Application of α-glucosidase activity and drug susceptibility tests to epidemiological studies on the fish pathogen <Emphasis Type="Italic">Nocardia seriolae</Emphasis>

This study determined the minimum inhibitory concentrations (MICs) for oxytetracycline hydrochloride (OTC) and erythromycin (Em), along with the α-glucosidase (α-glu) activities in 110 Nocardia seriolae strains isolated in Miyazaki and Kagoshima prefectures in 2008–2009. The strains were examined for the presence of the tet(K), tet(L), tet(M), tet(O), tet(S), erm(A), erm(B), mph(A), mef(A), and msr(D) genes. All the α-glu-positive strains (n = 15) were OTC resistant and Em sensitive, with MICs of 32–64 and <0.125 μg/ml, respectively. All the α-glu-negative strains (n = 95) were OTC sensitive, with MICs of 2–4 μg/ml, and most of them (93 of 95) were Em resistant, with MICs of >128 μg/ml. The MICs for Em in the remaining 2 α-glu-negative strains were <0.125 μg/ml. The 15 OTC-resistant strains possessed the tet(K) and/or tet(L) gene(s), and all of the 93 Em-resistant strains possessed both the mef(A) and msr(D) genes. The relationship between α-glu activity and drug sensitivity of the N. seriolae strains may explain the difference in prevalence of each phenotype. Nevertheless, the relationship should be further explored using N. seriolae isolates collected from more prefectures and farms.     

Immune response of largemouth bass, <Emphasis Type="Italic">Micropterus salmoides</Emphasis>, to whole cells of different <Emphasis Type="Italic">Nocardia seriolae</Emphasis> strains

Largemouth bass Micropterus salmoides were immunized with four different N. seriolae strains—two α-glucosidase-positive (961113, KU040801) and two α-glucosidase-negative (94260, OTTS) strains—along with Freund’s incomplete adjuvant. After primary immunization (week 0), a booster was administered at weeks 4 and 8. Nonspecific immune responses to multiple immunizations with the different N. seriolae strains were determined based on serum lysozyme activity and nitroblue tetrazolium (NBT)-positive cells in peripheral blood. The serum lysozyme activity and NBT-positive cells in peripheral blood were not significantly increased even after the two booster immunizations. Specific antibody responses against N. seriolae cells were investigated by enzyme-linked immunosorbent assay. At 4 weeks after immunization, all groups immunized with N. seriolae antigens showed significant increases in their specific antibody levels. The sera from fish immunized with different N. seriolae strains exhibited reactivity with N. seriolae sonicated antigens of 28, 30, 36 and 84 kDa by western blot analysis. After two boosters, fish were challenged with live N. seriolae to assess the vaccine’s efficacy; however, multiple injections of the N. seriolae strains did not reduce mortality, irrespective of the bacterin.     

Antifouling steroids isolated from red alga epiphyte filamentous bacterium Leucothrix mucor

Active antifouling steroids were isolated from the filamentous bacterium Leucothrix mucor. Bioactivity-guided isolation by silica gel column chromatography and high-performance liquid chromatography generated two compounds. The chemical constituents having the antifouling activities were identified as 17-(1,2-dihydroxyl-5-methyl-hexane)-2,3-dihydroxyl-cholest-4-en-6-one [compound (a)] and 13-acetate-17-(1,5-dimethylhexane)-cholest-7-en-3,5,6,15-tetraol [compound (b)] by interpreting one- and two-dimensional nuclear magnetic resonance and mass spectroscopy data. The two compounds were isolated from 780 mg of crude extract, yielding 12 and 14 mg, respectively. Antifouling activities were determined against a representative soft fouling macroalgae (Ulva pertusa), a biofouling diatom (Navicula annexa), and the bacteria Pseudomonas aeruginosa KNP-3 and Alteromonas sp. KNS-8. The two compounds were able to inhibit the spore settlement of Ulva pertusa zoospores with a low half maximal effective concentration (EC50) (1.2–2.1 μg/ml) and inhibit diatom growth with an EC50 of 5.2–7.5 μg/ml. Compound (a) showed activity with minimum inhibitory concentration values of 32–56 μg/ml and compound (b) showed an activity of 66–90 μg/ml. Among the two compounds, compound (a) is a new compound, whereas compound (b) was previously reported to have been isolated from a marine invertebrate.     

