Effect of temperature on α-tocopherol, fatty acid profile, and pigments of Diacronema vlkianum (Haptophyceae)

Diacronema vlkianum was grown in polyethylene bags at two different temperatures (18 and 26°C) in the laboratory. The biochemical composition level decreased when the temperature increased from 18 to 26°C. The maximum cell number at 18°C was 11.9 × 10⁶ cells ml⁻¹, while maximum cell number at 26°C was 1.6 × 10⁶ cells ml⁻¹. The maximum level of α-tocopherol was 257.7 ± 21.6 μg g⁻¹ dry weight (DW) at 18°C. The highest total carotenoids and chlorophylls were 6.5 mg g⁻¹ DW and 4.3 mg g⁻¹ DW, respectively, and the main pigments were determined as astaxanthin and lutein. Polyunsaturated fatty acids were found to be the predominant group, reaching 39.5% of the total fatty acids at 18°C. This comprised 20:5(n − 3) as the main polyunsaturated fatty acids (20.4%, at 18°C) followed by 22:6(n − 3) (4.8%, at 18°C). The results suggest that D. vlkianum can be successfully used as feed in shellfish hatcheries or aquaculture hatcheries, either as a substitute or in association with other microalgae, when this algae is cultured at 18°C.     

Effect of probiotic on reproductive performance in female livebearing ornamental fish

A probiotic bacterial strain, Bacillus subtilis, isolated from the intestine of Cirrhinus mrigala (Hamilton), was incorporated in fish feed at four different concentrations (5 × 10⁸ cells g⁻², 5 × 10⁷ cells g⁻², 5 × 10⁶cells g⁻¹ and 5 × 10⁵ cells g⁻¹) and fed to four species of livebearing ornamental fish, Poecilia reticulata (Peters), Poecilia sphenops (Valenciennes), Xiphophorus helleri (Heckel) and Xiphophorus maculatus (Gunther) for one year duration to observe the effect of dietary probiotic supplementation on their reproductive performance. Sixty virgin females of each species were stocked separately in circular FRP tanks (350 L) and fed diets with varying levels of probiotic cells and control feed. Broodstock performance was evaluated based on gonadosomatic index (GSI), fecundity and fry production of female broodstock. The results showed that supplementation of feed with probiotics increased significantly (P<0.05) the GSI, fecundity and fry production of spawning females and length and weight of fry in all the four species of fish. The number of dead and deformed fry were also significantly lower (P<0.05) in fish fed with the probiotic feeds. The use of higher concentration of the probiont in diet did not always lead to significantly improved reproductive performance of the spawners. Collectively, this study showed that female livebearers benefit from inclusion of probiotics in diet during their reproductive stages.     

Delivery of HUFA, probionts and biomedicine through bioencapsulated Artemia as a means to enhance the growth and survival and reduce the pathogenesity in shrimp Penaeus monodon postlarvae

One of the major problems in the shrimp culture industry is the difficulty in producing high-quality shrimp larvae. In larviculture, quality feeds containing a high content of highly unsaturated fatty acids (HUFA) and ingredients that stimulate stress and disease resistance are essential to produce healthy shrimp larvae. In the present study, Penaeus monodon postlarvae (PL15) were fed for 25 days on an unenriched Artemia diet (control; A) or on a diet of Artemia enriched with either HUFA-rich liver oil of the trash fish Odonus niger (B), probionts [Lactobacillus acidophilus (C1) or yeast-Saccharomyces cerevisiae (C2)] or biomedicinal herbal products (D) that have anti-stress, growth-promoting and anti-microbial characteristics. P. monodon postlarvae fed unenriched Artemia exhibited the lowest weight gain (227.9 ± 8.30 mg) and specific growth rate (9.95 ± 0.05%), while those fed the HUFA-enriched Artemia (B) exhibited the highest weight gain and specific growth rate (362.34 ± 12.56 mg and 11.77 ± 0.08%, respectively). At the end of the 25-day rearing experiment, the shrimp postlarvae (PL40) were subjected to a salinity stress study. At both low and high (0 and 50‰) salinities, the group fed the control diet (A) experienced the highest cumulative mortality indices (CMI) 935.7 ± 2.1 and 1270.7 ± 3.1, respectively. Those fed diet D showed the lowest stress-induced mortality, and CMI were reduced by 31.1 and 32.3% under conditions of low and high salinity stress, respectively. A 10-day disease challenge test was conducted with the P. monodon postlarvae (PL40–PL50) by inoculating the shrimp with the pathogen Vibrio harveyi at the rate of 10⁵–10⁷ CFU/ml in all rearing tanks. P. monodon postlarvae fed probiont-encapsulated Artemia diets (C1 and C2) exhibited the highest survival (94.3 and 82.3%, respectively) and lowest pathogen load (V. harveyi) in hepatopancreas (5.2 × 10² ± 9.0 × 10 and 4.6 × 10² ± 9.0 × 10 CFU g⁻¹, respectively) and muscle (2.0 × 10² ± 6 × 10 and 1.7 × 10² ± 8.6 × 10 CFU g⁻¹, respectively) tissues. The shrimp that were fed the unenriched Artemia (Control; A) showed the lowest survival (26.33%) and highest bacterial load in the hepatopancreas (1.0 × 10⁵ ± 5 × 10³ CFU g⁻¹) and muscle (3.6 × 10⁴ ± 6 × 10² CFU g⁻¹). The shrimp fed the herbal product (D)-enriched Artemia also exhibited enhanced survival and reduced V. harveyi load in the tissues tested compared to the control diet (A) group. The results are discussed in terms of developing a quality larval feed to produce healthy shrimp larvae.     

