Defining Patterns and Rates of Natural vs. Drought Driven Aquatic Community Variability Indicates the Ongoing Need for Long Term Ecological Research.

Most ecologists have used climate change, as an omnipresent pressure, to support their findings in researching the vulnerability of specific taxa, communities, or ecosystems. However, there is a widespread lack of long-term biological, biocoenological, or community data of periods longer than several years to ascertain patterns as to how climate change affects communities. Since the 1950s, southern Europe has faced an ongoing trend of drying and loss of precipitation. A 13-year research program in the Dinaric karst ecoregion of Croatia aimed to comprehensively track emergence patterns of freshwater insects (true flies: Diptera) in a pristine aquatic environment. Three sites, spring, upper, and lower tufa barriers (calcium carbonate barriers on a barrage lake system that act as natural damns), were sampled monthly over 154 months. This coincided with a severe drought event in 2011/2012. This was the most significant drought (very low precipitation rates for an extended period of time) in the Croatian Dinaric ecoregion since the start of detailed records in the early 20th century. Significant shifts in dipteran taxa occurrence were determined using indicator species analysis. Patterns of seasonal and yearly dynamics were presented as Euclidian distance metrics of similarity in true fly community composition compared at increasing time intervals, to ascertain the degree of temporal variability of similarity within the community of a specific site and to define patterns of similarity change over time. Analyses detected significant shifts in community structure linked to changes in discharge regimes, especially to the drought period.

Is the spatial distribution of lentic water mite assemblages (Acari: Hydrachnidia) governed by prey availability?

Predation is a biotic interaction that links water mites to different taxonomic groups of benthic invertebrates. Diptera larvae (Chironomidae) and microcrustaceans (Cladocera, Copepoda, Ostracoda) are known to be the most commonly preferred prey by water mites. Although these predatory interactions are known from observations and ex situ investigations, the distribution patterns and co-occurrence of water mites and their prey in littoral lentic habitats have been poorly studied. Our goal was to determine whether predation might serve as a significant factor in water mite assemblage composition and distribution. Samples were taken from littoral zones of 21 artificial lakes (reservoirs) in the Dinaric Western Balkan ecoregion of Croatia. At every site, 10 samples were collected with regard to shore slope in depth zones of up to 1 m. In total 490 samples were collected from April 2016 to July 2017. Data analysis showed that the spatial distribution of water mites was partially correlated with the environmental parameters we measured (explaining 45.1% of total water mite variation). A positive correlation between water mite abundance and diversity and depth gradient (favouring more stable conditions in deeper parts of the littoral zone) was also observed. Finally, water mite abundance and species richness variation were found to be best predicted by the abundance of potential prey groups. Predation was statistically determined to be a strong dispersion variable, most probably influencing both spatial distribution and composition of water mite assemblages.

Impact of habitat heterogeneity on zooplankton assembly in a temperate river-floodplain system.

Dissimilar life features of Rotifera, Cladocera and Copepoda enable these organisms to respond differently to changes in the hydrological regime which influence alterations in environmental characteristics. We investigated the effect of habitat heterogeneity (e.g. eupotamal, parapotamal, palaeopotamal) on individual zooplankton group assemblages and biodiversity indices (α, ß and γ diversity) during hydro regime change in floodplain waterbodies. Dissolved oxygen and organic nitrogen concentrations changed significantly among hydrological states while water depth was affected by both site and hydro regime replacement. Each studied site supported different zooplankton assemblage that highly depended on species-specific responses to hydro regime change. Also, individual zooplankton groups exhibited different correlations with specific environmental parameters regarding site change. Throughout the study, rotifers' local (α) and among-community (ß) diversities were susceptible to the site and inundation change while the microcrustacean biodiversity pattern diverged. Copepods highly discriminated different habitat types and hydrological phases at the regional scale (γ diversity), while we found a complete lack of biodiversity dependence on both site and hydrology for Cladocera. Our results show that heterogeneous environments support the development of different zooplankton assemblages that express the within-group dissimilarities. They also point to the importance of identifying processes in hydrologically variable ecosystems that influence biodiversity patterns at an individual zooplankton group level. Our results suggest the use of appropriate zooplankton groups as biological markers in natural habitats and stress the importance of proper management in preserving biodiversity in floodplain areas.

Cryptic diversity in the Western Balkan endemic copepod: Four species in one?

