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The study of metal bioaccumulation in the gills of European chub (Squalius cephalus) was conducted in September 2009 at the medium-sized rural river Sutla, characterized by agricultural and municipal type of water contamination. The concentration ranges were established for the first time in the soluble, metabolically available fractions of chub gills for 12 metals, which are environmentally extremely relevant and yet only seldom studied, as follows in a decreasing order: K, 225-895 mg L(-1); Na, 78-366 mg L(-1); Ca, 19-62 mg L(-1); Mg, 13-47 mg L(-1); Rb, 164-1762 μg L(-1); Sr, 24-81 μg L(-1); Ba, 13-67 μg L(-1); Mo, 1.3-16 μg L(-1); Co, 0.7-2.7 μg L(-1); Li, 0.4-2.2 μg L(-1); Cs, 0.2-1.9 μg L(-1); and V, 0.1-1.8 μg L(-1). The concentrations of Fe (1.6-6.4 mg L(-1)) and Mn (16-69 μg L(-1)) were also determined and were in agreement with previous reports. By application of general linear modelling, the influence of different abiotic (metal exposure level) and biotic parameters (fish sex, age, size and condition) on metal bioaccumulation was tested. It was established that bioaccumulation of many metals in fish depended on various physiological conditions, wherein Ba could be singled out as metal exhibiting the strongest association with one of biotic parameters, being significantly higher in smaller fish. However, it was also undoubtedly demonstrated that the concentrations of three metals can be applied as reliable indicators of metal exposure even in the conditions of low or moderate water contamination, such as observed in the Sutla River, and those were nonessential elements Li and Cs and essential element Fe. The results of our study present an important contribution to maintenance of high ecological status of European freshwaters, through enrichment of knowledge on the bioaccumulation of various metals in gills of European chub as frequently applied bioindicator species in monitoring of water pollution.

Vardic I., D. Kapetanovic, Z. TeskeredIic, E. TeskeredIic: First Record of Infectious Haematopoietic Necrosis Virus in Rainbow Trout Fry in Croatia. Acta Vet Brno 76, 2007: 65-70. The paper describes the first diagnosis of infectious haematopoietic necrosis virus (IHNV) in Croatia. The viral causative agent was detected in pooled organ samples from the imported rainbow trout fry on the fish farm. Reverse transcriptase - semi-nested polymerase chain reaction (RT- snPCR) was applied directly on the infected tissue for rapid virus detection. After isolation on cell cultures, IHNV isolate was characterised on the basis of the 303 nt region of the glycoprotein gene (Mid-G) sequence. Phylogenetic comparison to North American and European IHNV isolates revealed that this Croatian isolate belongs to the M genogroup, confirming the prediction of the M genogroup origin of European IHNV isolates. The introduction of the virus presents a threat of further spreading of the disease in Croatia, as the infected farm is in a direct contact with the open waters. Rainbow trout, IHNV, RT-snPCR, molecular epidemiology Infectious haematopoietic necrosis (IHN) is a world-wide disease problem in farmed rainbow trout and other cultured salmonid species. The disease is caused by a fish rhabdovirus, infectious haematopoietic necrosis virus (IHNV), which belongs to the genus Novirhabdovirus (Fauquet et al. 2001). IHN is typically characterized as an acute viremia with resulting haemorrhage and necrosis of major organ systems, although infected fish may not show clinical signs. Juvenile fish are more susceptible to the disease, and their mortality can reach over 90%, especially in hatcheries where the disease breaks out for the first time (Bootland and Leong 2003). The transmission of IHNV between fish is primarily horizontal, and the major cause of a wide geographical spreading of the virus is the movement of infected eggs and/or fry. IHNV was first identified in western North America, but soon spread to Japan and Europe. Although IHNV has been detected in neighbouring countries, for example Italy in 1987 (Bovo et al. 1987), and Slovenia in 1993 ( Jenaia et al.2002), there were no reported cases in Croatia previously (Orai c and Zrnaic 2005). Fast IHNV spreading and virus establishment among carrier fish require a rapid and reliable diagnostic method. Reverse transcriptase-polymerase chain reaction (RT- PCR), which detects specific parts of the IHNV genome, has been described (W inton and Einer-Jensen 2002) and approved by OIE as a valuable confirmation method for the IHNV diagnosis (OIE 2003). Further molecular characterisation, based on the sequence comparison of the glycoprotein (G) and non-virion (NV) genes from the different IHNV isolates proved to be useful in understanding and explanation of IHNV spreading, evolution and origin (Troyer et al. 2000; Kurath et al. 2003; Troyer and Kurath 2003; Garver et al. 2003). Phylogenetic analysis of partial G gene sequences (Mid-G, 303 nucleotide region coding antigenic determinants) from the North American IHNV isolates revealed 3 major virus genogroups. Those groups are designated U, M and L for the upper, middle and lower portions of IHNV

Here we report the genetic variability and presence of mtDNA-like sequences of Pomphorhynchus laevis from the chub, Squalius cephalus, caught at the sampling sites along the Sava River and its tributary the Sutla River in Croatia. Sequences of the cytochrome c oxidase subunit 1 (COI) gene of the recovered P. laevis specimens were used for haplotype network construction and phylogenetic analysis. These analyses showed that some specimens contained mitochondrial-like sequences, and they uncovered the existence of a Sava River basin strain different from known strains of P. laevis. This is the first time that P. laevis has been shown to contain mtDNA-like sequences, suggesting the need to exercise caution during COI analyses of P. laevis using universal primers. Highly conserved sequences of two nuclear markers, the ITS region and 18S rRNA, were not helpful for understanding genetic variability or differentiating strains. Furthermore, analysis of the dynamics of P. laevis infections in S. cephalus from the Sava and Sutla Rivers showed decreased prevalence and abundance at sites with inferior water quality, positive association of parasite abundance with fish size, and no clear association of parasite abundance with fish condition index or sex.

