measured in abdomens and elytra in 28 Stag Beetle populations and major land-cover types around the sampling sites.

Breaking down insect stoichiometry into chitin-based and internal elemental traits: Patterns and correlates of continent-wide intraspecific variation in the largest European saproxylic beetle.

Stoichiometric, trophic and ecotoxicological data have traditionally been acquired from patterns of variation in elemental traits of whole invertebrate bodies, whereas the critical issue of the extracellular origin of some portion of elements, such as those present in ingested food and internal organs, has been ignored. Here we investigated an unexplored, yet crucial, question relating to whether, and to what degree, metals from two major body fractions: exoskeleton (elytra) and internal (body organs with gut material present in abdomens), are correlated with each other in wild populations of the largest European saproxylic insect, the Stag Beetle Lucanus cervus, and how metals from these two fractions vary with insect size and local habitat conditions. We examined the continent-wide variation in the concentrations of 12 chemical elements (Ca, Mg, K, Na, Mn, Fe, Zn, Cu, As, Cd, Pb and Ni) measured in the elytra and abdomen of specimens from 28 populations inhabiting an urban-woodland habitat gradient across the species' entire distributional range from Spain to Russia. Across populations, elemental concentrations (except Ni and Pb) were 2-13 times higher in abdominal samples than in elytra, and the magnitude of these differences was related to both insect size and local habitat conditions. Smaller individuals from both woodland and urban habitat tended to have higher concentrations of trace elements (Zn, As, Cd, Pb and Ni). The concentration of only six elements (Mg, K, Na, Mn, Cd and Ni) was correlated in the elytra and abdomen at the individual and population levels, implying a limitation to the broader applicability of elytra as a surrogate for internal elemental pools. We highlight that in non-feeding adult saproxylic beetles, minerals, acquired during the larval stage, may be concentrated in the large quantities of residual body fat.

Species interactions and climate change: How the disruption of species co-occurrence will impact on an avian forest guild.

Interspecific interactions are crucial in determining species occurrence and community assembly. Understanding these interactions is thus essential for correctly predicting species' responses to climate change. We focussed on an avian forest guild of four hole-nesting species with differing sensitivities to climate that show a range of well-understood reciprocal interactions, including facilitation, competition and predation. We modelled the potential distributions of black woodpecker and boreal, tawny and Ural owl, and tested whether the spatial patterns of the more widespread species (excluding Ural owl) were shaped by interspecific interactions. We then modelled the potential future distributions of all four species, evaluating how the predicted changes will alter the overlap between the species' ranges, and hence the spatial outcomes of interactions. Forest cover/type and climate were important determinants of habitat suitability for all species. Field data analysed with N-mixture models revealed effects of interspecific interactions on current species abundance, especially in boreal owl (positive effects of black woodpecker, negative effects of tawny owl). Climate change will impact the assemblage both at species and guild levels, as the potential area of range overlap, relevant for species interactions, will change in both proportion and extent in the future. Boreal owl, the most climate-sensitive species in the guild, will retreat, and the range overlap with its main predator, tawny owl, will increase in the remaining suitable area: climate change will thus impact on boreal owl both directly and indirectly. Climate change will cause the geographical alteration or disruption of species interaction networks, with different consequences for the species belonging to the guild and a likely spatial increase of competition and/or intraguild predation. Our work shows significant interactions and important potential changes in the overlap of areas suitable for the interacting species, which reinforce the importance of including relevant biotic interactions in predictive climate change models for increasing forecast accuracy.

Correction to: Progress on bringing together raptor collections in Europe for contaminant research and monitoring in relation to chemicals regulation.

The correct affiliation of Sabrina Lo Brutto is shown in this paper.

Detection and Phylogenetic Analysis of Herpesviruses Detected in Wild Owls in Slovenia.

Herpesvirus (HV) was detected using PCR in the organs of eight of 55 wild owls (14.5%) from seven species that were found dead in various locations in Slovenia between 1995 and 2015. HV was detected in three species: the Eurasian eagle owl (Bubo bubo), Ural owl (Strix uralensis), and long-eared owl (Asio otus). Phylogenetic analysis of partial DNA polymerase gene nucleotide sequences showed that the detected HVs are similar to the avian and mammal alphaherpesviruses. Two sequences were very similar to known bird HV sequences. One sequence was identical to the columbid herpesvirus 1 (CoHV1) sequence, and the other was very similar to the gallid herpesvirus 2 (GaHV2) sequence. The phylogenetic tree revealed a lower similarity of the other six analyzed Slovenian sequences with the sequences of alphaherpesviruses of birds and mammals. This is the first study to report the detection of different HVs in owls.

