Content of cysteine, reduced and oxidized glutathione in spermatozoa of representatives of Acipenseriformes (Acipenser baerii and A. ruthenus) as well as teleosts (Perca fluviatilis and Sander lucioperca)

Glutathione belongs to a vital intra‐ and extra‐cellular protective antioxidant and is found almost exclusively in its reduced form. The ratio between its reduced and oxidized within cells is often used as a marker of cellular toxicity. The objectives of the study were to (i) determine both the reduced (GSH) and oxidized glutathione (GSSG) and cysteine (Cys) in the sperm of the Acipenser baerii and Acipenser ruthenus, as well as in Perca fluviatilis and Sander lucioperca, and (ii) to demonstrate the differences in concentration levels between representatives of acipenseriform and teleost species. High performance liquid chromatography with electrochemical detection was employed. The average content of the thiols determined in the sperm samples were as follows: Acipenser baerii (cysteine 55 ± 8 μg ml^?1; GSH 126 ± 19 μg ml^?1; GSSG 49 ± 7 μg ml^?1), Acipenser ruthenus (cysteine 62 ± 9 μg ml^?1; GSH 768 ± 115 μg ml^?1; GSSG 180 ± 16 μg ml^?1), Sander lucioperca (cysteine 251 ± 38 μg ml^?1; GSH 185 ± 28 μg ml^?1; GSSG 93 ± 14 μg ml^?1), Perca fluviatilis (cysteine 281 ± 42 μg ml^?1; GSH 496 ± 74 μg ml^?1; GSSG 138 ± 21 μg ml^?1). Based on the results obtained it can be concluded that this method is sensitive and selective for the determination of these compounds in real samples. Results revealed differences in cysteine content between species of the two systematic categories but also showed that ratios between GSH and GSSG can vary between species while potentially predict oxidative stress in fish sperm.     

Effects of administration of somatostatin-14 and immunoneutralization of somatostatin on endocrine and growth responses in rainbow trout

Injection of somatostatin‐14 (SS‐14) at 5 ng g^?1 body mass (BM) into rainbow trout Oncorhynchus mykiss decreased (P < 0·05, cubic, r² = 0·54) levels of growth hormone (GH) (1·5 ± 0·9 ng ml^?1v. 6·6 ± 0·6 ng ml^?1) over time when compared to controls. Somatostatin‐14 at 50 ng g^?1 BM also decreased (P = 0·064, quadratic; r² = 0·30) levels of GH (3·6 ± 2·1 ng ml^?1v. 6·6 ± 0·6 ng ml^?1) over time compared to controls. In a second study, passive immunization against SS‐14 (1 : 25 dose) increased (P = 0·10, cubic, r² = 0·12) levels of GH (11·0 ± 4·8 ng ml^?1v. 5·2 ± 1·4 ng ml^?1) over time. Passively immunizing against SS‐14 (1 : 50 dose) increased (P < 0·05, cubic, r² = 0·10) levels of GH (8·2 ± 2·3 ng ml^?1v. 5·2 ± 1·4 ng ml^?1) over time compared to controls. Overall, in the active immunization study there was no difference (P > 0·10) in specific growth rate (G) or feed conversion ratio (FCR) between the three treatment groups during the 9 weeks of the study. Only four of the fish immunized against SS‐14, however, developed antibody titres against SS. Compared to controls, these fish exhibited a G of 0·89 ± 0·09 v. 0·56 ± 0·09% per 3 weeks and FCR of 0·80 ± 0·04 v. 1·20 ± 0·05 g g^?1. In SS‐14 immunized fish, levels of GH decreased (P < 0·05) by day 63 while levels of insulin like growth factor‐I (IGF‐I) increased (P < 0·05) by day 42 and 63. These results indicate the hypothalamic hormone SS‐14 regulates GH secretion similarly in rainbow trout as it does in mammals. Active immunization against SS‐14 could improve growth performance in rainbow trout but enhanced G and FCR is dependent upon generation of antibody titres.     