Data on the microbiological quality of the freshwater systems under the anthropogenic influence, such as the Sava River, are of the major importance for the water resource management. Furthermore, analyses of the microbial quality of fish meat provide information of the fish as a valuable food resource from the investigated river basin. The health status of the fish, including dynamics of infection and biodiversity of endoparasites, is important bioindicator of changes in the ecosystem structure and function. For the ecosystem-based approach to the Sava River management, investigations of microbiological quality of the Sava River water and the meat of the European chub as the bioindicator organism, as well as dynamics of infection/biodiversity of intestinal parasites Acanthocephala, were performed. The survey comprised the data collected in periods 2005, 2006 and 2012. Microbiological investigation of water was performed in 2006 and 2012, while microbiological analyses of fish meat and ichthyo-parasitological investigation took place during 2005–2006. A high number of heterotrophic bacteria were recorded during 2006 survey, confirmed by the distinctly higher values of the three faecal indicators (total coliform, E. coli and enterococci), and indicated poor water quality downstream of the cities Zagreb and Velika Gorica, as a result of the municipal sewage outlets. The results from 2012 survey indicated the existence of moderate to critical faecal and organic pollution in all samples. Accumulation of the bacteria in the European chub meat was mainly uniform along the watercourse within standards and limitations for the human consumption. Sampling sites downstream cities of Zagreb and Velika Gorica were characterised with the lower prevalence and abundance of two common species of the chub intestinal acanthocephalan parasites, Pomphorhynchus laevis and Acanthocephalus anguillae. Poor microbiological quality of the water and lower distribution of chub intestinal parasites were related to the anthropogenic influence, downstream of the urban areas. Milačić R, Ščančar J, Paunović M, editors. The Sava River. Berlin, Heidelberg: Springer-Verlag; 2015. p 201-28.

Aeromonas hydrophila was recovered from fish living in lake Vrana on the Croatian island of Cres. The occurrence of the bacterium in the fish was assessed and related to gross signs of disease and findings at necropsy as a potential health hazard for fish. Isolated bacteria were subjected to morphological, physiological, biochemical and antibiotic susceptibility tests. A total of 26 A. hydrophila isolates were obtained. There was a clear seasonality, as no isolates were recovered in the summer months. Most of the isolates were sensitive to all the antimicrobials used in the study except novobiocin and penicillin G. Affected fish manifested haemorrhages over the skin, in the liver, kidney and swim bladder, spleen infarcts, fatty liver, ascitic fluid and swollen haemopoietic tissues. A. hydrophila does not appear to pose a major threat for the fish in the lake at present but under unfavourable and stressful conditions it could seriously compromise fish health.

In order to determine an unknown fish population from the Vrana Lake, mitochondrial cytochrome b gene and non-coding nuclear region Cyfun P were investigated. Stabile population of Bulldog rudd, Scardinius dergle Heckel & Kner, the endemic Croatian freshwater fish in the Krka River, was genetically characterized with the same markers in order to compare it with the material from the Vrana Lake. Genetic markers were sequenced and aligned with the similar ones obtained from the GenBank in order to determine taxonomic and phylogenetic position of these two species. A significant discrepancy between nuclear genetic markers of our specimens and the sequence from the GenBank was found. Phylogenetic analysis suggested that the specimens from the Vrana Lake belong to the species S. hesperidicus. Morphometric characteristics, the maximum length and body mass showed new maximum values for both S. dergle and S. hesperidicus.

In the present study, anisakids from: tuna (Thunnus thynnus) fattened in the Croatian farm in middle Adriatic Sea, three different feral fish species caught near tuna farm (Trachurus trachurus, Scomber japonicus and Oblada melanura) and fish marketed in Croatia (T. trachurus) were analysed by morphology and molecular methods. Larvae were identified to the species level by PCR restriction fragment length polymorphism and characterised by sequencing of nuclear (internal transcribed spacer) and mitochondrial (cytochrome c oxidase subunit 2) markers. The results revealed diverse Anisakidae community consisting of: Anisakis pegreffi, Anisakis simplex (s.s.), Anisakis typica and Hysterothylacium aduncum. This is the first report of A. typica in Adriatic Sea, and also the first record of this species in T. thynnus as host in Mediterranean Sea. Molecular identification of H. aduncum found in co-infection with Anisakis larvae type I expands our knowledge of the occurrence of these taxa in the Adriatic Sea. Zoonotic Anisakidae worms found in fish from the Adriatic Sea could represent a risk to acquire parasitic infection/allergies in Croatia.