Detección y análisis filogenético de herpesvirus detectados en búhos silvestres en Eslovenia Se detectaron virus herpes mediante PCR en los órganos de ocho de 55 búhos silvestres (14.5%) pertenecientes a siete especies y que se encontraron muertos en varios lugares de Eslovenia entre los años 1995 y 2015. Se detectaron virus herpes en tres especies: el búho real (Bubo bubo), cárabo uralense (Strix uralensis) y el búho chico (Asio otus). El análisis filogenético de las secuencias de nucleótidos parciales del gene de la polimerasa de ADN mostró que los virus herpes detectados son similares a los alphaherpesvirus aviares y de mamíferos. Dos secuencias fueron muy similares a las secuencias de virus herpes de aves conocidas. Una secuencia fue idéntica a la secuencia del herpesvirus 1 de palomas (CoHV1) y otra fue muy similar a la secuencia del herpesvirus 2 del pollo (GaHV2). El árbol filogenético reveló menores similitudes entre las otras seis secuencias analizadas de Eslovenia con las secuencias de alphaherpesvirus de aves y de mamíferos. Este es el primer estudio que informa la detección de diferentes virus herpes en búhos.

Novel, male-produced aggregation pheromone of the cerambycid beetle Rosalia alpina, a priority species of European conservation concern.

Several recent studies have demonstrated the great potential for exploiting semiochemicals in ecology and conservation studies. The cerambycid beetle Rosalia alpina represents one of the flagship species of saproxylic insect biodiversity in Europe. In recent years its populations appear to have declined substantially, and its range has shrunk considerably as a result of forest management and urbanization. Here, we collected volatile chemicals released by males and females of R. alpina. Analyses of the resulting extracts revealed the presence of a single male-specific compound, identified as a novel alkylated pyrone structure. In field bioassays in Slovenia, traps baited with the synthesized pyrone captured both sexes of R. alpina, indicating that the pyrone functions as an aggregation pheromone. Our results represent the first example of a new structural class of pheromones within the Cerambycidae, and demonstrate that pheromone-baited traps can provide a useful tool for sampling R. alpina. This tool could be particularly useful in the ongoing development of conservation strategies for the iconic but endangered Alpine longicorn.

Aphanomyces astaci isolate from latently infected stone crayfish (Austropotamobius torrentium) population is virulent.

Aphanomyces astaci infection is the cause of crayfish plague in European crayfish. Here the virulence of an A. astaci As strain isolated from apparently healthy stone crayfish (Austropotamobius torrentium) from Slovenia was compared to that of the Psl-Puujärvi A. astaci isolate in 3 crayfish species: noble crayfish (Astacus astacus), signal crayfish (Pacifastacus leniusculus) from Finland and stone crayfish from Slovenia. All 3 crayfish species were challenged with PsI-Puujärvi A. astaci and succumbed to crayfish plague, with both noble crayfish and stone crayfish showing 100% mortality, while 25% of the signal crayfish died during the challenge. In comparison, the As-Slovenia A. astaci isolate was pathogenic for noble crayfish but not for signal crayfish or stone crayfish. This finding suggests that A. astaci virulence could be species specific and a strain from latent A. astaci infection in one native European crayfish species could be detrimental to other native European crayfish species.

The role of metabolism in understanding the altitudinal segregation pattern of two potentially interacting lizards.

Sympatric species from the same ecological guild, that exhibit partial altitudinal segregation, can potentially interact in areas of syntopic occurrence. Besides general species' ecology, physiology can provide important answers about species interactions reflected in altitudinal patterns. Lizards Podarcis muralis and Iberolacerta horvathi exhibit partial altitudinal segregation, while they strongly resemble in overall morphology and ecology (diet, daily and seasonal activity pattern), but show certain degree of physiological dissimilarity. They have similar mean preferred body temperatures and patterns of seasonal and daily variations but differ in the magnitude of seasonal variation. Since an ectotherm metabolism is highly dependent on body temperature, thermoregulation is expected to directly affect their metabolism. We compared metabolic rates of adult males from an area of sympatry, measured under two temperature regimes (20°C and 28°C). Both species increased metabolic rates with temperature in a similar pattern. We also compared electron transport activity from tail tissues which provide values of species' potential metabolic activity (enzymatic capacity). Species clearly differed in potential metabolic activity; I. horvathi attained higher values than P. muralis. No difference was detected in how species exploited this potential (calculated from the ratio of electron transport activity and metabolic rates). However, we observed higher potential metabolic activity I. horvathi which together with the ability to thermoregulate more precisely could represent a higher competitive advantage over P. muralis in thermally more restrictive environments such as higher altitudes. Understanding of metabolism seems to provide valuable information for understanding recent distributional patterns as well as species interactions.

The thermal tolerance of crayfish could be estimated from respiratory electron transport system activity.