We use mitochondrial (mtCOI) and nuclear (nH3) sequence data to investigate differentiation of Eudiaptomus hadzici, a freshwater copepod endemic to the Western Balkans. E. hadzici has a disjunct distribution and morphological differences were observed at regional scale. In the current study 6 out of 7 known populations are included. We applied several species delimiting approaches, distance based methods (K2P p-distance and Automatic Barcode Gap Discovery, ABGD) using the mtCOI, Bayesian phylogeny and the Bayesian method implemented in bPTP and BPP programs using the concatenated sequences of both genes. Phylogenetic and species delimitation analyses all suggest that the nominal species E. hadzici consists of four isolated, cryptic evolutionary lineages in the Western Balkans. Each of the four lineages inhabits a single lake or a group of lakes in close proximity. They exhibit major differences in secondary sexual characters, e.g. right antennule in males. Denticulation of spine on 13th segment is substantially distinct among the four lineages, having different number and shape of tooth-like protrusions. Gene flow and dispersal are restricted to very small spatial scale, but with local differences, implying that diverse historical and contemporary processes are operating at small spatial scales in E. hadzici. In order to further examine spatial and temporal diversification patterns, we constructed a dated species tree analysis using (*)BEAST. Due to lack of reliable calibration points and taxa specific evolutionary rates, two evolutionary rates were applied and the faster one (2.6% myr) seems more plausible considering the geological history of the region. The divergence of E. hadzici lineages is dated from Early Miocene onwards with geographically close lineages diverging more recently, Late Miocene to Pleistocene and Pleistocene, respectively. Overall, our findings shed light on cryptic genetic complexity of endemics in one of European biodiversity hotspots. Moreover, this study represents one further example of integrative taxonomy, linking DNA methodology and classical taxonomy based on morphology. Therefore, it lays groundwork for future taxonomy and biogeography of freshwater microcrustaceans in the region.

Spatial distribution and seasonal changes of mayflies (Insecta, Ephemeroptera) in a Western Balkan peat bog.

Peat bogs are unique wetland ecosystems of high conservation value all over the world, yet data on the macroinvertebrates (including mayfly assemblages) in these habitats are still scarce. Over the course of one growing season, mayfly assemblages were sampled each month, along with other macroinvertebrates, in the largest and oldest Croatian peat bog and an adjacent stream. In total, ten mayfly species were recorded: two species in low abundance in the peat bog, and nine species in significantly higher abundance in the stream. Low species richness and abundance in the peat bog were most likely related to the harsh environmental conditions and mayfly habitat preferences. In comparison, due to the more favourable habitat conditions, higher species richness and abundance were observed in the nearby stream. Three of the recorded species, Caenis luctuosa from the peat bog, and Eurylophella karelica and Leptophlebia marginata from the stream are new records for the Croatian mayfly fauna. Typical Central European life cycle patterns were confirmed for several species (e.g. Baetis vernus, Nigrobaetis niger, Electrogena ujhelyii), while for several others (e.g. Habrophlebia fusca, Paraleptophlebia submarginata) some discrepancies were observed. Therefore, these results provide new and valuable information on the ecology of mayflies in peat bog habitats.

New records of Limoniidae and Pediciidae (Diptera) from Croatia.

New records are provided for Limoniidae and Pediciidae from Croatia, with new distribution records for species in 12 different genera. Four genera and 18 species are newly recorded for Croatia. Until now, including this data, 87 Limoniidae and eight Pediciidae have been recorded from Croatia. In this paper we confirm presence of Ormosia (Oreophila) bergrothi (Strobl, 1895) and we give the first records of Dicranota (Paradicranota) pavida (Haliday, 1833) and Molophilus (Molophilus) repentinus Starý, 1971 from Balkan Peninsula.

The impact of gypsum mine water: a case study on morphology and DNA integrity in the freshwater invertebrate, Gammarus balcanicus.

The aim of our study was to investigate how exposure to heavy metal-rich waters from gypsum mining affects the morphology and levels of primary DNA damage in Gammarus balcanicus. Chemical analysis revealed increased concentrations of metals in water and sediment collected at a site impacted by gypsum mine wastewaters. The specimens also showed elevated total tissue metal levels when compared with the organisms collected at the reference site. The most prominent increase was observed for strontium, followed by iron, nickel, vanadium, aluminium, and manganese. The major pathway of entry for these toxic substances was through the degraded exoskeleton as a consequence of excessive strontium input (unbalanced calcium/strontium ratio) and altered permeability. Disturbed exoskeleton integrity was observed only in individuals collected downstream of the gypsum mine, which was confirmed by electron microscopy. Levels of primary DNA damage were evaluated using the alkaline comet assay in the haemolymph of the specimens.

Cytotoxic and genotoxic effects of water and sediment samples from gypsum mining area in channel catfish ovary (CCO) cells.

Man-made activities such as mining generate certain amounts of metal contaminated wastes which can reach aquatic environment and cause the serious effects on different organisms and ecosystem. Chemical analysis of the environmental samples is the most direct approach to reveal their pollution status but it cannot always provide information on biological effects to different organisms, including fish. This study was aimed to investigate the in vitro cytotoxicity and genotoxicity of water and sediment samples from gypsum mining area using the channel catfish ovary (CCO) cell line. Results obtained by the WST-1 assay and alkaline comet assay revealed that exposure of CCO cells to the same concentrations of contaminated water and sediment samples caused significant decrease in cell viability and increased DNA damages. Chemical analysis of water and sediment samples showed that increased concentrations of strontium, aluminum and iron were mainly responsible for the observed cytotoxic and genotoxic effects in CCO cells. The study suggested that fish CCO cells could be useful biological test-system for water and sediment cytotoxicity and genotoxicity assessments.

Assessment of genotoxic potency of sulfate-rich surface waters on medicinal leech and human leukocytes using different versions of the Comet assay.