The study on medium size river Sutla in Croatia indicated considerable water contamination at specific sites during the baseflow period, probably associated to low flow-rate (0.73-68.8 m3 s(-1)), and consequently low dilution capacity of this river. Various aspects of contamination were observed: increased conductivity to 1,000 microS cm(-1), decreased dissolved oxygen level to 50%, 4-5 degrees C increased water temperature, increased concentrations of several dissolved trace elements (e.g., maximal values of Li: 45.4 microg l(-1); Rb: 10.4 microg l(-1); Mo: 20.1 microg l(-1); Cd: 0.31 microg l(-1); Sn: 30.2 microg l(-1); Sb: 11.8 microg l(-1); Pb: 1.18 microg l(-1); Ti: 1.03 microg l(-1); Mn: 261.1 microg l(-1); and Fe: 80.5 microg l(-1)) and macro elements (e.g., maximal values of Na: 107.5 mg l(-1); and K: 17.3 mg l(-1)), as well as moderate or even critical fecal (E. coli: 4,888 MPN/100 ml; total coliforms: 45,307 MPN/100 ml; enterococci: 1,303 MPN/100 ml) and organic pollution (heterotrophic bacteria: 94,000 cfu/ml). Although metal concentrations still have not exceeded the limits considered as hazardous for aquatic life or eventually for human health, the observed prominent increases of both metal concentrations and bacterial counts in the river water should be considered as a warning and incentive to protect the small and medium size rivers from the future deterioration, as recommended by EU Water Framework Directive.

Hydrostatic pressure or thermal shocks were administered to coho salmon eggs 20, 40, 60 and 80 min post-insemination. Pressure shocks ranged from 6.2 to 8.4×104 kPa, and lasted 4 min. Thermal shocks ranged from 24 to 28°C, and were applied for durations of 10 and 20 min. Triploid induction rate for each treatment group was evaluated by measurement of erythrocyte DNA content using flow cytometric analysis. Triploid rates of 100% were produced by pressure shocks of 6.9–8.3×104 kPa for eggs 20 min post-fertilization and by thermal trauma of 28°C for 20 min, 20 min after fertilization. Survival was inversely correlated with the intensity of treatments. Thus, triploid yields calculated as the product of the triploid rate and the numbers surviving to hatch, expressed as a percentage of the number of eggs originally present in the groups, were highest with a hydrostatic pressure treatment of 6.2×104 kPa for 4 min treatment, 20 and 40 min after fertilization or a heat shock of 26°C for 20 min treatment, 20 min after fertilization.

Summary The health status of rudd (Scardinius erythrophthalmus hesperidicus H.) in Lake Vrana, the largest Croatian karstic lake, was evaluated. Studies comprising parasitological, haematological and bacteriological surveys were conducted over a 2-year period. Parasitological examination revealed a light infestation of 27% of the examined fish, mostly Dactylogyrus and Ichthyophthirius species. Haematological studies showed that haematocrit values were lower than the physiological limit. A haematocrit coeAcient of correlation in all research periods was higher than 15%, indicating that the majority of fish in the study were susceptible to the development of bacterial and other diseases. Indeed, a diverse array of bacteria were isolated from rudd, mainly Flavobacterium spp. and Aeromonas spp., but also some specific fish pathogens, notably Pasteurella piscicida, Yersinia ruckeri, and Edwardsiella ictaluri, were identified. Under stress conditions, detected bacterial species can give rise to disease outbreaks.

The karyotype and major ribosomal sites as revealed by silver staining and CMA3-fluorescence of Croatian leuciscine cyprinid endemic to Cetina River, Telestes ukliva were studied. The diploid chromosome number was invariably 2n = 50. Karyotype consisted of eight pairs of metacentric, 13 pairs of submetacentric and four pairs subtelocentric chromosomes. The largest chromosome pair of the complement was subtelocentric, which is a characteristic cytotaxonomic marker for all representatives of the cyprinid lineage Leuciscinae. The nucleolar organizer regions (NORs) were detected in the telomeres of two pairs of medium-sized submetacentric chromosomes. Staining with CMA3 revealed four positive signals that corresponded to NOR sites. No heteromorphic sex chromosomes were found. The karyotype pattern of T. ukliva is nearly identical to that found in most other representatives of the Eurasian leuciscine cyprinids, while the multiple NOR phenotype appears to be more derived as opposed to a uniform one NOR-bearing chromosome pair, ubiquitous in this group.

