Physiological impact of sea lice on swimming performance of Atlantic salmon

Atlantic salmon Salmo salar were infected with two levels of sea lice Lepeophtheirus salmonis (0·13 ± 0·02 and 0·02 ± 0·00 sea lice g⁻¹). Once sea lice became adults, the ventral aorta of each fish was fitted with a Doppler cuff to measure cardiac output ( ̇ ), heart rate ( f _H) and stroke volume ( V _S) during swimming. Critical swimming speeds ( U _crit) of fish with higher sea lice numbers [2·1 ± 0·1 BL (body lengths) s⁻¹] were significantly lower ( P  < 0·05) than fish with lower numbers (2·4 ± 0·1 BL s⁻¹) and controls (sham infected, 2·6 ± 0·1 BL s⁻¹). After swimming, chloride levels in fish with higher sea lice numbers (184·4 ± 11·3 mmol l⁻¹) increased significantly (54%) from levels at rest and were significantly higher than fish with fewer lice (142·0 ± 3·7 mmol l⁻¹) or control fish (159·5 ± 3·5 mmol l⁻¹). The f _H of fish with more lice was 9% slower than the other two groups at U _crit. This decrease resulted in ̇ not increasing from resting levels. Sublethal infection by sea lice compromised the overall fitness of Atlantic salmon. The level of sea lice infection used in the present study was lower than has previously been reported to be detrimental to wild Atlantic salmon.     

Automated sperm morphology analysis in fishes: the effect of mercury on goldfish sperm

This study is the first to examine the morphology of fish sperm using automated sperm morphology analysis (ASMA). The technique was applied to investigate the effect of an environmental pollutant, mercury, on the sperm morphology of goldfish Carassius auratus , and the effects on sperm morphology were compared with those on sperm motility. Goldfish sperm flagellar length was significantly shortened after instant exposure to 100 mg l⁻¹ (368 µM) mercuric chloride, while curvilinear velocity (VCL) and the percentage of motile sperm were significantly decreased at mercuric chloride concentrations of 1 and 10 mg l⁻¹ (3·68 and 36·8 µM), respectively. After 24 h exposure to 0·001 mg l⁻¹ (0·0037 µM) mercuric chloride, flagellar length was significantly reduced in 38% of the spermatozoa. Following exposure to 0·1 mg l⁻¹ (0·37 µM) mercuric chloride for 24 h, however, the majority of spermatozoa (98%), had significantly shortened flagella and increased sperm head length, width and area. Sperm motility was also significantly decreased at 0·1 mg l⁻¹ (0·37 µM) mercuric chloride, probably due to the significantly reduced flagellar length at this concentration. This study shows that the morphological examination of fish sperm by ASMA provides, not only, an excellent tool for monitoring reproductive disruption caused by environmental pollution, but also has applications to other areas of fish reproductive biology, such as cryopreservation and aquaculture.     

Stimulation of biomethanation by Clostridium sp. PXYL1 in coculture with a Methanosarcina strain PMET1 at psychrophilic temperatures

Aim:  Bioaugumentation of low temperature biogas production was attempted by addition of cold-adapted Clostridium and a methanogen.
Methods and Results:  A psychrotrophic xylanolytic acetogenic strain Clostridium sp. PXYL1 growing optimally at 20°C and pH 5·3 and a Methanosarcina strain, PMET1, growing optimally on acetate and producing methane at 15°C were isolated from a cattle manure digester. Anaerobic conversion of xylose at 15°C with the coculture of the two strains was performed, and batch culture methane production characteristics indicated that methanogenesis occurred via acetate through 'acetoclastic' pathway. Stimulation studies were also undertaken to evaluate the effect of exogenous addition of the coculture on biogas yields at 15°C. Addition of 3 ml of PXYL1 at the rate of 12 × 10² CFU ml⁻¹ increased the biogas 1·7-fold (33 l per kg cowdung) when compared to control (19·3 l per kg cowdung) as well as increased the volatile fatty acid (VFA) levels to 3210 mg l⁻¹ when compared to 1140 mg l⁻¹ in controls. Exogenous of addition of 10 ml PMET1 inoculum at the rate of 6·8 ± 10² CFU ml⁻¹ in addition to PXYL1 served to further improve the biogas yields to 46 l kg⁻¹ as well as significantly brought down the VFA levels to 1350 mg l⁻¹.
Conclusions:  Our results suggest that the rate-limiting methanogenic step at low temperatures could be overcome and that biogas yields improved by manipulating the population of the acetoclastic methanogens.
Significance and Impact of the Study:  Stimulation of biomethanation at low temperature by coculture.     

