Effects of environmental parameters on spatial and temporal distribution of marine microbial communities in the southern Black Sea.

The Black Sea is a unique environment with strong and permanent vertical stratification, with a thin layer of oxic zone above and a permanent anoxic zone below. Few high-throughput genomic surveys have been conducted to examine microbiota in the Black Sea. Yet, there is no study on the seasonal and vertical variation in microbial community compositions, driving forces and mechanisms of community assembly. In this study, seasonal, vertical, and spatial microbial assemblages were studied in terms of diversity, abundance, and community structure using 16S rRNA metabarcoding. 16S rRNA metabarcoding confirmed seasonal changes in microbial communities and the presence of distinct microbial groups among different water layers. Taxa belonging to Cyanobiaceae contributed a large fraction of the total biomass and were the most abundant autotrophic bacteria found across the whole water column, including hydrogen sulfide-containing anoxic zone. Temperature, salinity, water density, conductivity, light, chlorophyll-a, O2, NO3, NH3, PO4, Si, and H2S had a significant influence on the vertical bacterial community assemblages. The copper mine discharge system at 180 m did not affect microbial community structure and composition. Temperature seemed to be a primary factor in the variance between shallow depths. In conclusion, the lack of light, low dissolved oxygen levels, and low temperature do not restrict microbial diversity, as proven by the higher diversity observed in deeper zones. Wastewater in Black Sea region may be discharged into the Black Sea to depth of 180 m or deeper without impacting microbial ecology.

Comparative susceptibilities and immune-related gene expressions of brown trout strains and their hybrids infected with Lactococcus garvieae and Yersinia ruckeri.

Brown trout are polymorphic salmonid species, and it is of importance to investigate whether hybridization affects disease resistance. In this study, susceptibility of brown trout (Salmo trutta Abant, Anatolian, Black Sea, and Caspius) strains and their hybrids to Lactococcus garvieae and Yersinia ruckeri as well as their immune-related gene expression profiles were studied. Results indicated that reciprocal hybridization did not affect disease resistance in brown trout strains. Purebred Black Sea strain of brown trout was the most resistant group against Y. ruckeri, followed by other Black Sea strain hybrids. On the other hand, purebred Anatolian strain was the most resistant group to L. garvieae, followed by other Anatolian strain hybrids. Expression pattern of target genes differed in families, but the overall gene expression was comparatively high in Y. ruckeri infected families. Upregulations were mainly significant at 7 and 28 d post infection while marginal regulations were observed 8 h after infection. Disease resistance status of strains was supported by high expression of immune-related genes such as major histocompatibility complex class I (MHC-I), immunoglobulin light chain (IgL), and antioxidant- and hemoglobin-related gene expression. Therefore, our findings suggest that Black Sea and Anatolian strains could be used to develop fish stock that are resistant for yersiniosis and lactocaccosis, respectively.

Genetic and molecular evidence that brown trout Salmo trutta belonging to the Danubian lineage are a single biological species.

We tested for reproductive isolation between Salmo trutta abanticus, S.t. labrax, S.t. caspius and S.t. fario by conducting crosses to produce F1 and F2 offspring. We also estimated the extent of genetic divergence between all three entities by examining sequence variation across the coI, d-loop and cytb mitochondrial genes. All of the F1 cross-types were successfully produced. After 2 years of culturing, F2 generation were produced as well. Fertilization, hatching and survival rates and hatching performance of F1 and F2 generations were evaluated. F2 generation had similar performance to their parent. Fertilization, hatching, larval survival rate and hatchery performance of F1 and F2 generation were similar except pure bred F2 S.t. abanticus. Purebred F1 individuals shared similar coloration patterns and spots with their parents but direction of the hybridization appeared to be decisive on morphology of hybrids. Some of the hybrids exhibit different morphological characters than their parents. Based on partial alignments of the three genes, phylogenetic analysis showed that these S. trutta are gathering within the same clade and appeared as monophyletic group. We found that there were some morphologic and genetic variation among S. trutta subspecies but the degree of variation does not warrant species level recognition. These findings indicate that the four subspecies constitute a single biological entity, corresponding to different morphs of the Danubian lineage. We therefore recommend that S. trutta belonging to Danubian lineage in Turkey be referred to as Salmo trutta and that strains be named according to location, such as Abant, Caspian, Black Sea and Anatolian.