Effect of a probiotic bacterium <Emphasis Type="Italic">Bacillus circulans</Emphasis> PB7 in the formulated diets: on growth,nutritional quality and immunity of <Emphasis Type="Italic">Catla catla</Emphasis> (Ham.)

Bacillus circulans PB7, isolated from the intestine of Catla catla, was evaluated for use as a probiotic supplement in the feeds for the fingerlings of Catla catla. The effect of supplement on the growth performance, feed utilization efficiency, and immune response was evaluated. Catla fingerlings (ave. wt. 6.48 ± 0.43 g) were fed diets supplemented with 2 × 10⁴ (feed C1), 2 × 10⁵ (feed C2), and 2 × 10⁶ (feed C3) B. circulans PB 7 cells per 100 g feed for 60 days at 5% of the body weight per day in two equal instalments in triplicate treatments. The control feed (CC) was not supplemented with the B. circulans. All the feeds were isocaloric and isonitrogenous. Fish fed with feed C2 displayed better growth, significantly (P ≤ 0.05) highest RNA/DNA ratio, a lower feed conversion ratio (FCR), and a higher protein efficiency ratio (PER) than the other experimental diets. Highest carcass protein and lipid was also observed in the fish fed C2 feed compared to the others. Significantly (P ≤ 0.05), highest protease was recorded in fish fed feed C2 (47.9 ± 0.016) and lowest in fish fed feed C3 (32.10 ± 0.009), where α-amylase activity did not differ significantly (P ≤ 0.05) beyond the lowest inclusion level. ALP, ACP, GOT, and GPT in the liver of Catla catla were the highest (P ≤ 0.05) in fish fed C2 feed. The highest TSP, albumin, and globulin was observed in fish treated with C2 feed after 60 days feeding trial, but the lowest glucose level was observed in the same treatment. After the feeding trial, the non-specific immunity levels and disease resistance of fish were also studied. Phagocytic ratio, phagocytic index, and leucocrit value were the highest in fish fed feed C2. After the feeding trial, the fish were challenged for 10 days by bath exposure to Aeromonas hydrophila (AH1) (10⁵c.f.u. ml⁻¹ for 1 h, and, after 7 days, 10⁷c.f.u. ml⁻¹ for 1 h). Highest survival percentage was observed in fish fed with feed C2 compared with only 6.66% in the controls, which indicated the effectiveness of B. circulans PB 7 in reducing disease caused by A. hydrophila.     