Response surface methodology to optimize the nutritional parameters for enhanced production of jasmonic acid by Lasiodiplodia theobromae

Aims:  To find out the cumulative effect of the nutritional parameters and to enhance the production of jasmonic acid (JA) in static fermentation by Lasiodiplodia theobromae using response surface methodology (RSM).
Method and Results:  Malt extract, sucrose, NaNO₃ and MgSO₄.7H₂O were analysed by a 30-trial central composite design using RSM for optimizing their concentrations in the medium and the effect of their mutual interaction on JA production. Sucrose and NaNO₃ were found highly significant in influencing the JA production. Malt extract and MgSO₄.7H₂O showed an effect on the JA production in interaction with other variables. When the optimum values of the parameters obtained through RSM (19·95 g l⁻¹ malt extract, 50 g l⁻¹ sucrose, 7·5 g l⁻¹ NaNO₃ and 3·51 g l⁻¹ MgSO₄.7H₂O) were applied, 32% increase in JA production (299 mg l⁻¹) was observed in comparison with 225 mg l⁻¹ of JA produced with same media components not analysed by RSM and subsequently validated the statistical model.
Conclusions:  Increase in JA production was achieved by optimizing the nutritional parameters.
Significance and Impact of the Study:  This is the first report of using RSM for optimizing a medium for JA production. It resulted in an increase in JA production without augmentation of costly additives.     

Polyaluminum chloride-enhanced concentration efficiency of poliovirus and f2 phage from sewage water

Aims:  To enhance the recovery of f₂ bacteriophage and poliovirus by an established method based on the adsorption to and elution from positively-charged Al(OH)₃-treated silica gel.
Methods and Results:  Polyaluminum Chloride (PAC) was added to water samples to neutralize the negatively charged materials, which can reduce virus recovery by providing a competing adsorption mode on the media surface. Using this improved process (PAC 30 mg l⁻¹, pH 6·5, temperature 20∼30°C), the recoveries of Poliovirus I and f₂ from small-volume sewage (100 ml) were 110·76 ± 36·0% and 92·06 ± 8·65%, respectively ( P  < 0·05 vs. traditional methods). Recovery from a 20-L volume of sewage averaged 85·65 ± 4·43% for f₂ and 88·73 ± 9·76% for poliovirus, significantly higher than the recoveries in the traditional methods ( P  < 0·05).
Conclusions:  PAC could enhance concentration efficiency of poliovirus and f₂ phage from sewage water.
Significance and Impact of the Study:  This method should significantly improve the recovery of viruses from sewage.     

Inhibition of the tempe mould, Rhizopus oligosporus, by ammonia

The hyphal extension rate of Rhizopus oligosporus NRRL 2710 was slowed in the presence of 0·42 and 0·84 mmol NH3 l⁻¹ and inhibited by 1·3 mmol l⁻¹. Sporulation was prevented at NH3 concentrations of 0·42 mmol l⁻¹ andabove. There was no evidence of toxicity due to NH⁺₄ at concentrations up to 300 mmol l⁻¹.Independent of the concentrations of NH3 or NH⁺₄, the lower the pH value, in therange 6·0–9·0, the higher was the rate of hyphal extension. It is suggested that accumulationof toxic levels of NH3 could be responsible for the cessation of mould growth in tempe.     