Total Mercury in Sediments,Macrophytes, and Fish from a Shallow Steppe Lake in Eastern Austria

During summer 2011, samples of sediment, macrophytes, and fish tissues from the shallow, slightly alkaline Lake Neusiedl, Austria, were evaluated for their total Hg content. This is the first report of Hg levels from this lake. Sediments displayed Hg contents between 0.025 and 0.113 μg g^?1 dw (dry weight), significantly correlating with the proportion of organic components pointing to a small anthropogenic impact on the lake's Hg content. Hg Levels in plants and fish were unexpectedly high: both investigated submerged plant species, Potamogeton pectinatus and Myriophyllum spicatum, showed mean values of 0.245±0.152 and 0.298±0.115 μg g^?1 dw, respectively. Biomagnification was evident when comparing muscle samples of the planktivorous fish species rudd Scardinus erythrophthalmus (n=10, mean=0.084 μg g^?1 ww (wet weight)) with the piscivorous perch Perca fluviatilis (n=21, mean=0.184 μg g^?1 ww) or pike‐perch Sander lucioperca (n=9, mean=0.205 μg g^?1 ww). Significantly lower values were found in the muscle of the piscivorous pike Esox lucius (n=25, mean=0.135 μg g^?1 ww), pointing to a specific Hg metabolism of this fish, presumably under the particular physicochemical properties of the lake. Hg Concentrations in fish could pose a risk to piscivorous birds in this protected wetland system.     

Dietary arginine requirement of fingerling Indian major carp,Labeo rohita (Hamilton) based on growth,nutrient retention efficiencies,RNA/DNA ratio and body composition

To quantify the optimum dietary arginine requirement of fingerling Indian major carp, Labeo rohita (4.10 ± 0.04 cm; 0.62 ± 0.02 g), an 8‐week growth trial was conducted in eighteen 70‐L indoor circular aqua‐coloured troughs provided with a flow‐through system at 28 ± 1°C. Isonitrogenous (40 g 100 g^?1 crude protein) and isocaloric (4.28 kcal g^?1 gross energy) amino acid test diets containing casein and gelatin as intact protein sources with graded levels of arginine (0.5, 0.75, 1.0, 1.25, 1.50 and 1.75 g 100 g^?1 dry diet) were fed to triplicate groups of fish to apparent satiation at 07:00, 12:00 and 17:30 hours. Growth performance of fish fed the above diets was evaluated on the basis of absolute weight gain (AWG), specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER), protein retention efficiency (PRE) and energy retention efficiency (ERE). Maximum AWG (2.61), SGR (2.80), best FCR (1.35), highest PER (1.85), PRE (37%) and ERE (76%) were recorded at 1.25 g 100 g^?1 dietary arginine. Maximum body protein (18.88 g 100 g^?1) and RNA/DNA ratio (5.20) were also obtained in a 1.25 g 100 g^?1 arginine dry diet. Except for the reduced growth performance in fish fed arginine‐deficient diets, no other deficiency signs were apparent. Based on the broken‐line and second‐degree polynomial regression analysis of the AWG, SGR, FCR, PER, PRE and ERE data, the optimum arginine requirement for fingerling Labeo rohita was found to be in the range of 1.22–1.39 g 100 g^?1 of the dry diet, corresponding to 3.05–3.47 g 100 g^?1 of dietary protein.     

Kocuria SM1 controls vibriosis in rainbow trout (Oncorhynchus mykiss,Walbaum)

Aims: To develop probiotics for the control of vibriosis caused by Vibrio anguillarum and Vibrio ordalii in finfish. Methods and Results: Kocuria SM1, isolated from the digestive tract of rainbow trout, was administered orally to rainbow trout (Oncorhynchus mykiss) for 2 weeks at a dose equivalent to c. 10⁸ cells per g of feed and then challenged intraperitoneally with V. anguillarum and V. ordalii. Use of SM1 led to a reduction in mortalities to 15–20% compared to 74–80% mortalities in the controls. SM1 stimulated both cellular and humoral immune responses in rainbow trout, by elevation of leucocytes (5·5 ± 0·8 × 10⁶ ml^?1 from 3·7 ± 0·8 × 10⁶ ml^?1), erythrocytes (1·2 ± 0·1 × 10⁸ ml^?1 from 0·8 ± 0·1 × 10⁸ ml^?1), protein (23 ± 4·4 mg ml^?1 from 16 ± 1·3 mg ml^?1), globulin (15·7 ± 0·2 mg ml^?1 from 9·9 ± 0·1 mg ml^?1) and albumin (7·3 ± 0·2 mg ml^?1 from 6·1 ± 0·1 mg ml^?1) levels, upregulation of respiratory burst (0·05 ± 0·01 from 0·02 ± 0·01), complement (56 ± 7·2 units ml^?1 from 40 ± 8·0 units ml^?1), lysozyme (920 ± 128·8 units ml^?1 from 760 ± 115·3 units ml^?1) and bacterial killing activities. Conclusions: Kocuria SM1 successfully controlled vibriosis in rainbow trout, and the mode of action reflected stimulation of the host innate immune system. Significance and Impact of the Study: Probiotics can contribute a significant role in fish disease control strategies, and their use may replace some of the inhibitory chemicals currently used in fish farms.     