Razvoj akvakulture, pa tako i tova tuna, u posljednjim je desetljećima, povećao zanimanje javnosti za moguće utjecaje na okoliš. Istraživanja utjecaja uzgoja gospodarski važnih ribljih vrsta su brojna, ali ne i u pogledu procjena mikrobioloških utjecaja, a napose ne u kontekstu tova tuna. Cilj ovog istraživanja bio je procijeniti utjecaj tova tuna (Thunnus thynnus) na okoliš na temelju praćenja mikrobioloških pokazatelja kakvoće morske vode na tovilištu tuna u srednjem Jadranu, te ostvarene rezultate usporediti s rezultatima istraživanja na tovilištu tuna u Mediteranu. Kakvoća morske vode analizirana je na dva lokaliteta, pri čemu je jedan lokalitet bio na tovilištu tuna, a drugi na udaljenosti oko 500m od tovilišta i poslužio je kao tzv. kontrolna točka. Uzorkovanje je provedeno u dvije sezone (proljeće/jesen), u 2007. i 2008. godini. Tijekom ovog istraživanja, analizirani su osnovni fizikalno-kemijski (temperatura, salinitet, prozirnost) i mikrobiološki parametri (ukupan broj bakterija, Vibrio, ukupni koliformi, Escherichia coli, fekalni koliformi, enterokoki) kakvoća morske vode. Za mikrobiološku analizu uzorci morske vode serijski su razrijeđeni s PBS otopinom (Merck). Za određivanje ukupnog broja heterotrofnih bakterija, uporabljene su metodom širenja razmaza dvije podloge, Marine agar (BBL) i Trypticase soy agar (BBL) s dodatkom 1% NaCl-a (Kemika), te supstrat metoda s uporabom SimPlate testa (IDEXX), sve u duplikatu. Za određivanje broja ukupnih koliforma i E. coli, odnosno enterokoka uporabljeni su supstrat testovi Colilert (IDEXX), odnosno Enterolert (IDEXX) u duplikatu. Utvrđene vrijednosti broja heterotrofnih bakterija, koje ukazuju na utjecaje tovilišta u vidu eutrofikacije mora, ukupno su manje od vrijednosti utvrđenih u istraživanjima poduzetim na tovilištima tuna u Mediteranu. Broj E. coli i enterokoka nije značajno različit između tovilišta i kontrolne točke, ali su te vrijednosti veće u odnosu na dosadašnja istraživanja na sličnim tovilištima u Mediteranu. Povećani broj enterokoka u uzorcima mora, i oko tovilišta, najvjerojatnija je indirektna posljedica nepravilne tehnologije hranidbe, koja privlači kolonije galebova. Ovi rezultati ukazuju da promjene kakvoće morske vode, a u svezi s tovom tuna, ovise o širem ekološkom kontekstu. Stoga, smještaj i procjenu utjecaja tova na okoliš valja razmatrati u sklopu šireg ekološkog konteksta područja., Aquaculture including tuna fattening, increased public interests in the possible impact on the environment in recent decades. Influences of cultivation of economically important fish species are numerous, but not in regard to assessment of microbial influences, particularly in the context of tuna. The aim of this study was to assess the effects of tuna fattening (Thunnus thynnus) in the sense of the environmental monitoring of microbial indicators of seawater quality at tuna farm in the central Adriatic, and to compare the achieved results with the results of research on a tuna farm in the Mediterranean. The quality of sea water was analyzed at two sites; one site was at the tuna farm, and the other at a distance of about 500m from the farm, the control point. Sampling was conducted in two seasons (spring / autumn), in 2007 and 2008. During this study, the basic physico-chemical (temperature, salinity, transparency) and microbiological parameters of seawater quality (total number of bacteria, Vibrio, total coliforms, Escherichia coli, faecal coliforms, enterococci) were analyzed. For microbiological analysis of seawater; samples were serially diluted with PBS solution (Merck). To determine the total number of heterotrophic bacteria, spread plate method was used on Marine agar (BBL) and Trypticase soy agar (BBL) supplemented with 1% NaCl-a (Kemika), as well as substrate method using SimPlate test (IDEXX), all in duplicate. To determinate total coliforms and E. coli, and enterococci substrate tests Colilert (IDEXX) and Enterolert (IDEXX) were used in duplicate. Number of heterotrophic bacteria, which indicate the effects of the farm on the eutrophication, was less than the number determined in similar study in the Mediterranean. Number of E. coli and enterococci were not significantly different between farm and control point, but these values are higher than those from research in the Mediterranean. Increased numbers of enterococci, on the farm and control point, were indirect consequence of improper nutrition technology, which attracted a colony of seagulls. These results suggest that changes in seawater quality in relation to tuna fattening, depend on the broader ecological context. Therefore, location and environmental impact of tuna fattening on the environment must be considered within the broader context of ecological areas.

Rijeka Sutla granična je rijeka između Hrvatske i Slovenije. Ukupna joj je dužina oko 91 km, od toga u Hrvatskoj 89 km. Istraživanje mikrofitobentosa provedeno je na šest lokaliteta duž rijeke Sutle. Uzorci su sabirani s određene površine karakterističnih staništa. Uz sakupljanje fitobentosa, izmjereni su osnovni fizikalno–kemijski pokazatelji: temperatura vode, pH i količina otopljenog kisika u vodi. Temperatura vode mijenjala se sukladno promjenama temperature okolnoga zraka i dubine rijeke, a kretala se od 5,1 do 6,3 ºC. pH–vrijednost bila je između 7,77 i 8,14, a koncentracija otopljenog kisika (mgO2 /L) na šest lokaliteta bila je između 8,6 i 14,9 mg/L. U kvalitativnom sastavu mikrofitobentosa utvrđeno je 87 vrsta mikrofita koji su pripadali sistematskim skupinama: Bacteriophyta, Cyanobacteria i Chrysophyta (Bacillariophyceae i Xanthophyceae). Najbrojnija su skupina bile dijatomeje ili Bacillariophyceae (76 vrsta ili 88,3 %), s dominantnim vrstama rodova Achnanthes, Cocconeis, Cymbella, Gomphonema, Navicula, Nitzschia i Surirella. Relativno malim brojem vrsta bila je zastupljena skupina Cyanobacteria (9 vrsta ili 10 %), a prevladavale su nitaste alge roda Phormidium. Od ukupnoga broja utvrđenih vrsta u mikrofitobentosu, 73 vrste ili 84 % bile su pokazatelji saprobnosti. Najviše je bilo beta–mezosaprobnih indikatora. Na osnovi indikatorskih vrijednosti nađenih vrsta u mikrofitobentosu na šest lokaliteta, P–B indeks saprobnosti kretao se od 1,8 do 2,0. Ove vrijednosti upućuju na kakvoću vode II. vrste na istraživanom dijelu rijeke Sutle., The Sutla river is a river along Croatian/Slovenian border. Its length is about 91 km, out of which 89 km in Croatia. Microphytobenthos investigations have been performed at six locations along the Sutla river on Croatian territory. Samples were collected from specific areas of characteristic habitats. Beside sample collection, basic physico–chemical parameters were measured: water temperature, pH values and quantity of water dissolved oxygen. Water temperature changed depending on air temperature and the depth of the river, ranging from 5.1ºC to 6.3ºC. pH values were between 7.77 and 8.14, and dissolved oxygen concentrations (mg/L O2) at the six locations ranged between 8.6 mg/L and 14.9 mg/L. Quantitative microphytobenthos composition comprised 87 microphythic species belonging to the systematic groups of Bacteriophyta, Cyanobacteria and Chrysophyta (Bacillariophyceae and Xanthophyceae). The most numerous group were the diatoms or Bacillariophyceae (76 species or 88.3%), with dominance of the species of the genera Achnanthes, Cocconeis, Cymbella, Gomphonema, Navicula, Nitzschia and Surirella. The group Cyanobacteria was represented with relatively small number of species (9 species or 10%), with the dominance of filamentous algae belonging to the genus Phormidium. From the total number of the determined microphytobenthic species, 73 species or 84% were indicators of saprobity. Most of them were beta–mezosaprobic indicators. Based on the indicator values of determined microphytobenthic species at six investigated locations, P–B saprobity index was in the range from 1.8 to 2.0. These values suggested that the water at the investigated part of the Sutla river belonged to the second class of Croatian Water Quality Directive.

