Effect of salinity on occurrence of notochord deformities in Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis> larvae

One of the difficulties to rear Anguilla japonica larvae is the frequent occurrence of notochord deformities. We tested the effect of salinity on the occurrence of the deformities, because we have been using 50 % diluted seawater (50 % SW) for glass eel production, on the basis of the fact that intermediate salinity saves energy due to lower cost for osmoregulation and contributes higher survival and growth rates. We reared 6-day-old larvae in 50 and 100 % SW for 85 days and observed their morphology. The occurrence rate of deformed larvae, including kyphosis and scoliosis, was significantly higher in 50 % SW (35.8 %) than in 100 % SW (25.4 %), while survival rate was significantly higher in 50 % SW (69.8 %) than in 100 % SW (32.3 %) and growth in 50 % SW (mean body depth: 7.9 ± 5.3 mm) was better than in 100 % SW (6.8 ± 4.6 mm). We speculate that the most of severely deformed larvae could not survive in the tougher condition of 100 % SW, showing the lower occurrence of deformed larvae. Eventually, the yield of normal larvae after 85 days rearing was 1.9-fold higher in 50 % SW than in 100 % SW, implying that the advantage of 50 % SW for rearing eel larvae outweighs the risk of deformities.     

Rapid identification of eels <Emphasis Type="Italic">Anguilla japonica</Emphasis> and <Emphasis Type="Italic">Anguilla anguilla</Emphasis> by polymerase chain reaction with single nucleotide polymorphism-based specific probes

ABSTRACT:   Standard molecular techniques, such as sequencing and restriction fragment length polymorphism analysis after polymerase chain reaction (PCR) amplification are relatively complicated, and species identification can take a long time when using such techniques. We established a quick method, using PCR with species-specific TaqMan Minor Groove Binder (MGB) probes based on single nucleotide polymorphism (SNP) to distinguish the two eel species Anguilla japonica and Anguilla anguilla . This method can be used in processed products. Partial sequences of the mitochondrial 16S rRNA gene were compared between A. japonica and A. anguilla to design a primer pair common to both A. japonica and A. anguilla and probes specific to A. japonica and A. anguilla . Different fluorescence intensities were produced in two PCR microtubes each containing A. japonica - and A. anguilla -specific probes for one target sample. We observed the fluorescence intensity of PCR products in microtubes under ultraviolet transillumination, with similar results to those obtained by real-time PCR. Therefore, SNP-based PCR is a powerful tool for identifying materials of processed foods from either A. japonica or A. anguilla .     

Occurrence of estuarine and sea eels <Emphasis Type="Italic">Anguilla japonica</Emphasis> and a migrating silver eel <Emphasis Type="Italic">Anguilla anguilla</Emphasis> in the Tokyo Bay area,Japan

Otolith microchemical analyses of the strontium (Sr) and calcium (Ca) concentrations in the eels Anguilla japonica and A. anguilla caught in Tokyo Bay were undertaken to reconstruct the eels’ migratory histories. A. japonica in the yellow stage (immature stage) were caught in a bay without any adjacent rivers or streams. A. anguilla was in the silver stage (early maturing stage), and the eel was confirmed to have just begun spawning migration to the open ocean from Tokyo Bay based on the otolith Sr:Ca ratios, which showed a typical catadromous life history with low Sr:Ca ratio values throughout the eel’s life after recruitment. The mean Sr:Ca ratios in A. japonica from the elver mark to the otolith edge indicated the eels belonged to several general categories of migratory histories, including sea eels (average Sr:Ca ratio ≥6.0 × 10⁻³) and estuarine eels (average Sr:Ca ratio 2.5 to 6.0 × 10⁻³) based on the criteria reported previously in A. japonica. All eels had a certain freshwater life period, although the period was highly variable among fish. These results indicate that A. japonica has a flexible pattern of migration, with the ability to adapt to various habitats and salinities.     

Anosmic Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis> can no longer detect magnetic fields

ABSTRACT:   Magnetosensitivity in anosmic Japanese eel Anguilla japonica was examined by conditioning and electrocardiography. Five eels from a freshwater farm in Kagoshima, Japan, were made anosmic by injection of boiling petroleum jelly (130–140°C) into the nasal cavity, a technique widely used in studies on olfaction and olfaction-related behavior of eels. Another five eels were not injected with boiling petroleum jelly and served as controls. Conditioning consisted of exposure of eels to an imposed magnetic field of 192 473 nT. After complete conditioning of the eels, the imposed magnetic field was reduced from 192 473 nT to 12 663 nT during the tests. The control eels showed a significant conditioned response (i.e. slowing of the heart beat) to the magnetic field after one set of conditioning runs. The anosmic eels did not respond to the magnetic field, presumably due to damage of the olfactory organ from the jelly injection. The results suggest that Japanese eels have a magnetosensitive organ in or around the nares. At 1 month after the anosmia treatment, there was no visible trauma to the olfactory lamellae of anosmic eels.     