Baseline Sensitivity of Populations of Phytophthora capsici from China to Three Carboxylic Acid Amide (CAA) Fungicides and Sequence Analysis of Cholinephosphotranferases from a CAA‐sensitive Isolate and CAA‐resistant Laboratory Mutants

During 2006–2008, 572 isolates of Phytophthora capsici were collected from seven provinces in China, and their sensitivities to three carboxylic acid amides (CAA), dimethomorph, flumorph and pyrimorph were determined. Of these isolates, 90 isolates without a history of exposure to CAA fungicides (CAAs) were used to set up the baseline sensitivity. Baseline EC₅₀ values ranged from 0.122 to 0.203 (mean ± SD, 0.154 ± 0.022) μg ml^?1 for dimethomorph, from 0.301 to 0.487 (mean ± SD, 0.373 ± 0.043) μg ml^?1 for flumorph and from 0.557 to 0.944 (mean ± SD, 0.712 ± 0.082) μg ml^?1 for pyrimorph, respectively. The other 482 isolates were tested with a single discriminatory dose and were completely inhibited at 0.5 μg ml^?1 of dimethomorph. Four CAA‐resistant mutants were generated by repeated exposure to dimethomorph in vitro. As compared to the parental wild‐type isolate, the four CAA‐resistant mutants showed similar fitness in hyphal growth, sporulation in vitro and pathogenicity in vivo. Mutants resistant to CAAs in the in vitro assay caused visible lesions on pepper stems or roots treated with the recommended dose of dimethomorph. Previous studies upon the mode of action of CAAs suggested that these fungicides maybe inhibit phospholipid biosynthesis and that the primary target could be the cholinephosphotranferase (CPT), which is referred to aminoalcoholphosphotransferases (AAPTs). We sequenced and analyzed two CPT (AAPT1 and AAPT2) genes in P. capsici. Based on the cDNA sequence, we found that the AAPT1 and AAPT2 gene span 1538 and 1459 bp and were interrupted by five and three introns, respectively. There was no difference between the parental wild‐type isolate and the four CAA‐resistant mutants in the amino acid sequences of AAPT1 and AAPT2 gene. So, it was assumed that the resistance to dimethomorph was not due to mutations in the amino acid sequence of these two possible target genes.     

Inactivation of Cronobacter spp. (Enterobacter sakazakii) in infant formula using lactic acid,copper sulfate and monolaurin

Aims: To investigate the effect of lactic acid (LA), copper (II), and monolaurin as natural antimicrobials against Cronobacter in infant formula. Methods and Results: The effect of LA (0·1, 0·2 and 0·3% v/v), copper (II) (10, 50 and 100 μg ml^?1) and monolaurin (1000, 2000, and 3000 μg ml^?1) suspended into tween‐80? or dissolved in ethanol against Cronobacter in infant formula was investigated. Reconstituted infant formula and powdered infant formula were inoculated with five strains of Cronobacter spp. at the levels of c. 1 × 10⁶ CFU ml^?1 and 1 × 10³ CFU g^?1, respectively. LA at 0·2% v/v had a bacteriostatic effect on Cronobacter growth, whereas 0·3% v/v LA resulted in c. 3 log₁₀ reduction. Copper (II) at the levels of 50 μg ml^?1 and 100 μg ml^?1 elicited c. 1 and 2 log₁₀ reductions, respectively. The combination of 0·2% LA and 50 μg ml^?1 copper (II) resulted in a complete elimination of the organism. Monolaurin exhibited a slight inhibitory activity against Cronobacter (c. 1·5 log₁₀ difference) compared to the control when ethanol was used to deliver monolaurin. Conclusions: A complete elimination of Cronobacter was obtained when a combination of sublethal concentrations of LA (0·2%) and copper (II) (50 μg ml^?1) was used. Significance and Impact of the Study: The use of the synergistic interactive combination of LA and copper (II) could be beneficial to control Cronobacter in the infant formula industry.     