Voda kao životni okoliš organizama koji u njoj žive svojom kvalitetom utječe na njihovo zdravstveno stanje. U dostupnoj je literaturi malo podataka o pokazateljima zdravstvenoga stanja klena, osobito iz rijeke Save. Zbog toga je svrha ovog istraživanja bila procijeniti zdravstveno stanje klena kao pokazatelja kvalitete vode, te odrediti odnos histoloških i parazitoloških pokazatelja prema broju bakterija u vodi. Istraživanje je provedeno na ukupno 289 klenova s pet postaja od slovensko–hrvatske granice do utoka rijeke Une u Savu. Uzorci organa klena uzeti su za parazitološku i histološku pretragu. Istodobno je uzorkovana voda i određen broj heterotrofnih i koliformnih bakterija. Razlike u količini bakterija u vodi bile su izražene među pojedinim postajama. Najviša je vrijednost utvrđena na postaji Oborovo, a najniža na postaji Otok Samoborski. Intenzitet histoloških promjena bio je u korelaciji s mikrobiološkom kakvoćom vode. U svim uzorkovanim organima utvrđen je barem jedan histološki pokazatelj u visokoj korelaciji s mikrobiološkom kakvoćom vode. Od parazitarnih invazija samo su ihtioftirijaza i trihodinijaza pokazale visoku negativnu korelaciju s mikrobiološkom kvalitetom vode. U sklopu parazitološke pretrage primijenjivane su i molekularne metode u svrhu identifikacije crijevnih parazita iz grupe Acanthocephala., Water quality has infuence on the health status of the organisms living in it. In the available literature, there is only a few reports on the health status of chub, particularly in the Sava river. The aim of this study was to estimate health status of chub as indicator of water quality. Furthermore we aimed at relating fish histology and parasitology to the number of bacteria in the water. We sampled 289 chubs for parasitological and histological examination. Fish were sampled on five sites from slovenian–croatian border through to the inflow of river Una in Sava. At the same time, water was sampled for the counting of coliforms and heterotrophic bacteria. The difference in the number of bacteria in the water was pronounced at different locations, with Oborovo and Otok Samoborski having the highest and the lowest values respectively. The intensity of histological parameters was in correlation with the number of bacteria in water. At least one histological parameter highly correlating with the water quality was found in every organ. Regarding parasite invasions, only ichthyophthiriasis and trichodiniasis showed high negative correlation with microbiological quality of the water. As part of parasitological examination, molecular methods were used for the identification of Acanthocephala.

In this paper we describe the bacterial community associated with salmonids from the Krka River. Diversity analysis demonstrated that majority of the recovered bacteria were related to Aeromonadaceae group. Bacterial analysis also revealed the presence of Shigella spp. and Pseudomonas fluorescens. Isolation of Aeromonas salmonicida from trout, presents first isolation of this bacteria Croatian rivers.