Enrichment of Artemia nauplii with the probiotic yeast Saccharomyces boulardii and its resistance against a pathogenic Vibrio

Enrichment of Artemia nauplii with a known probiotic yeast Saccharomyces boulardii (SB) and its role in enhancing resistance against the pathogen Vibrio harveyi was investigated. SB was cultured, then fed to instar II Artemia nauplii in three different treatments; 10² (T1), 10³ (T2) and 10⁴ (T3) colony forming units (CFU) per ml in triplicate. The algae Nanochloropsis sp. was used as control diet. Survival and total count of CFU nauplii⁻¹ was observed on different media (Sabouraud, for enumerating yeasts, thiosulphate citrate bile salts sucrose, for enumerating Vibrio and seawater agar, for enumerating total aerobic flora) for each replication. Enhanced survival of nauplii was observed in treatments as compared to control. Results indicated that enrichment of SB in Artemia nauplii proceeded in a linear fashion, and up to 3500 CFU of SB could be detected in one nauplii at 10⁴ CFU ml⁻¹ treatment. No conclusive trend could be observed in the count of Vibrio and total aerobic flora due to treatment. Enriched nauplii were then challenged with the pathogen V. harveyi for 24 and 48 h at a concentration of 6.1 × 10⁶ CFU ml⁻¹. The survival counts at 48 h showed that the resistance of the nauplii was significantly (P < 0.01) improved in those fed with 10⁴ CFU  ml⁻¹ SB (90% survival rate after 48 h of challenge versus less than 40% for the infected control group without SB and treatments T1 and T2). This study shows that SB, which has been used for the first time in an aquatic live feed organism, has a profound beneficial effect on the nauplii by increasing its resistance to a pathogenic Vibrio infection. This revised version was published online in August 2006 with corrections to the Cover Date.     

Influence of the antibacterial herbs, Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia on the survival, growth and bacterial load of Penaeus monodon post larvae

The indiscriminate use of antibiotics and chemicals in shrimp hatcheries has led to biomagnification and that in turn could lead to rejection of a whole consignment. The application of the bioencapsulation technique as a tool for curative treatment in shrimp larvae was investigated. Herbs having antibacterial properties such as Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia (methanolic extracts) were bioencapsulated in Artemia and fed to Penaeus monodon post larvae PL 1–25. The post larvae were reared in a medium inoculated with pathogenic bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi and Vibrio sp. Post larvae reared in the non-inoculated water and fed with non-enriched Artemia exhibited 90% survival, highest specific growth rate (12.43%) and reduced bacterial load. P. monodon reared in the bacterial inoculated water and fed with the non-enriched Artemia exhibited the lowest survival (10–30%), specific growth rate (8.42–9.1%) and increased bacterial load (2.86 × 10³ to 3.76 × 10⁵ cfu/g). The methanolic extracts of the herbs helped to increase survival and specific growth rate and reduced bacterial load in the P. monodon culture system. Among the three herbal extracts, P. corylifolia enriched Artemia fed post larvae showed the tendency to higher survival (>50%), growth rate (11.5 averaged) and low bacterial load (1.12 × 10⁵ cfu/g). This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization of Pseudomonas aeruginosa associated with diseased postlarval abalone in Shenzhen, China

Outbreaks of mass mortality of postlarval abalone, Haliotis diversicolor supertexta, have occurred in south China since 2002 and have forced many abalone farms to close. About 30 representative bacterial strains were isolated from a sample of five diseased postlarval abalone, taken 25 days post-fertilization during an outbreak of postlarval disease in Shenzhen, China, in October 2006. Bacterial challenge tests showed that the predominant strain, designated as strain 22, was highly virulent to postlarvae with an LD₅₀ value of 7.8 × 10⁴ colony forming units (CFU) ml⁻¹, while four of the other isolates were weakly virulent with LD₅₀ values ranging from 1 × 10⁶ to 1 × 10⁷ CFU ml⁻¹, and the remaining 25 isolates were classified as avirulent with LD₅₀ values greater than 1 × 10⁸ CFU ml⁻¹. By means of API 20NE and 16S rDNA and ITS sequencing analyses, strain 22 was identified as Pseudomonas aeruginosa. Antibiotic susceptibility tests showed that strain 22 exhibited around 75% of susceptibility to 16 various antibiotics tested. The results of this study show P. aeruginosa as one of the bacteria involved in the mortality of abalone postlarvae in Shenzhen, China.     