Antiviral activity of Distictella elongata (Vahl) Urb. (Bignoniaceae), a potentially useful source of anti-dengue drugs from the state of Minas Gerais, Brazil

Aims: To investigate the in vitro antiviral activity of Distictella elongata (Vahl) Urb. ethanol extracts from leaves (LEE), fruits (FEE), stems and their main components. Methods and Results: The antiviral activity was evaluated against human herpesvirus type 1 (HSV‐1), murine encephalomyocarditis virus (EMCV), vaccinia virus Western Reserve (VACV‐WR) and dengue virus 2 (DENV‐2) by the 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) colorimetric assay. LEE presented anti‐HSV‐1 [EC₅₀ 142·8 ± 5·3 μg ml^?1; selectivity index (SI) 2·0] and anti‐DENV‐2 activity (EC₅₀ 9·8 ± 1·3 μg ml^?1; SI 1·5). The pectolinarin ( 1 ) isolated from LEE was less active against HSV‐1 and DENV‐2. A mixture of the triterpenoids ursolic, pomolic and oleanolic acids was also obtained. Ursolic and oleanolic acids have shown antiviral activity against HSV‐1. A mixture of pectolinarin ( 1 ) and acacetin‐7‐O‐rutinoside ( 2 ) was isolated from FEE and has presented anti‐DENV‐2 activity (EC₅₀ 11·1 ± 1·6 μg ml^?1; SI > 45). Besides the antiviral activity, D. elongata has disclosed antioxidant effect. Conclusions: These data shows that D. elongata has antiviral activity mainly against HSV‐1 and DENV‐2, besides antioxidant activity. These effects might be principally attributed to flavonoids isolated. Significance and Impact of the Study: Distictella elongata might be considered a promising source of anti‐dengue fever phytochemicals.     

Renierol from marine sponge Haliclona.SP.: A natural inhibitor of xanthine oxidase with hypouricemic effects

The purpose of this study was to evaluate the inhibitory effect of renierol, extracted from marine sponge Halicdona.SP., on xanthine oxidase (XO) and its hypouricemic effect in vivo. Renierol and a positive control, allopurinol, were tested for their effects on XO activity by measuring the formation of uric acid and superoxide radical from xanthine. Renierol inhibited XO in a concentration-dependent and competitive manner. IC₅₀ value was 1.85 μg·ml^{? 1} through the measuring of uric acid and was 1.36 μg.ml^{? 1} through the measuring of superoxide radical. Renierol was found to have an in vivo hypouricemic activity against potassium oxonate-induced hyperuricaemia in mice. After oral administration of renierol at doses of 10, 20 and 30 mg.kg^{? 1}, there was a significant decrease in the serum urate level (4.08 ± 0.09 mg.dl^{? 1}, P < 0.01), (3.47 ± 0.11 mg.dl^{? 1}, P < 0.01) and (3.12 ± 0.08 mg.dl^{? 1}, P < 0.01), when compared to the hyperuricaemic control (6.74 ± 0.23 mg.dl^{? 1}). Renierol was a potent XO inhibitor with hypouricemic activity in mice.     

Experimental treatments with diflubenzuron and deltamethrin of sea bass, Dicentrarchus labrax L., infected with the isopod, Ceratothoa oestroides

Sea bass with approximate average weights of 5 and 20 g were treated against Ceratothoa oestroides infection with: (i) medicated pellets of diflubenzuron PC90 at a dosage of 3 mg kg^?1 body weight (BW) per day for 14 days. Lice were counted at the beginning of treatment and 19 days after treatment. The drug cleared all lice in the treated group; in the control group, infection remained high 30 days after beginning the experiment. It was concluded that medicated pellets containing 3 mg kg^?1 BW diflubenzuron effectively cleared pre‐adult and adult stages of the isopod parasite over a 14‐day period. No adverse effects were recorded in treated sea bass during the trials and no reinfection occurred 15 days after end of the treatment. (ii) Deltamethrin by means of bath treatments in infected sea bass kept in experimental tanks at 20°C. Before treatment, toxicity on healthy fish was preliminarily assessed by testing five fish from each size group at concentrations of 30, 10, 5, 3, 1, 0.1, 0.05 and 0.01 mg L^?1 for 30 min. The therapeutic concentrations tested were: 10, 5, 3, 0.15, 0.1, and 0.05 μg L^?1 and assessed at 1, 24 and 48 h. Best results were achieved with the 10 μg L^?1 (0.01 mg L^?1) dose, where prevalence was reduced from 100 to 0% over 24 h in both large and small fish. No parasite recovery was observed at 48 h. The dose of 5 μg L^?1 reduced prevalence from 100 to 11.7% and to 0% for small and large fish, respectively. Finally, with the 3 μg L^?1 dose, prevalence was reduced from 100 to 37.5% (small fish) and to 13.3% (large fish). Lower doses were ineffective on the parasites at either 24 or 48 h.     