Antimicrobial agents, triclosan, chloroxylenol, methylisothiazolinone and borax, used in cleaning had genotoxic and histopathologic effects on rainbow trout.

Triclosan (TRC), chloroxylenol (PCMX) and methylisothiazolinone (MIT) have been commonly used as an antimicrobial in soaps while borax (BRX) is used in household cleaning. After using these chemicals, they are washed down drains and getting into the aquatic ecosystem in which they may affect aquatic living organisms. In the present study, the chronic effects of TRC, PCMX, MIT and BRX on genotoxicity, gene expression and histopathology of rainbow trout (Oncorhynchus mykiss) were evaluated for 40 days under semi static condition. The comet assay results indicated that MIT, TRC and PCMX caused significant DNA damage to erythrocytes of the fish. Transcription of SOD, GPX1, GPX2, GSTA, HSP90BB, HSP90BA, CAT, and HSC70A genes were significantly regulated as a result of TRC, PCMX, MIT, and BRX exposure except PCMX exposed GSTA gene. Histological lesions were detected in gills, spleen liver, and trunk kidney of the fish. Lamellar fusion, hyperplasia and epithelial necrosis in gills, melanomacrophage centers and splenic necrosis in spleen, pyknotic nucleus, fat vacuoles, necrotic hepatocytes in liver, cloudy swelling in the tubules, renal tubule epithelial cells degeneration, glomerular capillaries dilation and glomerulus degeneration in kidney, were observed. Our study demonstrates the chronic toxic effect of TRC, PCMX, MIT, and BRX is high in rainbow trout. Therefore, we should be more careful when using these chemicals for cleaning in order to protect aquatic environment.

Protection of rainbow trout against yersiniosis by lpxD mutant Yersinia ruckeri.

Yersinia ruckeri is a Gram negative bacteria causing yersiniosis in freshwater and marine fish. Lipid A, important for pathogenesis of Gram negative bacteria, biosynthesis pathway requires nine enzyme catalyzed steps. Although there are nine genes encoding lipid A biosynthesis in bacteria, biosynthesis of lipopolysaccharides relies on lpxD gene that encodes the third pathway enzyme. The roles of LpxD in Y. ruckeri virulence have not been studied. In the present study, in-frameshift deletion of lpxD gene and their role in Y. ruckeri virulence in rainbow trout were determined. For this purpose, 92% of the Y. ruckeri lpxD genes were deleted by homologous recombination. After running in SDS-PAGE and staining with silver stain, no LPS was detectable in the Y. ruckeri ΔlpxD mutant. Virulence and immunogenicity of the Y. ruckeri ΔlpxD mutant (YrΔlpxD) were determined in rainbow trout. Rainbow trout immunized with YrΔlpxD with immersion, or intraperitoneal injection method displayed superior protection (relative percentage survival ≥ 84%) after exposure to wild type Y. ruckeri. In conclusion, our results indicated that deletion of the lpxD gene causes significant attenuation of Y. ruckeri in rainbow trout, and LPS deficient YrΔlpxD could be used as a live attenuated vaccine against Y. ruckeri in rainbow trout. This vaccine can protect fish and it can be applied to fish with different methods such as immersion or injection.

Succinate dehydrogenase mutant of Listonella anguillarum protects rainbow trout against vibriosis.