Myxosporean and microsporidian infections in cultured Pacific bluefin tuna <Emphasis Type="Italic">Thunnus orientalis</Emphasis> in Japan

During the parasitological survey of cultured juvenile Pacific bluefin tuna (PBT) Thunnus orientalis in 2007 and 2008, two myxosporeans and one microsporidian were found. Morphological and molecular analysis showed that the heart-infecting and brain-infecting myxosporeans are identified as Kudoa shiomitsui and K. yasunagai, respectively. This is a new host record for both species. High prevalence of infection (77–100%) with K. shiomitsui was observed in October to December (1–2 months post-transfer to sea cages), whereas only a few fish were infected with K. yasunagai. A microsporidian observed as white “cysts” in the trunk muscle of PBT had a resemblance to Microsporidium seriolae, the causative parasite of beko disease in yellowtail. Phylogenetic analysis showed that the microsporidian from PBT is closely related to but distinct from several other muscle-infecting species such as M. seriolae, Microsporidium sp. RSB, and Microsporidium sp. SH. Additionally, the spore dimension (2.7 × 1.5 μm on average) was remarkably smaller than the others. These results suggest that the present microsporidian is an undescribed species and designated Microsporidium sp. PBT. Prevalence and intensity of infection with Microsporidium sp. PBT reached a maximum of 100% and 20 cysts/fish, respectively. Although pathogenic effects of the two Kudoa species on fish health remain unknown, the microsporidian could be of concern to PBT aquaculture due to unsightly cysts in the musculature, reducing the market value of the fish.     

Potential of cinnamon (<Emphasis Type="Italic">Cinnamomum verum</Emphasis>) oil to control <Emphasis Type="Italic">Streptococcus iniae</Emphasis> infection in tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

In this study, four essential oils—cinnamon oil, leech lime oil, lemongrass oil, and turmeric oil—were examined for their antimicrobial activities against Streptococcus iniae, a bacterium that is pathogenic in fish, in which it causes streptococcosis. Cinnamon oil was the most potent antimicrobial agent among these oils, with a minimal inhibitory concentration (MIC) of 40 μg/ml. By using gas chromatography–mass spectrometry (GC–MS), it was found that the major components of cinnamon oil were cinnamaldehyde (90.24), limonene (2.42%), cinnamyl acetate (2.03%), linalool (1.16%), and α-terpineol (0.87%). Of these compounds, only cinnamaldehyde exhibited antimicrobial activity against S. iniae, with an MIC of 20 μg/ml. In an in vivo trial, no mortality was apparent in fish fed on fish diets supplemented with 0.4% (w/w) of cinnamon oil and with 0.1% (w/w) of oxytetracycline 5 days prior to infection with S. iniae. These results indicate that cinnamon oil had a protective effect on experimental S. iniae infection in tilapia, and thus has the potential to replace the antibiotics used to control this disease.     

Antibacterial activities of a new combination of essential oils against marine bacteria

The antibacterial activities of essential oils and herbal extracts have been demonstrated against a range of bacterial species. In this study, the antibacterial effects of a new combination of essential oils from the herbs Thymus vulgaris, Salvia officinalis, Eucalyptus globulus and Mentha piperita were assayed against common bacterial isolates (Staphylococcus aureus, Escherichia coli and Pseudomonas aeroginosa) and fifteen novel marine bacteria isolates. The sensitivity of different isolates to antibacterial activity of the essential oils was determined using well diffusion assays. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were quantified by micro dilution assays. The data demonstrate that the combination of essential oils had potent antibacterial effects and marine bacteria were more sensitive to growth inhibition (P < 0.05). MIC rates were 0.77–6.18 mg/ml, and MBC rates were 1.67–12.30 mg/ml. This indicates that the combined essential oils (CEO) can be a new source of antibacterial agents for use in marine aquaculture.     