Long-term ammonia exposure of turbot: effects on plasma parameters

Turbot juveniles were exposed to four ammonia concentrations [0·17 (L), 0·34 (M), 0·73 (MH) and 0·88 (H) mg l⁻¹ NH₃-N] for different exposure durations (28 days minimum to 84 days). Their physiological status and growth performances were compared to a control group [0·004 (C) mg l⁻¹ NH₃-N]. No growth was observed in the H group, and by day 57, mass increase in the MH group was only 15% of that in group C. During the first month growth in the L group was similar to that in control group while it was lower (33%) in the M group; afterwards the L and M groups had a similar growth (half that of controls). Accumulation of total ammonia nitrogen (TA-N) in plasma was dependent on ambient ammonia concentrations. Plasma urea levels in ammonia-exposed fish were lower, similar or greater than in controls (depending on ammonia concentration or exposure duration). Osmolarity, Cl^– and Na⁺ plasma concentrations were stable in the L and M groups. The increases in Na⁺, Cl^–, K⁺ and total Ca concentrations observed by the end of the experiment in the H and MH groups suggest that fish failed to adapt. There was an initial rise in plasma cortisol in all ammonia-exposed groups followed by a return to basal level (1·7–4 ng ml⁻¹) in the L and M groups. In group MH, plasma cortisol peaked at 42 ng ml⁻¹ by day 14, and after a decline at c . 1 month (14 ng ml⁻¹), it rose again.     

The antibacterial mechanism of carvacrol and thymol against Escherichia coli

Aims:  To investigate the antibacterial mechanism of carvacrol and thymol against Escherichia coli.
Methods and Results:  The time-kill curve results showed that carvacrol and thymol at 200 mg l⁻¹ could inhibit the growth of E. coli . Flow cytometry and fluorescent dyes were used to explore the effect of two components on membrane permeability and membrane potential. In membrane permeability experiment, the mean fluorescence intensity of cells treated with 200 mg l⁻¹ carvacrol or thymol were lower than nonexposed cells. The ratio of red to green fluorescence intensity of DiOC₂(3) reflected the change of membrane potential. Carvacrol and thymol at 200 mg l⁻¹ caused the ratio of red/green decreasing from 0·42 of control to 0·08 and 0·07, respectively.
Conclusions:  Carvacrol and thymol had desired antimicrobial effect on E. coli . The antibacterial effects were attributed to their ability to permeabilize and depolarize the cytoplasmic membrane.
Significance and Impact of the Study:  This study showed the potential use of flow cytometry as a suitable method to investigate the mode of antibacterial action of essential oil components.     

Effects of cadmium chloride on the development of rainbow trout Oncorhynchus mykiss early life stages

The sub-chronic (28–56 days) effects of exposure to low concentrations of cadmium (Cd; 0·05, 0·25, 0·50 and 2·50 μg l⁻¹) shortly following fertilization on embryos, larvae and juvenile rainbow trout Oncorhynchus mykiss were examined. Premature hatching occurred at lower concentrations (0·05 and 0·25 μg l⁻¹ Cd), however, delayed hatching was seen in the 2·50 μg l⁻¹ Cd group, with >90% of hatching occurring on the last day of the hatching period. Larval growth was negatively affected by Cd exposure in a concentration-dependent manner. Larvae exposed to 2·50 μg l⁻¹ Cd were 13·9 ± 0·8% shorter in total length ( L _T) and weighed 22·4 ± 3·5% (mean ± s . e .) less than controls at the end of the exposure period. Plasma sex steroid concentrations (oestradiol in juvenile females and 11-ketotestosterone in juvenile males) were elevated (four- to 10-fold over controls) in exposed fish in both males and females, following 28 days of exposure to 0·05, 0·25 and 0·50 μg l⁻¹ Cd, respectively. These results suggest that environmentally realistic concentrations (in the μg l⁻¹ range) of Cd can affect the development of O. mykiss impacting embryos, larvae and juvenile fish.     

Inactivation of oenological lactic acid bacteria (Lactobacillus hilgardii and Pediococcus pentosaceus) by wine phenolic compounds