Listonella anguillarum is a Gram-negative facultative anaerobic rod causing hemorrhagic septicemia in marine and rarely in freshwater fish. Succinate dehydrogenase (SDH) plays an important role in the tricarboxylic acid (TCA) cycle by oxidizing succinate to fumarate while reducing ubiquinone to ubiquinol. Recent studies indicate that central metabolic pathways, including the TCA cycle, contribute to bacterial virulence. However, the role of SDH in L. anguillarum virulence has not been studied. Here, we report in-frame deletion of the succinate dehydrogenase iron-sulfur protein (SDHB) and its role in L. anguillarum virulence in rainbow trout. To accomplish this goal, upstream and downstream regions of the L. anguillarum sdhB gene were amplified in-frame and cloned into a suicide plasmid. The chromosomal sdhB gene of L. anguillarum was deleted by homologous recombination. Virulence and immunogenicity of the L. anguillarum ΔsdhB mutant (LaΔsdhB) were determined in rainbow trout. Results show that LaΔsdhB was highly attenuated in rainbow trout, and fish immunized with LaΔsdhB displayed high relative survival rate after exposure to wild type L. anguillarum. These findings indicate SDH is important in L. anguillarum virulence in rainbow trout, and LaΔsdhB could be used as an immersion, oral, or injection vaccine to protect rainbow trout against vibriosis.

Occurrence of antibiotic resistance genes in culturable bacteria isolated from Turkish trout farms and their local aquatic environment.

Antibiotic resistance and presence of the resistance genes were investigated in the bacteria isolated from water, sediment, and fish in trout farms. A total of 9 bacterial species, particularly Escherichia coli, were isolated from the water and sediment samples, and 12 species were isolated from fish. The antimicrobial test indicated the highest resistance against sulfamethoxazole and ampicillin in coliform bacteria, and against sulfamethoxazole, imipenem, and aztreonam in known pathogenic bacteria isolated from fish. The most effective antibiotics were rifampicin, chloramphenicol, and tetracycline. The multiple antibiotic resistance index was above the critical limit for almost all of the bacteria isolated. The most common antibiotic resistance gene was ampC, followed by tetA, sul2, blaCTX-M1, and blaTEM in the coliform bacteria. At least one resistance gene was found in 70.8% of the bacteria, and 66.6% of the bacteria had 2 or more resistance genes. Approximately 36.54% of the bacteria that contain plasmids were able to transfer them to other bacteria. The plasmid-mediated transferable resistance genes were ampC, blaCTX-M1, tetA, sul2, and blaTEM. These results indicate that the aquatic environment could play an important role in the development of antibiotic resistance and the dissemination of resistance genes among bacteria.

Effects of chronic carbosulfan exposure on liver antioxidant enzyme activities in rainbow trout.

Catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), and selenium-dependent glutathione peroxidase (Se-GPx) activities in liver of rainbow trout (Oncorhynchus mykiss; 116.88±21.69g) were evaluated after exposing fish to sublethal concentrations (25µg/L) of carbosulfan in flow-through tanks for 60 days. During the experiment activities of CAT, SOD, GST, and Se-GPx and histopathological effects were determined once a week and once at the end of the 21 days of recovery period. All enzymes were affected by carbosulfan when compared to control fish. Fish had intracellular oedema, cell necrosis, pycnotic nucleus, and increase of sinusoidal space in the liver. After 21 days of the recovery period, all enzyme activities had returned to control levels and fish had no histological lesions in liver. Therefore all the changes observed during exposure were reversible. Results indicate that the liver CAT, SOD and GST enzymes are highly sensitive to carbosulfan as their activities altered significantly, suggesting they could be useful in predicting sublethal pesticide toxicity and useful as an indicator for assessment of pesticides in contaminated water.

Assessment of acute toxicity and histopathology of the fungicide captan in rainbow trout.

Acute toxicity of the fungicide, captan, to juvenile rainbow trout was evaluated under static-renewal test condition. Actual concentrations of captan ranged from 0.05 to 1.00 mg/L. The concentrations of captan that killed 50% of the rainbow trout (3.11±0.8 g) within 24 (24 h; LC(50)), 48, 72 and 96 h were 0.57±0.09, 0.49±0.10, 0.44±0.11 and 0.38±0.13 mg/L (95% confidence limits), respectively. None of the unexposed control fish died and the first fish died 6 h after exposure to captan (≥0.65 mg/L). Hypertrophy, separation of epithelium from lamellae, lamellar fusion, and epithelial cell necrosis were observed on captan exposed fish. Gills also had scattered areas of focal lamellar hyperplasia. Fish exposed to fungicide had inflammation and necrosis in liver, trunk kidney and spleen. In order, the most affected organs were gill, trunk kidney and liver.

Influence of bioassay volume, water column height, and octanol-water partition coefficient on the toxicity of pesticides to rainbow trout.