The aim of the present study was to investigate how exposure to sulfate-rich surface waters affects the level of primary DNA damage in hemocytes of leech Hirudo medicinalis. Samples of surface water were collected at two sites near a gypsum factory (Knin, Croatia) and two reference sites. In the laboratory, samples were subjected to detailed chemical analysis and used in toxicity testing. For that purpose, previously acclimatized individuals of H. medicinalis were sub-chronically exposed (for 28 days) to tested water samples. Levels of primary DNA damage were evaluated using the alkaline Comet assay in hemocytes collected on days 7, 14, 21 and 28 of exposure and compared with their baseline values. Genotoxic potency of the water sample with the highest sulfate concentration was further evaluated using the alkaline, neutral and hOGG1-modified Comet assay on human peripheral blood leukocytes exposed ex vivo for 30 min. The purpose was to explore which mechanisms are responsible for DNA damage. Chemical analysis revealed that sulfate concentrations in two water samples collected in Mali Kukar Lake (1630 mg/L SO4) and Kosovcica River (823.3 mg/L SO4) exceeded the WHO and US EPA defined limits for sulfate in drinking water. Increased levels of metals were found only in the water sample collected in Mali Kukar Lake. However, of the 65 elements analyzed, only nickel and titanium exceed the value legally accepted in Croatia for drinking water. The levels of DNA damage, estimated by the alkaline Comet assay in hemocytes of medicinal leech, increased with the duration of exposure to two sulfate-rich water samples. Since hemocytes responded sensitively to treatment, they could be used for biomonitoring purposes. As observed on treated human peripheral blood leukocytes, all versions of the Comet assay were effective in detecting DNA damage, which was measured in samples with sulfate concentrations equal to or higher than the legally accepted levels for drinking water. Based on the obtained results, it can be assumed that genotoxicity was a consequence both of direct (single- and double-strand DNA breaks) and indirect effects (oxidative damage) caused by the combined effects of all contaminants present in the tested water samples. Our results indicate the need for in situ monitoring and purification of gypsum mine water prior to its release in the natural environment.

Estimation of DNA integrity in blood cells of eastern mosquitofish (Gambusia holbrooki) inhabiting an aluminium-polluted water environment: an alkaline comet assay study.

To estimate the impacts of an Al-contaminated aquatic environment on DNA integrity in the blood cells of eastern mosquitofish Gambusia holbrooki Girard 1859 inhabiting Lake Njivice (Island of Krk, Croatia), an evaluation using the alkaline comet assay was carried out. Genome integrity was studied in parallel with the same fish species inhabiting the nearby, unpolluted Lake Ponikve. The amount of DNA damage in cells was estimated from three different parameters: comet tail length as the extent of genetic material migration, tail intensity (% DNA in the comet tail) and tail moment. The results indicate the loss of genome integrity in blood cells of mosquitofish inhabiting Lake Njivice and the genotoxicity of this aquatic environment. Using the same assay, acute genotoxicity of contaminated water and sediment was evaluated and confirmed on fish, mouse and human blood cells treated ex vivo. Results of the present study indicate that the alkaline comet assay applied to fish blood cells is a valuable tool for determining the potential genotoxicity of water pollutants and confirm its usefulness in the evaluation of DNA damage in fish living in Al-polluted waters.

Application of the comet assay and detection of DNA damage in haemocytes of medicinal leech affected by aluminium pollution: a case study.

This report describes an investigation of genotoxic effects in medicinal leech (Hirudo verbana) exposed to water and sediment of Lake Njivice (Krk Island, Croatia) contaminated by aluminium compounds. The levels of primary DNA damage in leech haemocytes and loss of DNA integrity caused by acute and chronic exposure to contaminated water and sediment were investigated using the alkaline comet assay. Genotoxic effects induced by acute exposure to contaminants were evaluated on leech haemocytes and blood cells of fish and mouse treated ex vivo. The effects of chronic exposure were assessed on haemocytes sampled from an animal kept under laboratory conditions on contaminated water and sediment for 180 days. The results indicate the DNA damaging potential of aluminium compounds present in an excess amount in tested samples.

Loaches in a long term study of the Drava River in Croatia.

The diversity, species composition and structure of the fish community were studied in the vicinity of three power plant reservoirs in the middle part of the Drava River in Croatia. Over a period of twenty years in a collection of 25,913 fishes, 56 different fish species belonging to 16 families were recorded. The family Cobitidae is represented by two species: Cobitis elongatoides and Misgurnus fossilis, while the family Balitoridae is represented by only one species, Barbatula barbatula. The presence of the Cobitidae family was recorded in eleven of seventeen investigated localities, while Balitoridae was found in six localities. In the studied fish community, the percentage of C. elongatoides is 1.41% of the total number of individuals, and B. harbatula is 1.02%. M. fossilis is a rare species (0.03 %). Both C. elongatoides and B. barbatula were not caught at the beginning of investigation. C. elongatoides and B. harhatula were first caught in 1985, while M. fossilis was first caught in 1996. The proportion of C. elongatoides in the entire community increased, while the proportion of B. barhatula decreased in later years.