Aims:  To investigate the inactivation properties of different classes of phenolic compounds present in wine against two wine isolates of Lactobacillus hilgardii and Pediococcus pentosaceus , and to explore their inactivation mechanism.
Methods and Results:  After a first screening of the inactivation potency of 21 phenolic compounds (hydroxybenzoic and hydroxycinnamic acids, phenolic alcohols, stilbenes, flavan-3-ols and flavonols) at specific concentrations, the survival parameters (MIC and MBC) of the most active compounds were determined. For the L. hilgardii strain, the flavonols morin and kaempferol showed the strongest inactivation (MIC values of one and 5 mg l⁻¹, and MBC values of 7·5 and 50 mg l⁻¹, respectively). For the P. pentosaceus strain, flavonols also showed the strongest inactivation effects, with MIC values between one and 10 mg l⁻¹ and MBC values between 7·5 and 300 mg l⁻¹. Observations by epifluorescence and scanning electron microscopy revealed that the phenolics damaged the cell membrane and promoted the subsequent release of the cytoplasm material into the medium.
Conclusions:  The antibacterial activity of wine phenolics against L. hilgardii and P. pentosaceus was dependent on the phenolic compound tested, and led not only to bacteria inactivation, but also to the cell death.
Significance and Impact of the Study:  New information about the inactivation properties of wine lactic acid bacteria by phenolic compounds is presented. It opens up a new area of study for selecting/obtaining wine phenolic preparations with potential applications as a natural alternative to SO₂ in winemaking.     

Efficacy of sodium hypochlorite and peracetic acid in sanitizing green coconuts

Aims:  To evaluate the efficacy of sanitizing green coconuts ( Cocos nucifera L.) through the treatment applied by juice industries using sodium hypochlorite and peracetic acid.
Methods and Results:  The surface of the fruits was inoculated with a mixture of five Listeria monocytogenes strains. The treatments consisted in immersing the fruits for 2 min at room temperature in sodium hypochlorite solution containing 200 mg l⁻¹ residual chlorine at pH 6·5, and 80 mg l⁻¹ solution of peracetic acid or sterile water. Bacterial populations were quantified by culturing on trypticase soy agar supplemented with yeast extract and Oxford selective culture medium; however, recovery was higher on the nonselective medium. Immersion in water produced a reduction in the L. monocytogenes population of 1·7 log₁₀ CFU per fruit, while immersion in sodium hypochlorite and peracetic acid solutions resulted in population reductions of 2·7 and 4·7  log₁₀ CFU per fruit respectively.
Conclusions:  The treatments studied are efficient to green coconuts.
Significance and Impact of the Study:  Sanitation of green coconut is one of the most important control measures to prevent the contamination of coconut water. This article provides information that shows the adequacy of sanitizing treatments applied by the juice industries.     

Effects of feeding and induction strategy on the production of BmR1 antigen in recombinant E. coli

Aim:  To investigate the effects of feeding and induction strategies on the production of Bm R1 recombinant antigen.
Methods and Results:  Fed-batch fermentation was studied with respect to the specific growth rate and mode of induction to assess the growth potential of the bacteria in a bioreactor and to produce high yield of Bm R1 recombinant antigen. Cells were grown at a controlled specific growth rate (μ_set) during pre-induction, followed by constant feeding postinduction. The highest biomass (24·3 g l⁻¹) was obtained during fed-batch process operated at μ_set of 0·15 h⁻¹, whereby lower μ_set (0·075 h⁻¹) gave the highest protein production (9·82 mg l⁻¹). The yield of Bm R1 was increased by 1·2-fold upon induction with 1 mmol l⁻¹ IPTG (isopropyl-β- d -thiogalactoside) compared to using 5 mmol l⁻¹ and showed a further 3·5-fold increase when the culture was induced twice at the late log phase.
Conclusions:  Combination of feeding at a lower μ_set and twice induction with 1 mmol l⁻¹ IPTG yielded the best result of all variables tested, promising an improved method for Bm R1 production .
Significance and Impact of the Study:  This method can be used to increase the production scale of the Bm R1 recombinant antigen to meet the increasing demand for Brugia Rapid^™, a commercial diagnostic test for detection of brugian filariasis.     

Fungal endophytes from Acer ginnala Maxim: isolation, identification and their yield of gallic acid

Aims:  The aim of the study was to isolate the endophytic fungi from Acer ginnala and screen isolates rich in gallic acid.
Methods and Results:  After epiphytic sterilization, 145 fungal endophytes were isolated from the stem, annual twig and seed of Acer ginnala . The endophytes were grouped into ten different taxa, Phomopsis sp., Neurospora sp., Phoma sp., Epicoccum sp., Penicillium sp., Alternaria sp., Fusarium sp., Trichoderma sp., Cladosporium sp. and a species of Pleosporales Incertae Sedis , by their morphological traits and ITS-rDNA sequence analysis. The content and yield of gallic acid of 141 isolates were determined by HPLC. On average, the species of Pleosporales Incertae Sedis had the highest content and yield of gallic acid (13·28 mg g⁻¹ DW; 119·62 mg l⁻¹), while Alternaria sp. had the lowest.
Conclusions:  Of 141 fungal endophytes from A. ginnala , Phomopsis sp. isolate SX10 showed both the highest content and the highest yield of gallic acid (29·25 mg g⁻¹ DW; 200·47 mg l⁻¹).
Significance and Impact of the Study:  Endophytic fungi isolated from A. ginnala may be used as potential producers of gallic acid and other compounds with biological activities, or functioned as elicitors to produce natural compounds.     

