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Pigmented macrophage aggregates (MAs) are known to change under influence of various factors, such as aging, season, starvation, and/or pollution. In this study, changes in the pigment content of the MAs in the spleen of Vardar chub (Squalius vardarensis, Karaman) (n = 129) collected in spring and autumn, from three rivers with different pollution impact was examined: Zletovska River (metals), Kriva River (metals and municipal wastewater), and Bregalnica River (municipal wastewater). Collected data revealed increased relative volume and number of MAs containing hemosiderin under the influence of metals, significant in autumn (p < .05). In chub exposed to metals combined with municipal wastewater, significant increase of lipochrome accumulation in MAs in autumn, melanin in MAs in fish captured in the spring season, and number of splenic MAs containing combination of melanin and lipochrome was noted. Volumes and number of MAs containing combination of hemosiderin and lipochrome increased in spleen of fish captured in autumn from both Zletovska River and Kriva River, most likely due to the contribution of hemosiderin and lipochrome, respectively. Values measured for the various pigments in splenic MAs in fish captured from Bregalnica River, were overall closer to the values measured for fish captured from Kriva River. Notably, melanin and lipochrome are more likely to be found in fish from waters influenced by municipal wastewater (organic pollution) and hemosiderin in fish spleen from water influenced by mining activity (heavy metals pollution).

The validity of four gudgeon species known for the ichthyofauna of North Macedonia has been a matter of different taxonomic disputes (except forGobio ohridanusKaraman, 1924). Recently, a restoration of the species status was proposed forGobio balcanicusDimovski et Grupče, 1977 andRomanogobio banarescui(Dimovski et Grupče, 1974). So far,Romanogobio stankoi(Karaman, 1974), has not been a part of any recent investigation and it is still considered as a junior synonym ofRomanogobio elimeius(Kattoulas, Stephanidis et Economidis, 1973) orR. banarescuidue to the lack of distinctive morphological characters. The osteological characters in the structure of the vertebral column, have significance to the systematics of the gudgeons, especially in elevation ofRomanogobioat the level of genus. To contribute to resolving the taxonomic status in these gudgeon species, the goal of this paper is to identify distinctive characters by presenting and analyzing the structure of their vertebral column. Samples were inspected from the fish collections in the Institute of Biology (G. ohridanus), the Institute of Animal Science (G. balcanicus), and the Macedonian Museum of Natural History (bothRomanogobiospecies). Fish samples were X-rayed as well as cleared and double-stained. Obtained data on the number of vertebrae were presented through the vertebral formula following Naseka (1996). Multiple Correspondence Analysis (MCA) was used to check the interrelation between taxa and the number of vertebrae in all units of the vertebral column.G. balcanicusis distinguished fromG. ohridanusmainly in the predorsal and preanal subregion (10 vs. 11 vertebrae and 1 vs. 0 vertebrae, respectively), whileR. stankoidistinguishes fromR. banarescuiby the number of vertebrae in the preanal and postanal subregion (2 vs. 4 vertebrae and 19 vs. 17 vertebrae, respectively). The structure of vertebral column offers distinctive morphological characters for the four gudgeon species from Ohird Lake and the Vardar River basin.

Piria, Marina/0000-0001-6324-9282; Simonovic, Predrag/0000-0002-4819-4962; WOS: 000418927100009 Fish introductions, particularly in areas of high biological diversity and endemism, represent a major threat for biodiversity. In the Balkan Peninsula, 60 fish species have been introduced to date, of which 36 have become naturalized in inland waters. Since the Balkans are one of the world's 35 biodiversity hot spots, this large presence of alien fish species poses a serious threat for the stability of freshwater ecosystems and the survival of the native ichthyofauna and of aquatic biodiversity in general. The motivation for the introductions, and the historical timeline, varies among the Balkan states. Despite recent attempts to implement and align legislation aimed at preventing the introduction of potentially invasive species, and the implementation of rigorous controls of introductions and increased protection of open waters, the majority of current introductions remain intentional, primarily via aquaculture. This review article provides a historical overview of freshwater fish introductions, the motivation behind them and the current distribution of alien freshwater fishes in the Balkans. The ecological implications and future perspectives concerning alien fish species in the region are also discussed. University of Zagreb, Croatia; Ministry of Education, Science and Technological Development of Serbia [173025] University of Zagreb, Croatia; Ministry of Education, Science and Technological Development of Serbia, Grant/Award Number: 173025