Whether electron transport system (ETS) activity could be used as an estimator of crayfish thermal tolerance has been investigated experimentally. Food consumption rate, respiration rates in the air and water, the difference between energy consumption and respiration costs at a given temperature ('potential growth scope', PGS), and ETS activity of Orconectes limosus and Pacifastacus leniusculus were determined over a temperature range of 5-30°C. All concerned parameters were found to be temperature dependent. The significant correlation between ETS activity and PGS indicates that they respond similarly to temperature change. The regression analysis of ETS activity as an estimator of thermal tolerance at the mitochondrial level and PGS as an indicator of thermal tolerance at the organismic level showed the shift of optimum temperature ranges of ETS activity to the right for 2° in O. limosus and for 3° in P. leniusculus. Thus, lower estimated temperature optima and temperatures of optimum ranges of PGS compared to ETS activity could indicate higher thermal sensitivity at the organismic level than at a lower level of complexity (i.e. at the mitochondrial level). The response of ETS activity to temperature change, especially at lower and higher temperatures, indicates differences in the characteristics of the ETSs in O. limosus and P. leniusculus. O. limosus is less sensitive to high temperature. The significant correlation between PGS and ETS activity supports our assumption that ETS activity could be used for the rapid estimation of thermal tolerance in crayfish species.

Multiple glacial refugia of the low-dispersal ground beetle Carabus irregularis: molecular data support predictions of species distribution models.

Classical glacial refugia such as the southern European peninsulas were important for species survival during glacial periods and acted as sources of post-glacial colonisation processes. Only recently, some studies have provided evidence for glacial refugia north of the southern European peninsulas. In the present study, we combined species distribution models (SDMs) with phylogeographic analyses (using mitochondrial DNA = mtDNA) to investigate if the cold-adapted, stenotopic and flightless ground beetle species, Carabus irregularis, survived the Last Glacial Maximum (LGM) in classical and/or other refugia. SDMs (for both a western European and for a Carpathian subgroup) were calculated with MAXENT on the basis of 645 species records to predict current and past distribution patterns. Two mtDNA loci (CO1 and ND5, concatenated sequence length: 1785 bp) were analyzed from 91 C. irregularis specimens to reconstruct the phylogeography of Central and eastern European populations and to estimate divergence times of the given lineages. Strong intra-specific genetic differentiation (inter-clade ΦST values ranged from 0.92 to 0.99) implied long-term isolation of major clades and subsclades. The high divergence between the nominate subspecies and the Carpathian subspecies C. i. montandoni points to two independent species rather than subspecies (K-2P distance 0.042 ± 0.004; supposed divergence of the maternal lineages dated back 1.6 to 2.5 million years BP) differing not only morphologically but also genetically and ecologically from each other. The SDMs also inferred classical as well as other refugia for C. irregularis, especially north of the Alps, in southeastern Europe and in the Carpathians. The coincidences between the results of both methods confirm the assumption of multiple glacial refugia for the studied species and the usefulness of combining methodological approaches for the understanding of the history of low-dispersal insect species.

Aphanomyces astaci in wild crayfish populations in Slovenia: first report of persistent infection in a stone crayfish Austropotamobius torrentium population.

All 5 crayfish species inhabiting Slovenian freshwaters, of which 3 are indigenous crayfish species (ICS: Astacus astacus, Austropotamobius pallipes, and A. torrentium) and 2 are non-indigenous (NICS: Pacifastacus leniusculus and Cherax quadricarinatus), were inspected for the presence of Aphanomyces astaci, the causative agent of crayfish plague. Wild crayfish populations showing no clinical signs of infection were inspected using A. astaci-specific real-time PCR. In addition, a conventional PCR assay was employed and confirmative sequencing was performed. Out of 88 analyzed crayfish, 15/27 (55.6%) specimens of A. torrentium from Borovniscˇ%%KERN_ERR%%ica Brook and 4/35 (11.4%) of P. leniusculus from the Mura River tested positive, showing low to moderate levels of infection (agent levels A1-A4 and A1-A3, respectively). Results revealed the presence of A. astaci not only in the resistant NICS but also in ICS, since the infected population of A. torrentium presumably had no contact with the NICS carrier and appeared to sustain A. astaci infection in the 2 sampling years. Although the A. astaci genotype has not yet been identified, a connection between the latent infection in ICS and a Group A strain of A. astaci, co-evolving with A. torrentium since its first introduction to Slovenia, is suggested as the most plausible conclusion. This is the first reported population of the genus Austropotamobius with persistent infection, in addition to the already known populations of the genus Astacus. Findings of the presumed co-evolution of A. astaci and ICS hosts open new perspectives, necessitating additional studies on the presence of A. astaci genotypes in the persistently infected ICS populations.