Growth rate of tank-reared Mediterranean amberjack, Seriola dumerili (Risso 1810) fed on three different diets

Mediterranean amberjack Seriola dumerili (Risso 1810) has aquaculture potential. The growth rate and food conversion ratio of S. dumerili on three different types of food (A: 100% frozen sardines; B: 50% frozen sardines and 50% pellet; C: 100% pellet) were measured, and analysed with respect to temperature, condition index and chemical composition of the fish fillet. Wild S. dumerili, average body weight 248 g and average total length 26.9 cm were caught in August and September 1994 in the South Adriatic Sea near Dubrovnik, Croatia and kept in three tanks (n=15 in each tank; duration of experiment, 226 days). The fish that were fed on diet A [initial weight, 252 ± 71 g; total length (TL), 24.3 ± 2.6 cm] started feeding immediately; however, fish assigned to diet C began to feed entirely on the pelleted diet 1 month after the start of the experiment. The mortality of fish fed on diet A was negligible, the registered growth rate was 313 ± 74 g (124.2%), specific growth rate was 0.32% day^?1 and the food conversion rate was 6.7. The fish fed on diet B (initial weight, 246 ± 74 g; TL, 28.2 ± 2.5 cm) started to feed on day 3 and achieved a total growth rate of about 98% (final weight gain, 241 ± 69 g) and specific growth rate of 0.24% day^?1 (feed conversion rate of 9.00 and mortality 13%). The fish fed on diet C (initial weight, 246 ± 74 g; TL, 28.2 ± 2.5 cm) started to feed on the pellets after 1 month and had a growth rate of 87% (weight gain 214 ± 85 g), a specific growth rate of 0.24% day^?1 and a food conversion rate of 10.6 with considerable mortality (27%). In all three diet groups the fish grew with considerable variation in food consumption and growth rate, depending on seasonal temperature variations of the ambient sea water supplied to the rearing tanks. Chemical analysis showed that the protein level (amount) in the fish meat exceeded 20% in all three fish fillet samples.     

Anesthesia of wild female Persian sturgeon,Acipenser persicus (Borodin, 1897) breeders during controlled propagation: effects on hematological parameters,stress response and reproductive performance

This study examined the effects of anesthesia on the hematological and biochemical parameters as well as the reproductive performance of wild female Persian sturgeon, Acipenser persicus, during controlled spawning. Fourteen mature females were divided into two groups: ‘anesthetized’ and ‘non‐anesthetized’. All activities including transportation, catheterizing and handling were performed with both groups: (i) under anesthesia (150 ppm clove oil), and (ii) without anesthesia. After 10 days storage and handling, blood samples were taken from all fish after anesthesia. No significant differences were found in the reproductive performance of either group. However, differences were found in the hematological parameters, with values being significantly higher in the non‐anesthetized group, including neutrophils (34.36 ± 6.33% vs 23.63 ± 5.22%), monocytes (2.84 ± 1.70% vs 1.27 ± 0.64%), mean corpuscular volume (321.3 ± 39.40 pg vs 228.0 ± 24.46 pg) and mean corpuscular hemoglobin (106.9 ± 15.70 fl vs 76.50 ± 7.50 fl). Significantly lower values were found in the non‐anesthetized group for lymphocytes (60.68 ± 7.25% vs 73.54 ± 4.80%), Hb (4.62 ± 0.74 mg dl^?1 vs 6.28 ± 1.21 mg dl^?1), Hct (13.86 ± 1.76% vs 18.84 ± 3.85%), red blood cell (0.43 ± 0.05 cell mm^?3 vs 0.85 ± 0.13 × 10⁶ cell mm^?3) and white blood cell (22 403 ± 2240 cell mm^?3 vs 35 318 ± 3084 cell mm^?3). The non‐anesthetized fish had significantly higher cortisol levels compared to the anesthetized group (62.33 ± 8.85 ng ml^?1 vs 46.12 ± 8.07 ng ml^?1). There was no difference in plasma glucose levels between groups. It is concluded that the use of clove oil as an anesthetic is suitable for handling of wild female Persian sturgeon in controlled propagation programmes. However, further research is needed to determine a standardized protocol for the safe application of anesthesia for use in sturgeons in general.     