Short‐term freshwater exposure benefits sea lice‐infected Atlantic salmon

Atlantic salmon Salmo salar were infected with sea lice Lepeophtheirus salmonis (0·08 ± 0·007 sea lice g⁻¹) over a period of 4 h. Both infected and non‐infected fish were swim tested in sea water (SW) and fresh water (FW). The ventral aorta of each fish was fitted with a Doppler cuff in order to measure cardiac output, stroke volume and heart rate during swim testing. Blood samples were taken at rest and after exercise. Critical swimming speed of infected fish in SW (2·14 ± 0·08 body lengths, bl s⁻¹) was significantly lower ( P  < 0·05) than infected fish switched to FW (2·81 ± 0·08 bl s⁻¹) and non‐infected fish in SW (2·42 ± 0·04 bl s⁻¹) and FW (2·61 ± 0·08 bl s⁻¹). Cardiac and blood results indicated infected fish exposed to FW did experience stress, but relief from osmotic and ionic distress probably reduces energy expenditure, allowing the increase in performance. As the performance of sea lice‐infected fish improved upon transfer to FW, it is likely that heavily infected salmonids do return to FW to restore compromised osmotic and ionic balance, and remove sea lice in the process.     

Haematology and leucocyte morphology of wild caught Thunnus maccoyii

The haematology of wild southern bluefin tuna Thunnus maccoyii was described using blood samples collected from fish immediately after they were caught. Cytology and cytochemistry revealed that the blood in peripheral circulation is comprised of erythrocytes, reticulocytes, ghost cells, lymphocytes, thrombocytes, eosinophilic granulocytes, neutrophilic granulocytes and monocytes. Reference ranges established were 41·09–55·50% for haematocrit, 0·62–3·00% for leucocrit, 13·25–17·92 g dl⁻¹ for haemoglobin and 2·1–2·9 million erythrocytes μl⁻¹ for erythrocyte count. Differential cell counts showed 94·58 ± 2·15% erythrocytes, 3·99 ± 1·44% leucocytes and 1·43 ± 1·03% thrombocytes (mean ±  s . d .). Normal ranges for differential leucocyte counts were 0·00–5·45% for neutrophils, 0·69–12·06% for eosinophils, 0·00–5·03% for monocytes, 46·97–74·32% for lymphocytes and 14·47–43·92% for thrombocytes. Erythrocyte indices, leucocyte types and cytochemistry were comparable to other species of scombrids. Packed cell volume was sensitive to the physiological state of the fish and to sample handling technique.     

Potential of Bacillus sp. to produce polyhydroxybutyrate from biowaste

Aim:  To test the Bacillus strains for their abilities to produce polyhydroxybutyrate (PHB) from different sugars and biowaste (Pea-shells).
Methods and Results:  Six Bacillus strains were checked for their ability to produce PHB from GM2 medium supplemented with different sugars at the rate of 1% (w/v) and from biowaste and GM2 (BW : M) combinations (3 : 7, 1 : 1, 7 : 3). Glucose supplemented GM2 medium resulted in maximum PHB production of 435 mg l⁻¹ constituting 31–62% w/w of the total cell dry mass. Substituting GM2 medium to the extent of 50% with biowaste (pea-shell slurry) resulted in 945–1205 mg l⁻¹ PHB (55–65% w/w). Optimization for additional nitrogen supplementation, inoculum size resulted in a final PHB production of 3010–3370 mg l⁻¹ equivalent to 300 g kg⁻¹ biowaste (dry wt).
Conclusion:  The Bacillus strains were able to produce PHB from biowaste (Pea-shells) as cheap source of substrate.
Significance and Impact of the Study:  This is the first report on usage of pea-shells as feed for PHB production, opening new possibilities for its use for production of PHB and Bacillus as potential candidate for the purpose.     