Identification and functional characterization of Histone‐like DNA‐binding protein in Nocardia seriolae (NsHLP) involved in cell apoptosis

Nocardia seriolae, a facultative intracellular bacterium, is the main pathogen of fish nocardiosis. Bioinformatic analysis showed that the histone‐like DNA‐binding protein (HLP) gene of N. seriolae (nshlp) encoded a secreted protein and might target the mitochondria in the host cell. To further study the preliminary function of HLP in N. seriolae (NsHLP), the gene cloning, extracellular products identification, subcellular localization, overexpression and apoptosis detection assay were carried out in this study. Mass spectrometry analysis of the extracellular products from N. seriolae showed that NsHLP was a secreted protein. Subcellular localization of HLP‐GFP fusion proteins mainly assembled in the nucleus, which indicated that the NsHLP was co‐located with the nucleus rather than mitochondria in fathead minnow (FHM) cells. Notably, the expression of NsHLP had changed the distribution of mitochondria into lumps in the FHM cell. In addition, apoptotic features were found in the transfected FHM cells by overexpression of NsHLP. Quantitative assays of mitochondrial membrane potential value, caspase‐3 activity and pro‐apoptotic genes mRNA (Bad, Bid and Bax) expression level demonstrated that the cell apoptosis was induced in the transfected FHM cells. All the results presented in this study provided insight on the function of NsHLP, which suggested that it may participate in the cell apoptosis regulation and play an important role in the pathogenesis of N. seriolae.     

Effects of temperature and salinity on oxygen consumption and ammonia excretion of juvenile miiuy croaker, <Emphasis Type="Italic">Miichthys miiuy</Emphasis> (Basilewsky)

The metabolic responses of the juvenile Miichthys miiuy in terms of oxygen consumption and ammonia excretion to changes in temperature (6–25°C) and salinity (16–31 ppt) were investigated. At a constant salinity of 26 ppt, the oxygen consumption rate (OCR) of the fish increased with an increase in temperature and ranged between 133.38 and 594.96 μg O₂ h⁻¹ g⁻¹ DW. The effect of temperature on OCR was significant (P < 0.01). Q₁₀ coefficients were 6.80, 1.41, 1.29 and 2.36 at temperatures of 6–10, 10–15, 15–20 and 20–25°C, respectively, suggesting that the juveniles of M. miiuy will be well adapted to the field temperature in the summer, but not in the winter. The ammonium excretion rates (AER) of the fish were also affected significantly by temperature (P < 0.01). The O:N ratio at temperatures of 6, 10, 15 and 20°C ranged from 13.12 to 20.91, which was indicative of a protein-dominated metabolism, whereas the O:N at a temperature of 25°C was 51.37, suggesting that protein-lipids were used as an energy substrate. At a constant temperature of 15°C, the OCRs of the fish ranged between 334.14 (at 31 ppt) and 409.68 (at 16 ppt) μg O₂ h⁻¹ g⁻¹ DW. No significant differences were observed in the OCR and AER of the juveniles between salinities of 26 and 31 ppt (P > 0.05). The OCR and AER at 16 ppt were, however, significantly higher than those at 26 and 31 ppt (P < 0.05), indicating salinity lower than 16 ppt is presumably stressful to M. miiuy juveniles.     

High water temperature tolerance in photosynthetic activity of <Emphasis Type="Italic">Zostera japonica</Emphasis> Ascherson &; Graebner seedlings from Ago Bay,Mie Prefecture,central Japan

Photosynthetic activity of Zostera japonica seedlings was measured using a gas volumeter at 0 and 6 days in culture under eight light (0–800 μmol photons/m²/s) and ten water temperature conditions (5–35°C). Seedlings from Ago Bay, Mie Prefecture were cultured in incubators accurately controlled at each test temperature for 1 week. After 1 week, maximum gross photosynthesis (P _maxg) appeared at 29°C and most seedlings cultured at 30–35°C bleached and withered. At the same time, the light compensation point (I _c) increased only at 30°C during the culture period. As a result, the upper critical water temperature for survival was 29°C in Z. japonica seedlings, which agrees well with that for the southern boundary of Z. japonica around Japanese coast. It is necessary to monitor this species around this boundary as a bio-indicator for seawater warming.     