Chemical composition and construction cost for roots of Mediterranean trees, shrub species and grassland communities

The construction cost of fine roots was studied in 23 woody species and two grassland communities, growing under natural conditions in southern Spain. Calculation of the energy (glucose) required for their synthesis was based on the quantification of chemical components present in tissues. Despite considerable differences in the chemical composition of the three life forms studied (trees, shrubs and herbaceous), detected differences in construction cost were non‐significant (mean value: 1·64 ± 0·13 g glucose g^?1). However, shrubs and herbaceous plants growing in more fertile habitats expended significantly less energy on root synthesis (1·58 ± 0·06 and 1·41 ± 0·05 g glucose g^?1, respectively) than those growing in less fertile areas (1·80 ± 0·06 and 1·57 ± 0·1 g glucose g^?1, respectively), because they contained smaller amounts of either waxes (shrubs) or lignins (herbaceous), both expensive to synthesize, and, proportionately, more cellulose; which is inexpensive to synthesize. Deciduous and evergreen tree species also differed mainly with regard to wax and cellulose contents, giving rise to a significantly higher construction cost in evergreens (1·57 ± 0·07 g glucose g^?1 versus 1·78 ± 0·02 g glucose g^?1). The differences observed in construction cost appeared to be due more to habitat‐induced differences in chemical composition than to any intrinsic difference between the species studied.     

Analysis of myosmine,cotinine and nicotine in human toenail,plasma and saliva

Myosmine is a minor tobacco alkaloid with widespread occurrence in the human diet. Myosmine is genotoxic in human cells and is readily nitrosated and peroxidated yielding reactive intermediates with carcinogenic potential. For biomonitoring of short-term and long-term exposure, analytical methods were established for determination of myosmine together with nicotine and cotinine in plasma, saliva and toenail by gas chromatography–mass spectrometry (GC/MS). Validation of the method with samples of 14 smokers and 10 non-smokers showed smoking-dependent differences of myosmine in toenails (66?±?56 vs 21?±?15?ng?g^?1, p?<0.01) as well as saliva (2.54?±?2.68 vs 0.73?±?0.65?ng ml^?1, p <0.01). However, these differences were much smaller than those with nicotine (1971?±?818 vs 132?±?82?ng g^?1, p <0.0001) and cotinine (1237?±?818 vs <35?ng?g^?1) in toenail and those of cotinine (97.43?±?84.54 vs 1.85?±?4.50?ng ml^?1, p <0.0001) in saliva. These results were confirmed in plasma samples from 84 patients undergoing gastro-oesophageal endoscopy. Differences between 25 smokers and 59 non-smokers are again much lower for myosmine (0.30?±?0.35 vs 0.16?±?0.18?ng?ml^?1, p <0.05) than for cotinine (54.67?±?29.63 vs 0.61?±?1.82?ng ml^?1, p <0.0001). In conclusion, sources other than tobacco contribute considerably to the human body burden of myosmine.     

Use of bacteriophage endolysin EL188 and outer membrane permeabilizers against Pseudomonas aeruginosa

Aims: To select and evaluate an appropriate outer membrane (OM) permeabilizer to use in combination with the highly muralytic bacteriophage endolysin EL188 to inactivate (multi‐resistant) Pseudomonas aeruginosa. Methods and Results: We tested the combination of endolysin EL188 and several OM permeabilizing compounds on three selected Ps. aeruginosa strains with varying antibiotic resistance. We analysed OM permeabilization using the hydrophobic probe N‐phenylnaphtylamine and a recombinant fusion protein of a peptidoglycan binding domain and green fluorescent protein on the one hand and cell lysis assays on the other hand. Antibacterial assays showed that incubation of 10⁶Ps. aeruginosa cells ml^?1 in presence of 10 mmol l^?1 ethylene diamine tetraacetic acid disodium salt dihydrate (EDTA) and 50 μg ml^?1 endolysin EL188 led to a strain‐dependent inactivation between 3·01 ± 0·17 and 4·27 ± 0·11 log units in 30 min. Increasing the EL188 concentration to 250 μg ml^?1 further increased the inactivation of the most antibiotic resistant strain Br667 (4·07 ± 0·09 log units). Conclusions: Ethylene diamine tetraacetic acid disodium salt dihydrate was selected as the most suitable component to combine with EL188 in order to reduce Ps. aeruginosa with up to 4 log units in a time interval of 30 min. Significance and Impact of the Study: This in vitro study demonstrates that the application range of bacteriophage encoded endolysins as ‘enzybiotics’ must not be limited to gram‐positive pathogens.     