Rast akvakulturne proizvodnje potiče i povećano zanimanje i brigu za njezin utjecaj na okoliš. Tijekom salmonidne proizvodnje na ribogojilištu u vodu se unose određene količine organske tvari. Količina visokoproteinske hrane i metabolički riblji proizvodi utječu na kakvoću vode koja se koristi u uzgoju riba. Bakterije imaju ključnu ulogu u razgradnji organske tvari. Prekomjerno obogaćivanje nutrientima dovodi do osiromašenja kisikom kroz mikrobiološku oksidaciju organske tvari i takvi uvjeti nepovoljno utječu na ukupnu kakvoću vodenog ekosustava. Cilj ovih istraživanja bio je procijeniti utjecaj salmonidnog uzgajališta na kakvoću vode na osnovi praćenja dinamike bakterijske populacije i fitobentosa u odnosu na fizikalno-kemijske parametre vode (temperatura, kisik, pH itd.). Istraživanja su provedena na pastrvskom ribogojilištu s godišnjom proizvodnjom oko 100 t ribe, kod protoka vode od oko 300 L/s. Uzorci vode za mikrobiološku obradu i uzorci fitobentosa sakupljeni su na 8 lokaliteta uz istovremeno mjerenje fizikalno-kemijskih parametara elektrosondom. Lokaliteti uzorkovanja odabrani su tako da se obuhvate sve uzgojne faze salmonidnog ribnjaka: ulaz u ribnjak, 4 lokaliteta na ribnjaku po pojedinim fazama uzgoja, na izlazu iz ribnjaka, te uzvodno i nizvodno od ribnjaka. Za određivanje ukupnog broja bakterija (cfu) uzorci vode su serijski razrijeđeni sa sterilnom Ringerovom otopinom pH 6,0 (Pliva) i inokulirani metodom razlijevanja po podlozi na mediju za izolaciju sukladno EN ISO 6222:1999. Nakon inkubacije do 5 dana na 22°C, prebrojane su izrasle bakterijske kolonije i rezultati su izraženi za 1 ml vode (cfu/ml). Dobiveni uzorci bentosa stavljeni su u boce i konzervirani s 4 %-tnim formalinom. Mikroskopska obrada uzoraka obavljena je u laboratoriju, pomoću mikroskopa «Opton» povećanja 12,5 x10; 12,5 x 25 i 12,5 x 40. Relativna zastupljenost vrsta fitobentosa određena je po Knöpp-u, od 1 do 7, (1954), a saprobne vrijednosti indikatorskih vrsta po Wegl-u (1983). Indeks saprobnosti na osnovi indikatorskih biljnih vrsta određen je prema Pantle-Buck-u (1955), te na osnovi dobivenih vrijednosti pokazatelja kakvo_e vode, istraživani lokaliteti svrstani su u određenu vrstu, prema Uredbi o klasifikaciji voda (1998). Fizikalno-kemijske karakteristike vode na istraživanim lokalitetima ne pokazuju u pravilu značajnije razlike. Gustoća populacije aerobnih bakterija pokazuje progresivne promjene na lokacijama unutar uzgajališta, a sukladno očitovanju obogaćenja nutrijentima. Tijekom istraživanja u fitobentosu najbrojnija skupina bile su dijatomeje ili Bacillariophyceae, tipični oblici fitobentoske zajednice. Od ukupnog broja utvrđenih vrsta, više od 50% pripadalo je indikatorima saprobnosti. Indikatorske vrste pripadale su oligosaprobnom i betamesosaprobnom stupnju. Vrijednosti izračunatog indeksa saprobnosti bile su 1,7 na lokalitetima prije i nakon izlaska iz ribogojilišta, te 1,8 na izlazu iz bazena II i III, gdje se nalazi konzumna riba. Vrijednosti P-B indeksa saprobnosti upućuju na kakvoću vode istraživanih lokaliteta na I odnosno II vrstu. Ova mikrobiološka i fitocenološka istraživanja upućuju na zaključak da sam proces uzgoja riba nema većeg utjecaja na kakvoću vode. Utvrđeno je da je voda koja izlazi iz ribnjaka bolje kakvoće od vode recipijenta., Aquaculture production is undergoing a rapid expansion including an increasing interest and concern for their potential impact on the environment. Salmon fish farming releases a substantial amount of organic matter in to the water. High protein fish food and fish excretion affecting the quality of water used in fish farming. Bacteria play the major role in decomposition of the organic matter. Excessive enrichment of nutrients leads to oxygen depletion in the oxidation of the organic matter. Such conditions have negative effects on aquatic environment. The aim of this study was to estimate the impact of salmon farming on the water quality, on the basis of bacterial population and phytobenthos monitoring in relation to physico-chemical water characteristics (temperature, dissolved oxygen, pH etc.). The investigation was carried out on a rainbow trout fish farm with an annual trout production of 100 tons, and water flow of 300 l/s. Water samples for microbiological and phytobenthos analyses were collected at eight locations. Physico-chemical parameters were measured by an electrosonde at the same time. Sampling locations were determined to include all stages of salmon production: entry to the fish farm, four locations on the fish farm at different stages of salmon production, outflow from the fish farm, and locations upstream and down stream from the fish farm. Water samples for the heterotrophic bacteria enumeration (cfu) were serially diluted with Ringer solution pH 6.0 (Pliva) and inoculated by spread plate method on the solid medium according to EN ISO 6222:1999. After incubation at 22 °C for five days colonies were counted and results expressed for 1 ml of water (cfu/ml). Samples for benthos analysis were collected in the bottles and conserved with 4 % formaldehyde. Samples were examined under ‘’Opton’’ light microscope with magnifications 12.5x10; 12.5 x 25 and 12.5 x 40 in the laboratory. Relative phytobenthos assemblage determination from 1 to 7 was performed according to Knöpp (1954), and saprobity measures of indicators species according to Wegl (1983). Index of saprobity was determined according to Pantle-Buck (1955). Examined locations were classified according to ‘’Uredba o klasifikaciji voda’’ (1998) on the basis of the obtained water quality values. Physico-chemical water characteristics were without significant differences on examined locations. Density of the heterotrophic bacteria showed progressive changes on the locations within the fish farm which was consistent with nutrient enrichment. The most frequent species in the phytobenthos were diatoms and Bacillariophyceae, which are typical forms of phytobenthic community. More than 50 % of all determined species belonged to saprobity indicators. Indicator species were characteristic for oligosaprobic and betamesosaprobic water. Saprobity indexes were 1.7 at locations upstream and downstream of the fish farm. At the escit from 2nd and 3rd pool, with consumer fish, saprobity indexes were 1.8. These the values of P-B saprobity index indicate first and second degree water quality of the examined locations. The results of these microbiological and phytocenological examinations lead to the conclusion that fish production has insignificant local impact on the water quality.