Identification of a mitochondrial‐targeting secretory protein from Nocardia seriolae which induces apoptosis in fathead minnow cells

Nocardia seriolae is the main pathogen responsible for fish nocardiosis. A mitochondrial‐targeting secretory protein (MTSP) 3141 with an N‐terminal transit peptide (TP) from N. seriolae was predicted by bioinformatic analysis based on the genomic sequence of the N. seriolae strain ZJ0503. However, the function of the MTSP3141 and its homologs remains totally unknown. In this study, mass spectrometry analysis of the extracellular products from N. seriolae proved that MTSP3141 was a secretory protein, subcellular localization research showed the MTSP3141‐GFP fusion protein co‐localized with mitochondria in fathead minnow (FHM) cells, the TP played an important role in mitochondria targeting, and only the TP located at N‐terminus but not C‐terminus can lead to mitochondria directing. Moreover, quantitative assays of mitochondrial membrane potential (ΔΨm) value, caspase‐3 activity and apoptosis‐related gene (Bcl‐2, Bax, Bad, Bid and p53) mRNA expression suggested that cell apoptosis was induced in FHM cells by the overexpression of both MTSP3141 and MTSP3141ΔTP (with the N‐terminal TP deleted) proteins. Taken together, the results of this study indicated that the MTSP3141 of N. seriolae was a secretory protein, might target mitochondria, induce apoptosis in host cells and function as a virulence factor.     

Enzyme-linked immunosorbent assay (ELISA) measurement of vitellogenin in plasma and liver histopathology in barfin plaice <Emphasis Type="Italic">Liopsetta pinnifasciata</Emphasis> from Amursky Bay,Sea of Japan

Vitellogenin (Vg) of the barfin plaice Liopsetta pinnifasciata was isolated and purified. In native polyacrylamide gel electrophoresis, Vg appeared as one band. After being subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE), Vg fraction produced several polypeptides with molecular masses of 180, 98, 70, 52, 41 and 37 kDa. MALDI–TOF mass spectrometry (MS) of the 180- and 98-kDa Vg polypeptides from the SDS–PAGE gel and de novo sequencing of their four peptide fragments based on MS/MS analysis confirmed that the purified proteins were vitellogenins, which shared high similarity with the Vgs of the barfin flounder Verasper moseri and Atlantic halibut Hippoglossus hippoglossus. The most part of the predicted sequences obtained from the L. pinnifasciata 180-kDa polypeptide has previously been found in the V. moseri vitellogenin type B, the sequences obtained from the 98-kDa polypeptide were found in V. moseri vitellogenin type A, so these findings allow us to propose that L. pinnifasciata has at least two different forms of Vg. Rabbit polyclonal antibodies against Vg were produced, and a quantitative enzyme-linked immunosorbent assay was developed. The concentration of Vg in barfin plaice from the moderately contaminated area of Amursky Bay in the Sea of Japan was detected based on the maturity stage of their gonads. In November 2008, the Vg concentration in the plasma of females with advanced oogenesis varied from 5.295 to 28.367 mg/ml (mean 16.38 ± 6.73 mg/ml, CV = 41.1%); in the plasma of males, the concentration ranged from non-detectable to 0.957 mg/ml (0.29 ± 0.42 mg/ml, CV = 127.9%). In October 2009, the Vg concentration in female plasma was lower than in November 2008 (2.21–13.87 mg/ml). High individual variability of plasma Vg was characteristic for maturing males (CV = 200.3%) and immature females (CV = 255.5%), and there was no significant difference between plasma Vg concentrations in males captured in November 2008 and October 2009 or in maturing males and immature females. Vacuolisation of hepatocytes was more typical for males with low plasma Vg concentrations and females with high plasma Vg concentrations. Necrosis and pyknosis of hepatocyte nuclei were more frequent in males with high Vg concentrations and in females with low plasma Vg concentrations.     

Epidemiological study on <Emphasis Type="Italic">Lactococcus garvieae</Emphasis> isolates from fish in Japan

In Japan, Lactococcus garvieae infection has been the main fish disease in aquaculture. Although commercial oral and injectable vaccines have been used to prevent L. garvieae infection in Japan, L. garvieae has been isolated not only from unvaccinated fish but also from vaccinated fish in which immunity induced by vaccination had diminished. In order to obtain epidemiological information on this fish pathogen, we conducted biased sinusoidal field gel electrophoresis (BSFGE) pattern analysis and phage typing of L. garvieae isolates (n = 427) from fish in Japan. These isolates were obtained from 13 different fish species between 1980 and 2007. In the BSFGE analysis, L. garvieae isolates were classified into 17 groups (S1–S17) based on the SmaI digestion patterns and into four groups (A1–A4) based on the ApaI digestion patterns. Phage typing revealed five different phage susceptibility profiles (A–E) in L. garvieae isolates. Since 2005, comparisons of the results of phage typing and BSFGE have indicated the presence of a novel genotype (S16/A4) with phage type E. All the strains belonging to this type showed lincomycin sensitivity.     