Substrate specificity of brain acetylcholinesterase and its sensitivity to carbamate insecticides in <Emphasis Type="Italic">Carassius auratus</Emphasis>

The substrate specificity of brain acetylcholinesterase (AChE) in adult Carassius auratus fish and its sensitivity to carbamate insecticides were investigated in vitro. The results showed that the order of four substrates hydrolyzed by brain AChE in C. auratus was acetylthiocholine iodide > β-methylthiocholine iodide > propionylthiocholine iodide > butyrylthiocholine iodide, and the maximum velocity (V _max) of AChE hydrolyzing acetylthiocholine iodide (ATCh) was the highest among the four substrates, and the V _max values were 0.067 and 0.082 mmol min⁻¹ mg⁻¹ for male and female fish respectively. But their Michaelis–Menten constants (K _m) were the lowest, only 0.071 and 0.072 mmol/l respectively. Compared with other carbamate insecticides, the sensitivity of brain AChE to carbofuran was the highest and the IC₅₀ values were 1.04 × 10⁻⁶ mol/l for females and 1.17 × 10⁻⁶ mol/l for males. The inhibitory tendencies of eserine, methomyl, and aldicarb to brain AChE were very similar, and the percentage inhibition increased with time at the concentration of 1 × 10⁻⁶ mol/l. The order of inhibition potential of the three inhibitors from the highest to the lowest was eserine, aldicarb, and methomyl.     

Effects of dimethoate and beta-naphthoflavone on selected biomarkers of Poecilia reticulata

The main objective of the present study was to investigate if a battery of enzymatic biomarkers was suitable for use as effect criteria in acute toxicity tests with Poecilia reticulata. To attain this objective, the in vivo effects of dimethoate and beta-naphthoflavone on acetylcholinesterase (AChE), cytochrome P4501A-dependent monooxygenase activity of 7-ethoxyresorufin O-deethylase (EROD), glutathione S-transferases (GST), lactate dehydrogenase (LDH) and Na⁺-K⁺-ATPase activities of P. reticulata were studied. After 96 h of exposure to sublethal concentrations (0.063 mg l⁻¹ to 1 mg l⁻¹) of the pesticide, an inhibition of the enzymes AChE and GST, as well as an induction of LDH was observed. The compound beta-naphthoflavone significantly induced both EROD and GST. The remaining enzymes analysed were not significantly altered by the exposure to beta-naphthoflavone (0.82 mg l⁻¹ to 1.7 mg l⁻¹). These results suggest that in vivo toxicity tests based on the biomarkers used in this study are sensitive and present advantages to conventional acute tests based on mortality, since they were able to detect sublethal effects in a short-period of time (96 h) indicating target and/or detoxification mechanisms. This revised version was published online in July 2006 with corrections to the Cover Date.     

Age and growth of Gerres sp. (Perciformes: Gerreidae) in Okinawa Island of Southern Japan

A total of 518 Gerres sp. were collected around Okinawa Island, Japan, from November 2002 to July 2005, with monthly sampling where the standard length of females (n=218) were 56.2–147.1 mm, and males (n=149) were 62.2–139.4 mm. The maximum ages observed for females were 5⁺ years and males were 4⁺ years, estimated by transverse sectioned sagittal otoliths. Mean marginal increment indicated that opaque rings were formed once a year during April to July. The standard length (SL; mm) — body wet weight (BW; g) relationships were described as BW=(3.26×10⁻⁵) SL^2.97 and BW=(3.13×10⁻⁵) SL ^2.98 for females and males, respectively, and the standard length at age described by von Bertalanffy growth function for females, L _t=137.1(1−e^{−0.80[t+0.80]}) and males, L _t=127.3(1−e^{−0.82[t+0.93]}).     

Ammonia autointoxication of common carp: case studies

Three case studies of ammonia autointoxication of the common carp (Cyprinus carpio L.) are described. In the first case, carp yearlings with a full digestive tract were transferred during the growing period from pond water (22°C) to tap water (17°C). In the second case, marketable carp were transferred from the fishing ground of a pond (18°C) to a storage pond (10–12°C). Harvest was performed in late September when the fish were still ingesting natural feed. In the third case, marketable carp after storage for 1 month were transferred to storage ponds with markedly lower water temperature. Stress because of harvest and handling also occurred in this case. In all cases, highly increased concentrations of ammonia were found in the blood plasma of the fish (mean ± SD 1760 ± 350 μmol L⁻¹ in the first case, 870 ± 540 μmol L⁻¹ in the second case, and 880 ± 150 μmol L⁻¹ in the third case). Highly congested, dark-red coloured, oedematous gills were observed for all specimens. We can avoid similar cases of ammonia autointoxication by protection of fish from sudden changes of temperature during rearing, harvesting, and handling.     