Welfare status of cultured seabass (Dicentrarchus labrax L.) and seabream (Sparus aurata L.) assessed by blood parameters and tissue characteristics

Seabass (SBS) and seabream (SBM) juveniles were reared with the goal of obtaining three different final densities (SINT = 40 kg m^?3; INT = 20 kg m^?3; SEM = 0.2 kg m^?3) to ascertain the effects thereof on the welfare of the fish. Significant blood metabolites and hepatic glycogen were determined every 3 months and at harvest. Trials lasted 18 months in seabass and 17 months in seabream. At the end of experiment the main biometric productive parameters and quality of body composition were also recorded. Regarding intensively reared seabass (SINT‐SBS, INT‐SBS), the plasma triglycerids, total cholesterol and transaminases (AST, ALT) were always significantly higher than in semi‐intensively maintained fish (SEM‐SBS). At the final sampling in the SINT‐SBS batch, the total protein and glucose were also markedly increased. Conversely, at harvest the liver glycogen content decreased in SINT‐SBS (34 ± 8 mg g^?1 liver) with respect to INT‐SBS (57 ± 12 mg g^?1 liver) and SEM‐SBS (63 ± 11 mg g^?1 liver). No differences among groups were observed for creatine kinase (CK) and lactate dehydrogenase (LDH). With regard to seabream, SINT‐SBM and INT‐SBM constantly showed plasma triglycerids and total cholesterol as being significantly higher when compared to SEM‐SBM. In the final two blood samplings, SINT‐SBM exhibited the most elevated values for LDH. At harvest, AST, ALT, total protein and glucose markedly increased in SINT‐SBM, whereas liver glycogen content was reduced (22.5 ± 9 mg g^?1 liver), more than in INT‐SBM (70 ± 16 mg g^?1 liver) and SEM‐SBM (75 ± 20 mg g^?1 liver). In both seabass and seabream, body composition was very similar in the different stocking densities, except for total cholesterol. Total n‐3 polyunsaturated fatty acid (PUFA) in seabream was significantly different from fish of the semi‐intensive groups; however, nutritional values and fatty acid profiles were equally good. The intermediate final density of seabass and seabream at 20 kg m^?3 seemed to give the best results in terms of their well being when compared to fish reared at 40 kg m^?3. The absence of differences in blood metabolites and hepatic glycogen levels between the intensive batch and the semi‐intensive groups until harvest was a reference to the positive status of the fish. A density of 20 kg m^?3 can be considered acceptable for farm strategy planning for raising healthy on‐growing seabass and seabream juveniles.     

Characterization of glycolipid biosurfactant from Pseudomonas aeruginosa CPCL isolated from petroleum‐contaminated soil

Aims: To isolate and characterize the biosurfactant‐producing micro‐organism from petroleum‐contaminated soil as well as to determine the biochemical properties of the biosurfactant. Methods and Results: A novel rhamnolipid‐producing Pseudomonas aeruginosa (GenBank accession number GQ241355 ) strain was isolated from a petroleum‐contaminated soil. Surface active compound was separated by solvent extraction of the acidified culture supernatant. The extract was able to reduce the surface tension of water from 72 to 44 mN m^?1 at a critical micelle concentration of 11·27 ± 1·85 mg l^?1. It showed better activity (based on microdilution method) against Gram‐positive (≤ 31 mg ml^?1) bacteria and filamentous fungi (≤ 50 mg ml^?1) than Gram‐negative bacteria (≥ 125 mg ml^?1) with mild toxicity (HC₅₀– 38 ± 8·22 μg ml^?1) to red blood cells. Fourier transform infrared spectroscopy revealed the presence of aliphatic chain, hydroxyl groups, ester and glycosidic bonds. Presence of nineteen rhamnolipid homologues with variation in chain length and saturation was revealed from liquid chromatography coupled to mass spectrometry with electrospray ionization. Conclusion: The results indicate that the isolated biosurfactant has a novel combination of rhamnolipid congeners with unique properties. Significance and Impact of the Study: This study provides a biosurfactant, which can be used as a biocontrol agent against phytopathogens (Fusarium proliferatum NCIM 1105 and Aspergillus niger NCIM 596) and exploited for biomedical applications.     