Effects of water volume and water column height on toxicity of cypermethrin, carbaryl, dichlorvos, tetradifon, maneb, captan, carbosulfan endosulfan and HgCl2 to juvenile rainbow trout (Oncorhynchus mykiss, 3.2 ± 0.7 g) were evaluated in different glass aquaria under static conditions. When fish were exposed to the chemical compounds in 23 cm water column height (25 L), their mortality ranged between 0% and 58%. At the same water volume, but lower water column height (9 cm), mortality of fish increased significantly and was in a range from 60% to 95%. At the same water column height, toxic effects of chemicals were significantly higher in 25 L water volume than that of 8.5 L, water except maneb which has lowest (-0.45) octanol-water partition coefficient value. Mortality rates ratio of 9 and 23 cm water column height ranged between 1.12 and 90 while mortality rates ratio of 9 and 25 L water volume ranged between 1.20 and 4.0. Because actual exposure concentrations were not affected by either water volume or water column height, we propose that increased pesticides' toxicity was related to an increase in bioassay volume, since more pesticide molecules were able to interact with or accumulate the fish. However, there seem to be no relationship between the effects of water volume, water column height and Kow value of chemicals with regard to toxicity in juvenile rainbow trout.

Effects of some pesticides on the vital organs of juvenile rainbow trout (Oncorhynchus mykiss).

Gill, trunk kidney, spleen, and liver of rainbow trout (Oncorhynchus mykiss) were examined after exposure to different sublethal concentrations of carbosulfan (25, 50 and 200 µgL(-1)), propineb (3, 6 and 24 mgL(-1)), and benomyl (2, 5 and 20 mgL(-1)) for 14 days. Lesions were observed in gill, trunk kidney, spleen, and liver of rainbow trout exposed to either concentration of pesticides. The most important lesions were determined in the highest concentrations of pesticides. Lamellar fusion, lamellar hyperplasia, epithelial lifting, vacuolization of epithelial tissue, epithelial necrosis, hypertrophy and sloughing of epithelium were observed on fish exposed to carbosulfan, propineb and benomyl. Fish had cell necrosis, degeneration and oedemas in liver, trunk kidney and spleen. None of these lesions were seen in control fish.

Histopathological changes induced by maneb and carbaryl on some tissues of rainbow trout, Oncorhynchus mykiss.

Acute toxicity of the pesticides, maneb and carbaryl, to juvenile rainbow trout were evaluated under static-renewal test conditions. Actual concentrations of maneb ranged from 0.10mg/L to 2.00mg/L and carbaryl ranged from 0.20mg/L to 3.90mg/L. The concentrations of maneb that killed 50% of the rainbow trout (3.27+/-0.9g) within 24-h (24-h; LC(50)), 48-h, 72-h and 96-h were 1.19+/-0.12, 1.04+/-0.11, 0.92+/-0.12 and 0.81+/-0.14mg/L (95% confidence limits), respectively. LC(50) values of carbaryl for 24-h, 48-h, 72-h and 96-h were 2.52+/-0.71, 2.16+/-0.63, 1.71+/-0.46 and 1.39+/-0.15mg/L, respectively. None of the unexposed control fish died and the first fish died 6h after exposure to maneb (>or=1.30mg/L), and carbaryl (>or=2.60mg/L). Lamellar edema, separation of epithelium from lamellae, lamellar fusion, swelling of the epithelial cells and epithelial cell necrosis were observed on maneb and carbaryl exposed fish. Gills also had scattered areas of focal lamellar hyperplasia. Fish exposed to pesticides had inflammation and focal necrosis in liver, trunk kidney and spleen. Maneb and carbaryl had similar histopathological lesions. In order, the most affected organs were gill, trunk kidney and liver.

Histopathological changes in rainbow trout (Oncorhynchus mykiss) after exposure to sublethal composite nitrogen fertilizers.