Comparative growth of Pacific oyster (Crassostrea gigas) postlarvae with microfeed and microalgal diets

Two trials were carried out in the laboratory in order to assess the effect of microparticulated feed (F) and live (Thalassiosira pseudonana, M) diets on the growth of recently set (396 ± 13 μm shell height) and 2 mm Crassostrea gigas postlarvae. Different proportions of M and F (100:0, 75:25, 50:50; 25:75, 0:100) were delivered in a single dose of 3 h d⁻¹ in trial 1. Dietary M:F proportions of 100:0, 50:50, and 0:100 were delivered as a single pulse of 8 h d⁻¹ (P1) or two pulses of 4 h⁻¹ (P2) in trial 2. Maximal daily M ration was 296 cells μl⁻¹ d⁻¹ (trial 1), 150 M cells μl⁻¹ d⁻¹ (trial 2), or their equivalent F dry weight. Shell height (SH), dry (DW), and organic weight (AFDW) were evaluated weekly. Oysters from trial 1 significantly increased their size after 28 days, and exhibited no significant dietary differences in terms of DW (1.21 ± 0.15 to 2.01 ± 0.28 mg) or AFDW (0.091 ± 0.022 to 0.166 ± 0.029 mg). Newly set postlarvae (trial 2) also exhibited significant growth after 25 days. No dietary differences were observed in trial 2, yet P2 oysters attained significantly higher shell heights (825–912 μm) than P1 oysters (730–766 μm) after 25 d. Pulse effects were marginally not significant in terms of AFDW and growth rate. Together, these findings showed that balanced microfeeds have a practical potential for the culture of early C. gigas postlarvae, when they are delivered in pulse-feeding schemes     

Effect of in vitro exposure to Vibrio vulnificus on hydroelectrolytic transport and structural changes of sea bream (Sparus aurata L.) intestine

The everted gut sac technique has been used to investigate the effect of Vibrio vulnificus on water and electrolyte (Na⁺, K⁺, Cl⁻, HCO₃ ⁻) transport on the intestine of sea bream (Sparus aurata L.). Both the anterior and the posterior intestine were incubated in a medium containing 10⁸ V. vulnificus cells ml⁻¹ at 25°C for 2 h. The presence of V. vulnificus resulted in a significant reduction (P < 0.05) of water absorption in the anterior intestine, while sodium absorption in the anterior (P < 0.01) and posterior (P < 0.05) intestine was elevated. Chloride absorption was increased, but the changed was not significant, while potassium absorption decreased significantly (P < 0.05), but only in the posterior intestine. Incubation the sea bream intestine with V. vulnificus did not affect carbonate secretion in the anterior segment, whereas high secretion was stimulated in the posterior segment (P < 0.01). Histological evaluations demonstrated damage in the anterior intestine of sea bream that was characterized by the detachment of degenerative enterocytes, alterations in the microvilli, and the presence of a heterogenous cell population, indicating inflammation. Based on our results, we conclude that V. vulnificus caused cell damage to the intestine of sea bream and that the anterior intestine is more susceptible than the posterior part of the intestine. Several hypotheses are suggested to explain our observations, such as the presence of higher numbers of villosities in the anterior intestine than in the posterior one and/or the presence of endogenous bacteria in the posterior intestine which may have a protector role.     

Purification and characterization of α<Subscript>1</Subscript>-proteinase inhibitor and antithrombin III: major serpins of rainbow trout (<Emphasis Type="Italic">Oncorhynchuss mykiss</Emphasis>) and carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>) blood plasma

The main serine proteinase inhibitors of rainbow trout (Oncorhynchuss mykiss) and common carp (Cyprinus carpio) blood plasma were isolated and purified. The investigated inhibitors, α₁-proteinase inhibitor (α₁-PI) and antithrombin III (AT III), act by forming stable complexes with target proteinases. The association rate constants k _on for the interaction of fish plasma inhibitors with several serine proteinases have been determined: k _on for both carp and rainbow trout α₁-PI were >10⁷ M⁻¹ s⁻¹ for human neutrophil elastase, and in the case of bovine trypsin and chymotrypsin k _on values were 2.0–5.2 × 10⁶ M⁻¹ s⁻¹. Association rate constants k _on for the interaction of carp and rainbow trout AT III with bovine trypsin and thrombin were about 1.3 × 10⁴–7.9 × 10⁵ M⁻¹ s⁻¹ without and >10⁷ M⁻¹ s⁻¹ in presence of heparin; so antithrombins require the presence of heparin to become effective proteinase inhibitors. The high degree of homology of the estimated amino acid sequences of fish inhibitors reactive site loops confirms their similarity with other proteinase inhibitors from the serpin family.     