A lipid peroxidation product, malondialdehyde (MDA), was studied in Vardar chub (Squalius vardarensis Karaman) as an indicator of oxidative stress, using native fish from three rivers in northern Macedonia: the mining-impacted Zletovska and Kriva rivers and the agriculturally impacted Bregalnica River. MDA concentrations were measured in the intestine in the spring and autumn of 2012 and in the gills in autumn. The aims of the study were to establish the type of contamination which provokes a more pronounced MDA increase, as well as the organ which more reliably reflects the occurrence of oxidative stress. MDA levels in the intestine in spring amounted to 3.29–155.8 nmol g−1 and in autumn to 4.85–111.1 nmol g−1, whereas MDA concentrations in the gills in autumn were 7.69–147.5 nmol g−1. Stronger influence of organic contamination on development of oxidative stress was observed in both organs, as seen from higher median MDA concentrations in autumn in fish from the highly pesticide-contaminated Bregalnica River (gills 78.4 nmol g−1; intestine 23.5 nmol g−1) compared to the highly metal-contaminated Zletovska River (gills 15.9 nmol g−1; intestine 17.4 nmol g−1). The response of the gills to contamination was twice stronger than that of the intestine. The majority of fish from the pesticide-polluted river had increased MDA in the gills, in contrast to only sporadically increased MDA in the intestine. Our results indicated that development of oxidative stress strongly depends on the selected fish organ and that the gills seem to be a better choice for monitoring oxidative stress than the intestine, due to their continuous and direct exposure to polluted river water.

Pollution with heavy metals may influence the immune system of fish, leading to impairment of their health or even increase their mortality. The fish kidney is one of the first fish organs to be affected by water contamination. Amounts of kidney macrophages (MACs), which are involved in fish immune response, as well as the qualitative and quantitative changes in the pigmented MACs in fish kidney, are used as biomarkers of pollution. Therefore, in this study, we have evaluated relative and total volumes of trunk kidney pigmented MACs, and analyzed the pigments accumulated within them. Fish were sampled from two mining impacted rivers, Kriva and Zletovska, highly contaminated with heavy metals, and from one reference river, Bregalnica, in spring and autumn of 2012. We have observed that main pigments found in kidney MACs of Vardar chub were melanin and lipofuscin/ceroid, as well as that relative volumes of MACs ranged from 0.56 to 1.68%. Moreover, the results showed that relative volumes of pigmented MACs were higher in metal contaminated rivers, especially in autumn season in the Zletovska River, concurrently with extremely high metal exposure. In addition, condition factors and kidney somatic indices were found significantly lower in the Zletovska River in both seasons, autumn and spring, possibly also as a consequence of high water pollution. Our data confirm that increase in relative volumes of pigmented MACs may serve as warning sign of potential heavy metal pollution in aquatic environment.

Integrated chemical and biomarker approaches were performed to estimate if there is ongoing toxicity in the River Bregalnica, namely connected with the presence of metals. The study was performed in water, sediment, and barbel (Barbus peloponnesius), collected in two seasons, from two suspected polluted and one reference zones. The water analyses revealed higher mean values in polluted sites for most of the examined physicochemical parameters. Metal concentrations (Zn, Cu, Cd, Mn, Pb, and Fe) in water were more or less constant, whereas in sediment, they were higher at the two polluted locations. Condition factor (CF), as a general health indicator, revealed better overall condition in barbel from the reference site. In general, blood parameters revealed higher values in the polluted localities. Irrespective of sex and/or season, the frequency of micronuclei (MN) and vacuolated nuclei (VN) were with higher rates in polluted sites. Similarly, the frequencies of the leucocytes (Le), binuclei (BN), and irregularly shaped nuclei (ISN) were also significantly increased in the polluted localities, but they seemed prone to be influenced by sex and/or season. However, strong positive correlations between blood biomarkers and most water physicochemical parameters and metal in sediment were estimated. Our data support that the River Bregalnica's lower course receives significant genotoxic pollution, likely via metal industry effluents, agricultural runoff, and domestic sewage, and reinforced the utility of MN and other nuclear abnormalities as sensitive and suitable biomarkers for genotoxicity when used in monitoring studies.