Subchronic toxicity of composite inorganic fertilizers such as ammonium sulfate [fertilizer A; (NH(4))(2)SO(4); 21% NH(4)-N)], composite fertilizer 15-15-15 (fertilizer B; commercial formulation: 15% NH(4)-N, 15% phosphorus, and 15% potassium oxide), and composite fertilizer 25-5-10 (fertilizer C; commercial formulation: 25% NH(4)-N, 5% phosphorus, and 10% potassium oxide) on the skin, liver, kidney, pancreas, and gills of the juvenile rainbow trout (Oncorhynchus mykiss) was studied in two-week toxicity tests under static-renewal test conditions. Fish exposed to sublethal concentrations of fertilizers did not show any behavioral abnormality compared to control groups. Histological lesions were observed in skin, gills, liver, pancreas, and trunk kidney of the fish. In the epidermis, degenerated/vacuolated epithelial cells, microcystic dilatations, and intracellular edema of mucus cell were observed. Liver had swollen and degenerated hepatocytes without losing adenoid structure. Hematopoietic tissues had necrosis and vacuolar degeneration on proximal tubules of the kidney. In order, the most affected organs were skin, liver, and kidney.

Development of multiplex PCR assay for simultaneous detection of five bacterial fish pathogens.

A multiplex polymerase chain reaction (PCR) method was designed for the simultaneous detection of the five major fish pathogens, Aeromonas hydrophila, Aeromonas salmonicida subsp. salmonicida, Flavobacterium columnare, Renibacterium salmoninarum, and Yersinia ruckeri. Each of the five pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The detection limits of the multiplex PCR was in the range of 2, 1, 1, 3, and 1CFU for A. hydrophila, A. salmonicida, F. columnare, R. salmoninarum, and Y. ruckeri, respectively. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria. The multiplex PCR assay was useful for the detection of the bacteria in naturally infected fish. This assay is a sensitive and specific and reproducible diagnostic tool for the simultaneous detection of five pathogenic bacteria that cause disease in fish. Therefore, it could be a useful alternative to the conventional culture based method.

Histopathology of rainbow trout exposed to sublethal concentrations of methiocarb or endosulfan.

Liver, spleen, trunk kidney, gills, and brain of rainbow trout (Oncorhynchus mykiss) were examined histologically after exposure to different concentrations of methiocarb (2.5 and 3.75 mg/L) or endosulfan (0.6 and 1.3 micro g/L) for 21 days. Histological recovery was also studied by maintaining the pesticide-exposed fish in a freshwater system for an additional 30 d. Lesions were not evident in liver, kidney, spleen, or brain of fish exposed to either concentration of methiocarb for 21 d. Lesions were observed in gills, liver, spleen, and trunk kidney (but not brain) of rainbow trout exposed to either concentration of endosulfan. There was no concentration-related effect observed on the histopathological lesions. After 30 days of recovery, fish had no histological lesions in gills, kidney, spleen, liver, or brain. Therefore all the changes observed during exposure were reversible.

Effects of water quality and fish size on toxicity of methiocarb, a carbamate pesticide, to rainbow trout.

The acute toxicity of methiocarb in juvenile rainbow trout (Oncorhynchus mykiss, 3.25±0.79g) was evaluated in glass aquaria under static conditions. Nominal concentrations of methiocarb in the toxicity test ranged from 1.25 to 7.50mgL(-1). The concentrations of methiocarb that killed 50% of the rainbow trout within 24-h (24-h LC(50)), 48-h LC(50), 72-h LC(50), and 96-h LC(50) were 5.43±0.19, 5.04±0.18, 4.95±0.19, and 4.82±0.21mgL(-1) (95% confidence limits), respectively. Mortality of fish increased with increasing water temperature. Increasing alkalinity from 19mgL(-1) as CaCO(3) to 40, 60, or 90mgL(-1) as CaCO(3) significantly decreased mortality of fish. Total hardness ranging from 50mgL(-1) as CaCO(3) to 147mgL(-1) as CaCO(3) did not affect mortality of fish exposed to methiocarb. Fish exposed to methiocarb had histological alterations such as lamellar edema, separation of epidermis from lamellae, and lamellar fusion. Methiocarb exposed fish had necrosis between molecular and granular layer of cerebellum where Purkinje cells present. Results indicate that alkalinity, temperature, and fish size affect methiocarb toxicity of rainbow trout.