Bacterial flora associated with the giant freshwater prawn Macrobrachium rosenbergii, in the hatchery system

The objective of this study was to understand the microbial flora associated with the hatchery system of giant fresh water prawn, Macrobrachium rosenbergii during an entire rearing cycle. Bacteriological and physico-chemical analysis was done for different samples of water, larvae, and Artemia. The total bacterial load in well water, seawater and inlet water varied from 10¹ to 10⁵ cfu ml^− 1 with higher counts seen in larval rearing tank (LRT) water. The Vibrio count ranged between 10¹ to 10³ cfu ml^− 1. Larval samples harboured a bacterial load of 10⁶ to 10⁷ cfu/10 larvae. The bacterial load in Artemia hatching water ranged from 4.90 × 10⁴ to 5.63 × 10⁶ cfu ml^− 1 while Artemia had a load ranging from 1.08 × 10⁷ to 2.09 × 10⁹ cfu g^− 1. Vibrio count in the LRT water ranged from 10¹-10³ cfu ml^− 1 while the count in larvae ranged from 10² to 10⁴ cfu/10 larvae. The bacterial genera were predominantly Gram-negative and comprised of Aeromonas spp., Pseudomonas spp., Vibrio spp. and Bacillus spp. and non-spore formers (NSF) were the dominant Gram-positive bacteria. This study documents the bacterial flora associated with Macrobrachium hatchery system during a regular normal run. Knowledge of the qualitative and quantitative aspects of bacterial flora in the hatchery would help to understand disturbances, if any, brought about during disease outbreaks.     

Migration history of Sakhalin taimen <Emphasis Type="Italic">Hucho perryi</Emphasis> captured in the Sea of Okhotsk,northern Japan,using otolith Sr:Ca ratios

Sakhalin taimen Hucho perryi populations have decreased drastically to near extinction. It is urgent to establish an effective conservation strategy based on an understanding of the characteristics of migration and habitat use of this species. We examined the migration history of anadromous Sakhalin taimen captured off the Sarufutsu coast, northern Hokkaido, Japan, using otolith Sr:Ca ratios and also examined the relationship between their otolith Sr:Ca ratios during freshwater and seawater residence in a rearing experiment. Otolith Sr:Ca ratios of some fish from the Sarufutsu coast showed freshwater levels (0.5–4.0 × 10⁻³) near the core, which thereafter increased to brackish water levels (4.0–6.0 × 10⁻³), and then to seawater levels (6.0–10.0 × 10⁻³) in the outermost regions. Those findings indicate that specimens from the Sarufutsu coast migrated to the brackish water region or the sea and spent most of their lives there. The anadromous migration pattern including the timing of downstream migration seems to be flexible among individuals in the species. They migrate between freshwater and seawater or brackish water several times during their lives, showing extensive habitat use. It is essential to secure the continuity among the freshwater, brackish water, and seawater areas for their effective conservation.     

Age and growth of two gizzard shads, <Emphasis Type="Italic">Nematalosa come</Emphasis> and <Emphasis Type="Italic">N. japonica</Emphasis>, in coastal waters around Okinawa Island,southwestern Japan

The age and growth of two Nematalosa species around Okinawa Island were examined using sectioned otoliths collected from September 2003 to April 2006. Monthly changes in the frequency of the appearance of a translucent band on the outer margin of the otoliths indicated that ring formation occurred once a year from January to July for Nematalosa come and from January to March for Nematalosa japonica. The von Bertalanffy growth equations for both species were as follows: N. come: L _t  = 365.5{1 − exp[−0.111 × (t + 0.288)]} for females and L _t  = 214.7{1 − exp[−0.700 × (t – 1.110)]} for males; N. japonica: L _t  = 205.1{1 − exp[−1.068 × (t − 1.180)]} for females and L _t  = 195.5 {1 − exp[−1.293 × (t − 1.269)]} for males. The maximum ages observed for N. come and N. japonica were 11 and 6 years old, respectively. The growth of these species was characterized by the slow growth of N. come over many years, resulting in a larger size than N. japonica.     