Information on the bacterial population associated with cultured tuna are scarce, particularly for bluefin tuna (Thunnus thynnus). The aim of this work was to investigate the initial microflora of bluefin tuna in the Adriatic Sea. Four bluefin tunas weighing 6.5 to 15.0 kg were obtained from tuna farm in the Middle Adriatic. Fish samples were taken in spring and autumn from 2003 to 2005. Samples for bacterial isolation were collected from skin, gills, spleen, liver and kidney, and cultured on Tryptic soy agar (BBL) supplemented with 1 % NaCl. Isolated strains were further characterized using the API kits (bio Merieux, France) and identified by the APILAB Plus identification software (bio Merieux, France). Gram-negative bacteria from genera Moraxella, Pasteurella, Brevundimonas, Weeksella, Klebsiella and Pseudomonas dominated the microflora on examined tuna, whereas Staphylococcus was the only one Gram- positive isolated genus.

Izlovljavanje jakovske kapice (Pecten jacobaeus Linnaeus, 1758.) iz prirodnih populacija u Hrvatskoj obavlja se sporadično i neorganizirano, uglavnom kao sekundarna djelatnost pri ulovu ribe, a kontrolirani uzgoj ne postoji. Istraživanja gonadnog indeksa odraslih jakovskih kapica, prihvata ličinki, stupnja preživljenja i rasta juvenilnih stadija provedena su radi utvrđivanja mogućnosti kontroliranog uzgoja i uvođenja nove vrste u akvakulturnu proizvodnju u Hrvatskoj. Sposobnost obnavljanja populacije varira u vremenu i prostoru, ovisno o hidrografskim značajkama ušća rijeke Krke. Najpovoljnije razdoblje za kolektiranje mlađi jest ožujak/lipanj, a najpovoljnija dubina 6–8 m iznad morskoga dna. Vertikalni prihvat mlađi obrnuto je proporcionalan salinitetu. Najniži salinitet na kojem su utvrđeni prihvat i preživljenje ličinki iznosi oko 20 x 10–3, a uspješno kolektiranje moguće je uglavnom pri salinitetima višima od 30 x10–3. U pokusnom uzgoju jakovske kapice starosti 6 mjeseci, srednje veličine 33,1±5,5 mm i srednje mase 8,8 g, nakon godinu dana, postigle su srednju veličinu 83,8±9,5 mm i srednju masu 80,6 g. Ukupni mortalitet za vrijeme istraživanja iznosio je 33%., The capture of scallops (Pecten jacobaeus Linnaeus, 1758) from natural populations in Croatia is sporadic and unorganizaed, as a secondary activity in fishing, while no controlled cultivation of this species exists. Research of the gonad index of adults, larvae attachment, survival and growth of juvenile scallops was conducted with the aim of determining the possibility of the establishment controlled cultivation and in this way the introductes new species in Croatia’s aquaculture production. The capability to replace the population varies in time and space, and is dependent upon the hydrographical characteristics of the river mouth. The most favourable period for collecting juveniles is March–June between 6 to 8 meters depth above the sea bottom. The vertical attachment of juveniles is inversely related to salinity. The lowest salinity for attachment and juveniles survived was about 20 x 10–3, while successful collection is possible at salinities above 30 x 10–3. Shellfish aged 6 months, mean size of 33.1±5.5 mm and with a mean mass of 8.8 g reached mean sizes of 83.8±9.5 mm and mean mass of 80.6 g at one year. Total mortality during this study was 33%.

Kontrolirani uzgoj male kapice (Chlamys varia Linnaeus, 1758) u Hrvatskoj ne postoji, a iz prirodnih se populacija izlovljava sezonski, sporadično i neorganizirano. Istraživanja indeksa kondicije adultnih malih kapica, prihvata ličinki, stupnja preživljenja i rasta juvenilnih školjkaša provedena su radi utvrđivanja mogućnosti njezina kontrolirana uzgoja. Rezultatima istraživanja utvrđeno je da mala kapica ima dualni tip mriješćenja, s primarnim maksimumom u proljeće (ožujak/svibanj) i sekundarnim u jesen (listopad). Najpovoljnije razdoblje za kolektiranje mlađi jest srpanj/rujan, a najpovoljnija dubina 2–10 m iznad morskoga dna. Najniži salinitet na kojem su utvrđeni prihvat i preživljenje ličinki jest oko 20 x 10–3, a uspješno kolektiranje moguće je uglavnom pri salinitetima višima od 30 x 10–3. U pokusnom su uzgoju male kapice, srednje veličine od 16,4±2,1 do 17,6±2,2 mm i srednje mase 0,95 do 1,35 g, nakon godinu dana postigle srednju veličinu od 42,7±3,2 do 47,5±3,0 mm i srednju masu od 12,1 do 17,5 g. Ukupna smrtnost za vrijeme istraživanja iznosila je od 25 do 57%., In Croatia, the capture of scallop (Chlamys varia Linnaeus, 17583) from natural populations are sporadic and unorganized, while there is no controlled cultivation of this species. To determine the possibility of the establishment of their commercial cultivation, the research comprehended the condition index of adults, larval attachment, juvenile survival and growth in Šarina draga Bay, in a locality with dense scallop populations. The changes of condition index shoved on main spawning season in spring (March–May) and second one, but less intensive, in autumn (October). According to results, the best season for juvenile collection is recommended the period from July to September at depths between 2 to 10 m, and salinities above 30 x 10–3. Low larval settlement at 2.5 m depth is surely result of salinity decrease by Krka river intensive fresh water inflow. Shellfish mean size from 16.4±2.1 to 17.6±2.2 mm and with a mean mass from 0.95 to 1.35 g reached mean size from 42.7±3.2 to 47.5±3.0 mm and mean mass from 12.1 to 17.5 g in one year. Total mortality during this study was ranged from 25 to 57%.