There are so many reports of introduction of non-native fish species in Europe but reports including introduction of species from Serrasalmidae, especially from piranhas fish are scarce. This paper documents the first record of the occurrence of the exotic red bellied piranha, in Dojran Lake, the southern part of Republic of Macedonia. The morphological taxonomy revealed that the specimen is Pygocentrus nattereri Kner, 1860.

Many natural freshwater ecosystems, especially in the north eastern Macedonia, are polluted with heavy metals, which are released by active mines. Long-term exposure to high levels of dissolved metals might result in increased metal bioaccumulation in organs of aquatic organisms, and consequently might cause various sub-toxic and toxic effects. The aim of this study was to assess the health of Vardar chub (Squalius vardarensis) inhabiting mining impacted rivers Zletovska and Kriva, in comparison with chub from the reference Bregalnica River. It was done by use of indicators of tissue damage (histopathology of liver and gonads) and general indicators of exposure to environmental stressors (condition factor, organo- somatic indices and external/internal macroscopic lesions). Histological assessment of gonads revealed good reproductive health in all three rivers, indicating high tolerance of gonads to contaminant exposure. Contrary, several external/internal lesions were more pronounced in chub from severely metal contaminated Zletovska River. Prevalence of hepatic lesions was also higher in mining impacted rivers (in Kriva, 70% ; in Zletovska, 59%) compared to Bregalnica River (38%). The spectrum of histological lesions observed in chub liver varied from non-specific minor degenerative conditions, such as lymphocyte infiltration, fibrosis, parasites, granulomas and lipidosis, to extensive and/or more severe changes such as bile duct proliferation, necrosis, megalocytosis, light-dark hepatocytes and hepatocytes regeneration. The results of histopathological investigation for all three rivers showed clear signs of water contamination, especially prominent in mining influenced rivers. More research efforts should be devoted to study of environmental conditions and metal contamination in the mining impacted rivers worldwide, especially of their effects on health of local ichtyofauna.

Nutritional manipulation and genetic selection for egg size and production may lead to changes in egg components. This experiment was carried out to analyze the egg structure parameters of eggs produced by Hisex Brown laying hens fed with diet with different supplements inclusion. The intensity of egg production was significantly higher in the groups fed with enriched feed with iodine (90.00%), vitamin E (90.00%) and selenium (91.98%), and significantly lower in the group fed with DHA inclusion feed (76.00%) in respect to the control group (82.00%), confidence interval of 95%. Concerning diet supplemented with selenium and diet supplemented with vitamin E, the egg yolk weight was statistically different compared with the control group (confidence interval of 95%). The yolk weight averaged 1.80 g and 1.29 g more than yolk weight in the control eggs, respectively. The egg shell weight was statistically different in diet enriched with iodine compared with the control. The egg shell weight averaged 1.48 g less per egg for the eggs enriched with iodine with 17.45% less underweight than the control eggs. Enriched eggs offer consumer a variety of value-added options for their egg purchase. Although enriched eggs may provide consumers with a specific quality attribute or healthful ingredient, they do not appear to provide quality and value in a traditional sense as defined by the standards of quality and grade.

Freshwater contamination with mining waste can result with high concentrations of toxic metals in the water and in fish organs. In North-Eastern Macedonia, several rivers (e.g., Zletovska, Kriva) are exposed to acid mine drainage from active Pb/Zn mines. Previous studies confirmed high concentrations of dissolved metals in their water. This study was performed in liver and gills of Vardar chub (Squalius vardarensis Karaman, 1928) from three Macedonian rivers (Bregalnica, Kriva and Zletovska) in spring and autumn 2012. The aim was to establish if increased exposure to certain metals have resulted with their increased bioaccumulation. The concentrations of 19 elements were measured in cytosolic tissue fractions, to obtain information on metabolically available metal species. The following ranges of cytosolic concentrations of highly toxic elements were measured in the Vardar chub liver (in μg/L): Cd, 1.18–184; Cs, 0.25–25.4; Tl, 0.02–5.80; Pb, 0.70–61.1. Their ranges measured in the gills (in μg/L) were the following: Cd, 0.24–59.2; Cs, 0.39–24.4; Tl, 0.01–1.00; Pb, 0.65–87.2. Although the water of the mining impacted Zletovska River was highly contaminated with several essential metals, especially with Mn and Zn, the majority of essential elements (Na, K, Ca, Mg, Co, Cu, Fe, Mn, Mo, and Zn) did not reflect the exposure level. In contrast, seven nonessential elements reflected the level of exposure in the water. Significantly increased hepatic and gill concentrations of Cs, Rb, Sr, and Tl were detected in Vardar chub from the Zletovska River compared to the other two rivers, of Cd and Pb in the Zletovska and Kriva River compared to Bregalnica, and of V in the Bregalnica River compared to Zletovska and Kriva rivers. Observed significant metal bioaccumulation, in particular of highly toxic elements, as a consequence of exposure to water contaminated with mining waste points to necessity of intensified supervision of mining impacted rivers.