Evolution of drug resistance and minimum inhibitory concentration to enrofloxacin in <Emphasis Type="Italic">Tenacibaculum maritimum</Emphasis> strains isolated in fish farms

The in vitro susceptibility of 63 isolates of Tenacibaculum maritimum from four fish farms to eight chemotherapeutic agents used for the treatment of bacterial diseases in fish were assessed. The results indicated that all strains were resistant to oxolinic acid and susceptible to amoxicillin, nitrofurantoin, florfenicol, oxytetracycline and trimethoprim-sulphamethoxazole. However, some isolates presented resistance to enrofloxacin and flumequine, ranging from 10 to 30%, and from 25 to 60%, respectively, depending on the farm sampled. These data were used in an attempt to predict whether the resistance to enrofloxacin was static or evolved during the time of sampling from 2003 to 2004. A relationship between the use of enrofloxacin and levels of resistance was detected in the studied farm, increasing significantly from no resistant isolates in 2003 to 44.8% resistant strains in 2004, the year in which this drug was commonly employed. This result was accompanied by a marked decline of about 29.2% of the inhibition zone sizes for the T. maritimum strains in comparison to the initial values (average 21.5 mm). Minimum inhibitory concentration (MIC) of enrofloxacin for 100 T. maritimum strains was determined by the microdilution method. Twenty isolates were resistant to enrofloxacin (> 256 μg ml⁻¹), while the remaining strains showed a bimodal distribution, which ranged from 0.5 to 32 μg ml⁻¹. Our interpretation of the enrofloxacin MIC data suggests that the breakpoint for T. maritimum should be 4 μg ml⁻¹. However, similar studies in other laboratories are necessary to validate this breakpoint value.     

Acute toxicity of nitrite on tilapia (Oreochromis niloticus) at different external chloride concentrations

Tilapias (Oreochromis niloticus) juveniles (total length 4.9 ± 0.2 cm and weight 1.8 ± 0.2 g) were exposed to several nitrite concentrations (0, 10, 18, 32, 56 and 100 mg l⁻¹) for 96 h, using a semi-static renewal method at chloride levels of 35.0 and 70.0 mg l⁻¹. At the end of the 96-h period, the median lethal concentration (LC₅₀) of nitrite was 28.18 mg l⁻¹ in water with low chloride content (35.0 mg l⁻¹) and 44.67 mg l⁻¹ with high chloride content (70.0 mg l⁻¹, respectively). It indicated that high concentrations of chloride ions could reduce the toxicity of nitrite. During the toxicity experiments, the behaviour and clinical signs of tilapias were also observed. Furthermore, the test of toxic mechanism was designed taking five test concentrations, viz., 5, 10, 15 and 20 mg l⁻¹ and a nitrite-free control. Nitrite exposure produced high levels of methaemoglobin (MHb) but did not seem to cause mortality, as surviving tilapias showed high levels (85.37 ± 2.23 and 53.82 ± 3.44 at 35.0 and 70.0 mg l⁻¹ chloride, respectively). The percentage of MHb exposed to nitrite was significantly higher (P<0.05) than the control (0 mg l⁻¹ nitrite) and increased with the increasing nitrite concentration. However, the percentage of MHb decreased with the increasing chloride concentration.     

Toxicity of Diazinon 60 EC for Cyprinus carpio and Poecilia reticulata

Diazinon 60 EC (chemical insecticide, organophosphate, active substance diazinon at a concentration of 600 g l⁻¹) is used in fish-farming in well-founded cases as a biocide to suppress excessive propagation of coarse Daphnia zooplankton. At the concentration of 10 μg l⁻¹ (i.e., 100 g per 1 ha at a mean depth of the pond of 1 m), Diazinon 60 EC can highly selectively eliminate Daphnia zooplankton, causes no harm to fish, and relatively quickly decomposes in the aquatic environment. This has been corroborated by results of acute toxicity tests in fish: the 96 h LC50 is 3 mg l⁻¹ for the guppy (Poecilia reticulata) and 10–25 mg l⁻¹ for the common carp (Cyprinus carpio). As demonstrated by embryonic and larval tests of toxicity for the common carp (C. carpio), Diazinon 60 EC at the concentration of 10 μg l⁻¹ causes no harm to the tested, individuals even in these sensitive (critical) stages of ontogeny.