Inhibitory activities of the edible brown alga <Emphasis Type="Italic">Laminaria japonica</Emphasis> on glucose-mediated protein damage and rat lens aldose reductase

Increased aldose reductase (AR)-related polyol pathway activity and the subsequent formation of advanced glycation end-products (AGEs) have been implicated in the onset of diabetic complications. We have evaluated the inhibitory effects of the methanolic extract and its fractions of Laminaria japonica on rat lens AR (RLAR) and AGE formation. The CH₂Cl₂ fraction was separated by high-performance liquid chromatography to yield active porphyrin derivatives, including pheophorbide a (1) and pheophytin a (2), which were assayed for their anti-diabetic complications and yield. Compound 1 exhibited potent inhibitory activities against both AGE formation and RLAR, with respective half-maximal inhibitory concentration (IC₅₀) values of 49.4 and 12.3 μM. Conversely, compound 2 was found to be active against AGE formation, with an IC₅₀ of 228.7 μM. For further elucidation of the structure–inhibitory activity relationship of the porphyrin derivatives, the inhibitory activities of four commercially available porphyrin derivatives on AGE formation and RLAR were measured. The presence of a carboxyl group and the absence of a phytyl group at the C-17² position of the porphyrin were found to contribute to the inhibitory effects towards both AGE formation and RLAR. These results suggest that the L. japonica and its porphyrin derivatives may represent a potential functional food resource for further prevention of diabetic complications.     

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.     

Homogeneity of <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> from farmed amberjack <Emphasis Type="Italic">Seriola dumerili</Emphasis> in Japan

Streptococcus dysgalactiae strains have been isolated from cultured amberjack Seriola dumerili and yellowtail Seriola quinqueradiata in Japan. To characterize the fish isolates, we performed genetic analysis and compared the biochemical properties of these isolates with those of the S. dysgalactiae subsp. dysgalactiae and S. dysgalactiae subsp. equisimilis strains isolated from mammals. The genetic analysis revealed that the fish isolates were genetically very similar to each other with high DNA–DNA relatedness (>95.4%) and sequence homology. Meanwhile, the DNA relatedness between mammalian isolates and the fish isolates was 73.4–82.6%. In biased sinusoidal gel electrophoresis (BSFGE) analysis, the restriction patterns of mammalian isolates were different from those of fish isolates. The fish isolates did not show streptokinase activity in plasminogen obtained from mammals. These characteristics enabled us to distinguish between the fish isolates and the Sdd and Sde strains isolated from mammals. In order to obtain epidemiological information on the fish isolates, BSFGE patterns from 284 S. dysgalactiae strains from fish in Japan were examined. Based on the results of BSFGE analysis, the fish isolates were classified into 16 groups (AP1–AP16) with restriction enzyme ApaI. The dendrogram based on BSFGE analysis indicated that all fish isolates using in this study were closely related.     

An increase in the concentration of hepatic iron during the metamorphosis of the lamprey Geotria australis is accompanied by increased superoxide dismutase activity

