Mišel Jelić

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.

Two distinct evolutionary lineages of the Astacus leptodactylus species-complex (Decapoda : Astacidae) inferred by phylogenetic analyses

Abstract. Narrow-clawed crayfish (Astacus leptodactylus Eschscholtz, 1823 species-complex) is one of five European freshwater crayfish species. Even though widely distributed, it hasn’t been frequently studied and its taxonomy and systematics are unresolved. The results of a recent comparative morphometric character study revealed that morphometry of Asian and European populations differ significantly. In this research, for the first time, mitochondrial molecular markers (16S rRNA and COI) were used with the aim of elucidating the phylogenetic relationship between European and Asian populations of the narrow-clawed crayfish. Analyses included crayfish from Croatia, Bulgaria, Armenia, Russia, Poland and Turkey, and three different optimality criteria were applied. Phylogenetic relationships were reconstructed using the COI dataset, as well as the concatenated one (COI + 16S rRNA). For both datasets, congruent topologies were obtained and trees were characterised by the existence of two well supported phylogroups, one that included European populations, and the other Asian. Results indicate the presence of distinct evolutionary lineages within the A. leptodactylus species-complex, and corroborate previous results obtained using morphometric analyses.

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.

Species-specific differences in dynamics of agonistic interactions may contribute to the competitive advantage of the invasive signal crayfish (Pacifastacus leniusculus) over the native narrow-clawed crayfish (Astacus leptodactylus)

Abstract Species-specific differences in dynamics of agonistic interactions may influence the outcome of interspecific competition and potentially contribute to competitive advantage of one species over another. In this study, we compared the dynamics of agonistic interactions of one of the most successful crayfish invaders of European freshwater ecosystems, the signal crayfish (Pacifastacus leniusculus) and the widespread native European species currently undergoing range expansion in Croatia and Europe, the narrow-clawed crayfish (Astacus leptodactylus). Comparisons between P. leniusculus pairs and the A. leptodactylus pairs demonstrated significant differences in frequency and duration of agonistic encounters: P. leniusculus dyads engaged in fewer fights, but their duration was significantly longer. In staged interactions between size-matched interspecific pairs, agonistic behaviour of P. leniusculus individuals translated into dominance over their A. leptodactylus counterparts. This indicates that the success of P. leniusculus in agonistic encounters with the native competitor might stem from its readiness to continue fighting for a longer time period, and could lead to ecological advantages during niche competition even when facing a successful native crayfish species.

First observation of Posthodiplostomum cuticola (Nordmann, 1832) metacercariae in cypriniformes from Croatia

SummaryDuring an ichthyological study of the Ilova River, rudd (Scardinius erythrophthalmus) infected with Posthodiplostomum cuticola was observed for the first time in Croatia. Later, a more detailed study was made at the same sampling point and the distribution of P. cuticola in the fish species present was examined. The condition of the hosts and parasitological indices such as prevalence, mean abundance and mean intensity of all hosts examined were investigated. Metacercariae of P. cuticola were most prevalent (100 %) in Rutilus rutilus, Pseudorasbora parva, Leuciscus leuciscus and Alburnus alburnus, slightly lower (93.94 %) in Leuciscus cephalus, substantially lower in Rhodeus amarus (66.67 %) and Barbatula barbatula (50 %) while the lowest prevalence was found in Gobio gobio (47.83 %). The correlation coefficient between number of parasites and total and standard length, weight and Fulton’s condition index of sampled specimen was significant only for L. cephalus.

Distribution of endangered Italian gudgeon Romanogobio benacensis (Cypriniformes, Cyprinidae, Gobioninae) with remarks on distinguishing morphological characters

Abstract Distribution data on many freshwater fish species in Croatia are scarce and species identifications are difficult, requiring further detailed studies. This paper presents a report of the Italian gudgeon Romanogobio benacensis from the Mirna River in the Istra Peninsula in Croatia, in the south-east from its previously known distribution range. The identification of R. benacensis in Croatia was supported by a morphological comparison with R. benacensis from Italy and Slovenia, the common gudgeon Gobio gobio, and the Danubian gudgeon Gobio obtusirostris from geographically close locations. A combination of character states (number of scales between anus and anal-fin origin, branched pectoral-fin rays, lateral-line scales, total, abdominal, and caudal vertebrae, and the size and number of lateral blotches) distinguishes R. benacensis from both G. gobio and G. obtusirostris. The phylogenetic analyses using mitochondrial sequences of cytochrome b gene confirmed that specimens from the Mirna River belong to R. benacensis. Also, Reka River system (Adriatic Sea basin) in Slovenia is inhabited by a possibly introduced Danubian gudgeon, G. obtusirostris, and not by R. benacensis.