The aims of this study were to determine the influence of the factors affecting fin damage under different rainbow trout production systems and to compare the findings with the known experimental reports. The study was based on a questionnaire that included information about the main factors i.e. oxygen level in exit water, water temperature, stocking density, daily feed ration, number of meals and grading frequency on seven rainbow trout farms. Standard multiple regression analysis, based on a previously published fin damage dataset, was used to assess the relationship between the level of fin damage per fin and the factors. Daily feed ration received the strongest weight in the model for the caudal, anal and both pectoral fins, whereas number of meals received the strongest weight in the model for both pelvic fins. Grading frequency received the strongest weight only in the dorsal fin model. Lower levels of daily feed ration and number of meals combined with higher water temperature increased the level of fin damage, whereas stocking density had no effect. The results conform to the experimental research on fin damage in rainbow trout. The research model contributes to the overall assessment of fish welfare and the regression analysis used in this study could be used on rainbow trout farms to evaluate the effect of the main factors on the level of fin damage.

Many bioindicators have not yet been well characterized regarding their tendency to bind trace elements by different cytosolic biomolecules in response to trace element exposure. Accordingly, our principal aim was to define the cytosolic distributions of Cd, Co, Cu, Fe, Mn, Mo, Se, and Zn among the biomolecules of different molecular masses in liver and gills of Vardar chub (Squalius vardarensis Karaman), a representative fish species of Macedonian rivers, and to determine distribution changes which occur as a consequence of increased exposure to specific trace elements. Additionally, we aimed to confirm the presence of heat-stable biomolecules in chub hepatic and gill cytosols. Distribution profiles were obtained by separation of cytosols and heat- treated cytosols using size-exclusion high performance-liquid chromatography, and by offline determination of trace element concentrations using high resolution inductively coupled plasma- mass spectrometry. Distribution profiles of trace elements were mainly characterized by several peaks encompassing different ranges of molecular masses, as a sign of incorporation of trace elements in various biomolecules within hepatic and gill cytosols. Especially interesting finding was probable binding of Fe to ferritin, which was especially pronounced in the liver, as a sign of important liver function in Fe storage. Furthermore, association with heat-stable proteins, metallothioneins (MT), was indicated for Cd, Cu, and Zn in the hepatic cytosol, as well as for Cd in the gill cytosol, whereas a sign of Zn- MT association was not observed in the gills. The presence of Mo- and Se-binding heat-stable compounds of very low molecular masses (

Changes in fish macrophage aggregate centres (MACs) are a useful indicator of environmental stress. In this study we evaluated stereologically the values of these cells in the polluted sites compared to the clean site in the River Bregalnica. MACs aggregates were randomly distributed in spleen and often showed irregular form. Dominant pig-ment in them was hemosiderin. Relative volume of MACs aggregates showed significant higher value in brook barbel from polluted site. Total volume showed the same pattern, but differences were not significant. Our data confirm that increase in MACs aggregates may serve as to warn on potential pollution in aquatic ecosystems. However, the pre-caution is strongly suggested, as results were based on quantitative approach.