The concentration of total non-haem iron and its ferritin iron component, and the activity of the enzyme superoxide dismutase (SOD), were measured in the livers of ammocoetes, metamorphosing animals (stages 1–7) and recently metamorphosed downstream migrants of the lamprey Geotria australis. Total non-haem iron in the liver rose significantly from 0.15–0.55 μg.mg wet weight⁻¹ in ammocoetes and metamorphosing stages 1–3 to 2.2–2.9 μg.mg⁻¹ in stages 5–7 and to 8.8 μg.mg⁻¹ in downstream migrants. The comparable values for ferritin iron were 0.06–0.26, 1.4–2.0, and 5.3 μg.mg⁻¹. Superoxide dismutase activity fell sharply from 0.39 μg.mg⁻¹ in large ammocoetes to between 0.07 μg.mg⁻¹ in stage 1 and 0.15 μg.mg⁻¹ in stage 6, before rising significantly to 0.26 μg.mg⁻¹ in stage 7 and 0.35 μg.mg⁻¹ in downstream migrants. The sharp fall in SOD activity at the beginning of metamorphosis is assumed to be related to the marked decline in plasma iron which occurs at the onset of this non-trophic phase in the life cycle. It is proposed that the subsequent increase in SOD activity in the liver of G. australis with increasing iron represents a mechanism aimed at reducing the potentially toxic effects of iron accumulation. This view is consistent with the significant and positive correlation found between both total non-haem and ferritin iron and SOD activity in the liver of non-trophic animals.     

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.     

Effect of gamma-aminobutyric acid and porcine growth hormone on survival of the euryhaline rotifers <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> sp. complex preserved at low temperature

Gamma-aminobutyric acid (GABA) at 50 μg/ml and porcine growth hormone (GH) at 0.025 IU/ml were tested to see whether these chemicals would reduce the stress experienced by euryhaline rotifers Brachionus plicatilis species complex (L-, S- and SS-morphotypes) during low temperature (4–12°C) storage. Rotifers cultured at 25°C were transferred to 4–12°C for 10–30 days and transferred back to 25°C for recovery. GABA or GH were added to the rotifers at three different time points: 6 h before transfer from 25°C to low temperature (6h−), on day 7 after preservation at low temperature (7d+) and on the first day of recovery. For L-type rotifers, the GH treatment before the transfer to 4°C for 30 days was effective for better survival, while the GABA treatment was most effective for the S-type preserved at 10°C for 14 days. For the SS-type, the chemical treatments were not effective when the rotifers were preserved at 12°C for 14 days. After low temperature preservation, GABA treatments with the S- and SS-type rotifers just after their transfer to 25°C induced a relatively faster recovery of the rotifer population.     

Growth and feed utilization of gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>, L.) fed to satiation and restrictively at increasing dietary energy levels

Three isoproteic (47% protein) diets were formulated to contain graded levels of crude fat (diet D16:16%, diet D24: 24% and diet D32: 32%). Each diet was fed to satiation in three and to 80% satiation in two replicate groups of gilthead sea bream (Sparus aurata), having an initial body weight of 72–74 g. The trial lasted 81 days. Groups fed to satiation showed higher final body weight (FBW; 238.8–252.3 g vs. 218.0–229.3 g) and daily growth index (DGI; 2.49–2.65%/day vs. 2.27–2.34%/day) than those fed to 80% satiation. Feed intake was significantly different both for feeding level and for diet composition. Fish fed to satiation had higher feed conversion rate (FCR) compared to the 80% satiation groups (1.33–1.44 vs. 1.13–1.17; P ≤ 0.001). Within satiation groups, FCR was significantly lower in fish fed D16 compared to fish fed D32 (1.33 vs. 1.44, P ≤ 0.05), whereas no statistical differences were found within the 80% satiation groups. The increase in dietary lipid level did not improve growth performance, feed efficiency and protein utilization but decreased gross lipid efficiency. Conversely, a reduction in ration from satiation to 80% satiation decreased DGI, thus improving FCR. Feed costs were influenced by dietary energy level and feeding ratio, the lowest energy diet at 80% satiation being the most profitable combination among the variables.     

Sequencing of 16S−23S rRNA internal transcribed spacer and its application in the identification of Nocardia seriolae by polymerase chain reaction

A method for the diagnosis of nocardiosis in yellowtail (Seriola quinqueradiata), using polymerase chain reaction (PCR), was developed in this study. Primers specific for Nocardia seriolae were synthesized based on the alignment of 16S?23S rRNA internal transcribed spacer region sequences of N. seriolae. The primers did not amplify specific PCR product from other fish pathogens. However, two and three fishes could be diagnosed as infected with N. seriolae by clinical signs and bacterial isolation. PCR amplification of N. seriolae by specific primers detected six infected fishes. Thus, the primers used in this study are useful in detecting nocardiosis in fish.