Effect of alternative treatments on seed-borne Didymella lycopersici in tomato

Aims:  To quantify the phytotoxicity and effect of alternative seed treatments based on acidified nitrite and elicitors of plant resistance (Tillekur and Chitosan) against seed-borne inocula of Didymella lycopersici .
Methods and Results:  Treatments tested were: nitrite [sodium nitrite in citric acid buffer (pH 2)] at 30, 100 and 300 mmol l⁻¹ and three exposure times (10, 20 and 30 min); Tillekur (in water) at 12·5, 25, 50, 100 and 200 mg ml⁻¹; Chitosan (in 0·05% acetic acid) at 2·5, 5, 10 and 50 mg ml⁻¹. Efficacy of treatments was determined in growth chamber experiments. Nitrite at 300 mmol l⁻¹ was completely effective, as was the fungicide, at controlling disease when applied for less than 20 min. Tillekur was as effective as the fungicide postemergence, but proved to be phytotoxic pre-emergence. Chitosan was significantly less effective than the other treatments.
Conclusions:  The high efficacy and low cost of acidified nitrite indicates that it is a suitable alternative to fungicides.
Significance and Impact of the Study:  There is currently a lack of effective seed treatments that can be used in organic and low-input crops. Treatments identified in this study can be considered as an effective alternative to chemical control against seed-borne fungal pathogens.     

The linamarase of Mucor circinelloides LU M40 and its detoxifying activity on cassava

Mucor circinelloides LU M40 produced 12·2 mU ml⁻¹ of linamarase activity when grown in a 3 l fermenter in the following optimized medium (g l⁻¹ deionized water): pectin, 10·0; (NH₄)₂SO₄,
1·0; KH₂PO₄, 2·0; Na₂HPO₄, 0·7; MgSO₄.7H₂O, 0·5; yeast extract, 1·0; Tween-80,
1·0, added after 48 h of fermentation. The purified linamarase was a dimeric protein with a molecular mass of 210 kDa; the enzyme showed optimum catalytic activity at pH 5·5 and 40 °C and had a wide range (3·0–7·0) of pH stability. The enzyme substrate specificity on plant cyanogenic glycosides was wide; the K_m value for linamarin was 2·93 mmol l⁻¹. The addition, before processing, of the fungal crude enzyme to cassava roots facilitated and shortened detoxification; after 24 h of fermentation, all cyanogenic glycosides were hydrolysed.     

Susceptibility to chlorine of Aeromonas hydrophila strains

The susceptibility of five Aeromonas hydrophila strains and one Escherichia coli strain to chlorine was studied under carefully controlled laboratory conditions. Of the Aer. hydrophila strains, two were from untreated water, two from tap water (immediately downstream of a water treatment plant) and one from the DSM collection. The study included disinfectant concentration (0·1, 0·2 and 0·5 mg l⁻¹), pH (6, 7 and 8) and temperature (4, 21 and 32 °C) as controlled variables. The results indicated that the untreated water strains, the DSM strain and the E. coli strain were inactivated within 1 min of chlorine treatment. The strains from chlorinated water (TW11 and TW27) showed a different susceptibility to chlorine disinfection, the rate of inactivation being greater at pH 6 than at pH 8 for both strains. Under the standard conditions of temperature 21 °C, pH 7 and chlorine concentration 0·2 mg l⁻¹, an increase or decrease of approximately 1 log unit in the number of bacteria did not affect the kill rate of the strains TW11 and TW27.     

Bactericidal activity of catechin-copper (II) complexes on Escherichia coli ATCC11775 in the absence of hydrogen peroxide

Washed Escherichia coli ATCC11775 cells were killed by (–)-epigallocatechin (EGC) in the presence of a non- lethal concentration of Cu²⁺ (1 μmol l⁻¹) without additional H₂O₂, but not by (–)-epicatechin (EC). EGC alone (< 0·1 mmol l⁻¹) did not reduce the viability of the cells. The survival curve obtained in the presence of EGC and Cu²⁺ was similar to that obtained in the presence of (–)-adrenaline (EN) and Cu²⁺.