Recent field studies have shown that the survival of Zingel balcanicus (Karaman, 1937) was enabled in an isolated area along the Treska River. The presence of two opercular spines in Z. balcanicus, compared to only one in Zingel streber, has attracted our interest for investigating more details about osteological characters of Z. balcanicus in order to provide more useful anatomical data that could be of osteo-taxonomic value. A complete osteological description of Z. balcanicus is presented with some comparative aspects discussed. Zingel balcanicus shares a large number of osteological characteristics with other members of Zingel. However, the articular processes of the premaxillary bone, teeth in the palatine bone, opercular spination and number of precaudal centra were emphasised as stable osteo-taxonomic characters. On the other hand, the architecture of the hyomandibula, spine-like extensions of infraorbital bones, gill rakers, and independent cartilage plates and presence of “Spreitzer vertebra” were noticed as peculiar characteristics that never have been described for the genus Zingel.

A high level of freshwater fish endemism in the Balkans Region emphasizes the need for non-native species risk assessments to inform management and control measures, with pre-screening tools, such as the Fish Invasiveness Screening Kit (FISK) providing a useful first step. Applied to 43 non-native and translocated freshwater fishes in four Balkan countries, FISK reliably discriminated between invasive and non-invasive species, with a calibration threshold value of 9.5 distinguishing between species of medium and high risk sensu lato of becoming invasive. Twelve of the 43 species were assessed by scientists from two or more Balkan countries, and the remaining 31 species by a single assessor. Using the 9.5 threshold, three species were classed as low risk, 10 as medium risk, and 30 as high risk, with the latter category comprised of 26 moderately high risk, three high risk, and one very high risk species. Confidence levels in the assessments were relatively constant for all species, indicating concordance amongst assessors.

Inter-population variability within Sabanejewia populations from the western Balkans, and their phylogenetic position in comparison to other European populations were investigated. Of 79 samples analysed, 51 unique haplotypes were identified. Network analysis divided thirteen populations from five river basins into two clusters: cluster I was composed of populations from the Vardar drainage and tributaries of the neighbouring River Morava (River Danube basin), while cluster II contained the River Timok (eastern Serbia) and all the River Sava populations. The only locality that housed haplotypes of both clusters was the River Kutinska reka in the upper Morava basin. When compared with the haplotypes reported in former studies, both clusters fell within the ‘Danubian-Balkanian complex’. Cluster II was included in the most heterogeneous sub-lineage S. montana — S. bulgarica — S. balcanica (III), while cluster I was related to the sub-lineages S. doiranica — S. balcanica (II) and S. balcanica (VI). Recently published haplotypes from the Croatian Sava (rivers Petrinjcica and Rijeka) and Drava (rivers Drava and Vocinska) basins, as well as Czech and Slovak individuals from the Danube and Tisza river basins were included. The River Drava showed the same population subdivision as the River Kutinska reka.

Quantification of histopathological alterations in the gills of Vardar chub (Squalius vardarensis Karaman) was performed in 2012 in rivers of north- eastern Macedonia, with the aim to examine the effects of water quality in the rivers (Zletovska and Kriva River - impacted by active Pb/Zn mines ; Bregalnica River - contaminated by agricultural waste). The biological alterations in chub were classified as: circulatory disturbances, regressive and progressive changes, but their severity differed. Altogether the mildest changes were observed in the gills of chub from the Bregalnica River, a less polluted river, whereas mining impacted rivers were characterized by more severe alterations. In the gills of chub from the Zletovska River, which is highly contaminated with numerous metals, sulphates and chlorides, the highest lesion indices were found for the regressive changes of both epithelium and supporting tissue, with typical lesions referring to atrophy, thinning and lifting of epithelial cells, necrosis of epithelium and chloride cells, as well as deformations of lamellar cartilaginous base. Gill damages of chub from the Kriva River were overall milder compared to the Zletovska River, in accordance with pollution status. In the gills of chub from that river, progressive changes were more pronounced, specifically severe hyperplasia of mucous cells and epithelium in the interlammellar space, leading to fusion of lamellae, as well as hypertrophy of chloride cells. The comparison between seasons indicated higher intensity of progressive changes in all three rivers in autumn, when water level was very low, and consequently, water contamination was more pronounced due to concentration effect. The pattern and severity of histopathological alterations in the chub gills reflected differences in contamination levels and type of contaminants in different rivers and sampling periods, and thus have been proven as a valuable indicator of water quality.