U dostupnoj literaturi nema podataka o strukturi bakterijske flore kod ličinki i mlađa kalifornijske pastrve u prvim danima života. Zadatak je našeg rada bio pratiti bakterijsku floru od 3. do 8. tjedna života. Tijekom 35 dana pokusa utvrđivana je bakterijska flora kalifornijske pastrve uz praćenje fizikalno–kemijskih parametara kvalitete vode i njezina utjecaja na zdravlje. Uzorci za bakteriološku pretragu uzimani su sa škrga, srca i područja bubrega i inokulirani na podloge. Bakterijske su kolonije pregledane makroskopski, razmazi su obojeni po Gramu, a zatim su načinjeni biokemijski testovi. Pri identifikaciji primjenjivan je APILAB Plus program (bio Mérieux, France). Bakterijska flora ličinki i mlađa kalifornijske pastrve mijenjala se je sa starošću. Fizikalno–kemijske karakteristike vode kretale su se unutar optimalnih vrijednosti. Od inokuliranog materijala najviše je izraslo bakterijskih kolonija s izolata škrga (64,4 %), zatim iz srca (21,8 %) i iz područja bubrega (13,8 %). Bakterijsku floru ličinki u inkubatoru većim su dijelom tvorile gram–pozitivne bakterije (64,7 %), i to vrste: Renibacterium salmoninarum (23,5 %), Lactobacillus spp. (11,8 %), Staphilococcus spp. (17,6 %) i Corynebacterium aquatile (11,8 %). Premještanje ličinki iz inkubatora u bazene reduciralo je bakterijsku floru (–66,7 %) nakon 45 minuta boravka u bazenu. Gram–negativne bakterijske vrste koje su u ličinki u inkubatoru bile zastupljene u niskom postotku (35,3 %), premještanjem ličinki u bazene postale su dominantne i činile su više od 95 % bakterijske flore ličinki, odnosno mlađa kalifornijske pastrve. Flavobacterium, Acinetobacter i Yersinia bili su pretežiti gram–negativni rodovi u ličinki u inkubatoru, dok su Aeromonas, Pseudomonas, Flavobacterium i Pasteurella glavni izolati iz ličinki, odnosno mlađa u bazenima do kraja pokusa. Bakterijska flora ličinki u inkubatoru većinom se sastoji od gram–pozitivne flore, a u ličinaka i mlađa kalifornijske pastrve u bazenima od gram–negativne bakterijske flore., There are no information in available literature about the structure of bacterial flora in rainbow trout larvae and fry in the first days of their lives. The objective of our work has been to follow bacteroflora between the third and the eighth week of their lives. During 35 days of experiment bacteroflora of rainbow trout has been examined, along with following physico–chemical characteristics of water quality as well as it’s influence on health. Samples for bacteriological examination were taken from gill, heart and kidney areas and innoculated on the plates. Bacterial colonies were examined macroscopically, slides with Gram staining, and afterwords biochemical tests were performed. For identification, APILAB Plus programme (bio Mérieux, France) was used. Bacterial population of rainbow trout larvae and fry changed in dependence with their age. Physico–chemical characteristics of water ranged within optimal values. Most of bacterial colonies originated from gill isolates (64,4 %), than from heart (21,8 %) and kidney areas (13,8 %). The bacterial flora of larvae in incubator was composed mostly of Gram–positive bacteria (75,1 %), genera: Renibacterium (25 %), Lactobacillus (16,7 %), Staphilococcus (16,7 %) and Corynebacterium (16,7 %). The transfer of larvae from incubator into the pools resulted in reducing bacterial flora (–66,7 %) after 45 minute stay in the pool. Gram–negative bacteria, which have been represented in larvae in incubator with low percent (24, 9 %), after the transfer of larvae to the pools became dominant and represented more than 95 % of rainbow trout larvae and fry bacterial flora. Flavobacterium, Acinetobacter and Yersinia were the predominant Gram–negative genera in larvae in incubator, whereas Aeromonas, Pseudomonas, Flavobacterium and Pasteurella were the main isolates from rainbow trout larvae and fry until the end of experiment. Bacterial flora of larvae in incubator mostly consists of Gram–positive bacteria, whereas in rainbow trout larvae and fry in pools Gram–negative bacterial flora prevail.

A complex survey has been conducted in the norhern Adriatic Sea over a period of one year that included comparative parasitological, bacteriological, virological, histological, and biochemical studies of the cultured and wild sea bass (Dicentrarchus labrax L.). The results showed that parasite infestations were due mainly to ectoparasitic monogenea Diplectanum sp. in both cultured and wild sea bass. Philometra sp. and Lerneae sp. were detected in wild sea bass while Triaenophorus sp. and Eimeria sp. were found in reared sea bass. Bacterial pathogens isolated from both reared and wild sea bass belong to Pseudomonadaceae (Pseudomonas sp., P. fluorescens) and unknown Gram-negative bacteria. Moraxellaceae (Acinetobacter sp.), Vibrionaceae (Chewanella putrefaciens) , Enterobacteriaceae (Pantoea agglomerans) and Flavobacterium sp. were isolated from reared fish only. Virological examinations were negative. Histological analysis revealed "fatty liver" (fatty infiltration and degeneration) in the cultured fish. Triglyceride, cholesterol and glucose levels were higher in cultured sea bass (2.55 +- 1.77 mmol/l, 3.68 +-1.43 mmol/l and 9.97 +- 3.33 mmol/l, respectively) than in wild fish (0.80 +- 0.57 mmol/l, 2.95 +- 0.77 mmol/l and 4.79 +- 3.29 mmol/l, respectively). The present paper contributes to establishing a relationship between disease and pathophysiological conditions in wild and cultured fish.