Dietary protein requirement for juvenile Chinese sucker (Myxocyprinus asiaticus)

This study was conducted to determine the dietary protein requirement for juvenile Chinese sucker, Myxocyprinus asiaticus. Six fishmeal‐based experimental diets containing various crude protein levels ranging from 300 to 500 g kg^?1 were fed to triplicate groups of 20 fish each (initial weight 13.5 ± 1.1 g) for 56 days at a temperature of 28 ± 1°C (tank size 400 × 45 × 40 cm, linked to a recirculation system). Survival was not affected by dietary protein level (overall survival 71 to 90%). Weight gain (WG) and specific growth rate (SGR) increased with an increasing dietary protein level up to 460 g kg^?1. The feed conversion rate (FCR) generally showed a decline at higher protein levels (from 1.62 in 300 g protein kg^?1 to 1.13 in 500 g protein kg^?1 feed). Protein efficiency ratio (PER) showed gradual improvements with increasing dietary protein up to 460 g kg^?1. A similar trend was found for the protein productive value (PPV). Among the proximate compositions of the fish, crude protein content increased significantly with increasing dietary protein levels. Based on broken‐line regression analysis of SGR against dietary protein levels, the optimal dietary protein requirement for juvenile Chinese sucker was estimated to be close to 465 g kg^?1.     

Mitigating stress effects during transportation of matrinxã (Brycon amazonicus Günther, 1869; Characidae) through the application of calcium sulfate

This study verified the effects of CaSO₄ on physiological responses of the tropical fish matrinxãBrycon amazonicus (200.2 ± 51.1 g) in water containing CaSO₄ after a 4‐h transportation at concentrations of: 0, 75, 150, and 300 mg L^?1. Blood samples were collected prior to transportation (initial levels), immediately after packaging, at arrival, and 24 h and 96 h after transportation (recovery). Cortisol levels increased after packaging (118.2 ± 14.2 ng ml^?1), and decreased slightly after transportation in water containing CaSO₄ (106.8 ± 14.1), but remained higher than initial levels (21.0 ± 2.6 ng ml^?1). Fish kept at 150 mg L^?1 CaSO₄ reached the pre‐transportation levels at 24 h of recovery. Blood glucose increased after transportation in all treatments (8.2 ± 0.2 mmol L^?1) and declined after full recovery to values below initial levels (4.8 ± 0.1 mmol L^?1). Chloride levels did not change in CaSO₄ treatments; serum sodium concentrations decreased after packaging and after transportation. Serum calcium levels did not differ among treatments, but decreased after packaging and increased at 96 h of recovery. Hematocrit and the number of red blood cells were higher in all treatments after packaging and arrival, except in fish exposed to 300 mg L^?1 CaSO₄. Mean corpuscular volume increased in 75 mg L^?1 CaSO₄, which reached the higher VCM after transportation. Hemoglobin levels increased only after transportation, regardless of calcium sulfate levels. Handling before transportation and transportation itself were both stressful to fish; calcium sulfate at concentrations tested in the present work had a moderate influence in the reduction of stress responses.     

Identification of an Antifungal Compound in Unripe Avocado Fruits and its Possible Involvement in the Quiescent Infections of Colletotrichum gloeosporioides1

A new antifungal compound was isolated from peel and flesh of unripe avocado fruits and identified as 1-acetoxy-2,4-dihydroxy-n-heptadeca-16-ene. The maximal concentration of the anti-fungal monoene in unripe fruits was about 800 μg. g^?1 fr.wt. During ripening the monoene decreased to 40 μg. g^?1 fr.wt. concomitantly with the appearance of disease symptoms. The concentration of the previously described antifungal diene, 1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene (Prusky et al. 1982), in avocado peel was 1,600 μg. g^?1 fr.wt. in unripe fruits, decreasing during ripening to 120 μg. g^?1 fr.wt. At 750 μg. ml^?1 the inhibition of germ tube elongation of germinated conidia by the antifungal monoene and the antifungal diene was 15 % and 44 %, respectively. A 1: 1 mixture of both antifungal compounds in concentrations ranging from 50 to 750 μg. ml^?1, showed synergistic activity and increased the percent of inhibited germ tubes of germinated conidia up to 15 % over the sum of activities of the separate compounds. The results are discussed in relation to the hypothesis that the antifungal diene and the antifungal monoene are involved in the quiescence of the germinated appressoria of Colletotrichum gloeosporioides in unripe avocado fruits.