Effect of combination feeding of <Emphasis Type="Italic">Nannochloropsis</Emphasis> and freshwater <Emphasis Type="Italic">Chlorella</Emphasis> on the fatty acid composition of rotifer <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> in a continuous culture

ABSTRACT:   A continuous culture of rotifer was conducted to investigate the effect of combination feeding of both a high density of Nannochloropsis oculata (N) and condensed freshwater Chlorella (FC) on the fatty acid composition of L-type rotifer Brachionus plicatilis in a continuous culture system. The algal feeding of the rotifers was carried out in three successive steps: N-feeding → N+FC-feeding → FC-feeding. The culture was conducted at 24°C and 25–27 psu in a 2000 mL bottle with 50% of water exchanged daily. The combination N+FC-feeding was effective in increasing rotifer density. The rotifers fed on N+FC (N+FC-R) had more non-polar lipids than polar ones, similar to those on N (N-R), opposite to the rotifers fed on FC (FC-R). N+FC-R contained higher levels of 16:2, 18:2n-6 (linoleic acid [LA]) and 20:2n-6, but lower levels of 18:1, 20:4n-6 (arachidonic acid), 20:5n-3 (eicosapentaenoic acid [EPA]) and 22:5n-3 (docosapentaenoic acid [DPA]) compared with N-R. Whereas N+FC-R contained higher levels of 16:1n-7, EPA and DPA, but lower levels of 16:2 and LA compared with FC-R. N+FC-R had more DPA in polar lipids than in non-polar ones. The Σn-6/Σn-3 ratio in N+FC-R was 0.9–1.0, significantly different from those in N-R (0.4) and FC-R (6.6–8.4). Therefore, it is inferred that the fatty acid profile of the N+FC-R cultured in a continuous culture system was affected by both N and FC. Also, the combination N+FC-feeding may be effective in manipulating the Σn-6/Σn-3 ratio in continuously cultured rotifers.     

Growth and survival of grouper <Emphasis Type="Italic">Epinephelus</Emphasis><Emphasis Type="Italic">coioides</Emphasis> (Hamilton) larvae fed free-living nematode <Emphasis Type="Italic">Panagrellus redivivus</Emphasis> at first feeding

The free-living nematode, Panagrellus redivivus, was tested as live food for grouper Epinephelus coioides larvae during the first feeding stage. A series of experiments were conducted to determine the acceptability of the free-living nematodes in grouper larvae at first feeding, the optimum nematode density and the response of the larvae to nutritionally enriched nematode. All experiments were conducted in 200-L conical tanks filled with 150-L filtered seawater and stocked at 15 larvae L⁻¹. Duration of feeding experiments was up to day 21 (experiment 1) and 14 days (experiment 2 and 3). Brachionus plicatilis and Artemia (experiment 1) and Brachionus plicatilis alone (experiment 2 & 3) was used as the control treatment. Observations indicated that the grouper larvae readily fed on free-living nematodes as early as 3 days posthatching, the start of exogenous feeding. Optimum feeding density for the larvae was 75 nematodes ml⁻¹. The enrichment of cod liver oil or sunflower oil influenced the total lipids and n-3 highly unsaturated fatty acids of P. redivivus, which in turn influenced those of the grouper larvae, however, growth and survival of the larvae were not affected (P > 0.05). The results from this investigation showed that the nematode, P. redivivus, can be used as first live food for grouper larvae from the onset of exogenous feeding until they could feed on Artemia nauplii.     

Spatial learning-induced <Emphasis Type="Italic">egr</Emphasis>-<Emphasis Type="Italic">1</Emphasis> expression in telencephalon of gold fish <Emphasis Type="Italic">Carassius auratus</Emphasis>

The immediate-early gene (egr-1) expression was used to examine the neuron’s response in telencephalon of goldfish during spatial learning in small space. Fishes were pre-exposed in the experimental apparatus and trained to pick food from the tray in a rectangular-shaped arena. The apparatus was divided into identical compartments comprising three gates to provide different spatial tasks. After the fish learned to pass through the gate one, two more gates were introduced one by one. Fish made more number of attempts and took longer time (P < 0.05) to pass through the first gate than the gate two or three. This active learning induces the expression of egr-1 in telencephalon as established by western blot analysis. Subsequently, the fish learn quickly to cross the similar type of second and third gate and make fewer errors with a corresponding decline in the level of egr-1 expression. As the fish learned to pass through all the three gates, third gate was replaced by modified gate three. Interestingly, the level of egr-1 expression increased again, when the fish exhibit a high exploratory behavior to cross the modified gate three. The present study shows that egr-1 expression is induced in the telencephalon of goldfish while intensively acquiring geometric spatial information to pass through the gates.     

Influence of the dietary inclusion of <Emphasis Type="Italic">Gracilaria cornea</Emphasis> and <Emphasis Type="Italic">Ulva rigida</Emphasis> on the biodiversity of the intestinal microbiota of <Emphasis Type="Italic">Sparus aurata</Emphasis> juveniles

Partial substitutions of fish meal by 5, 15, or 25 % of Gracilaria cornea or Ulva rigida in experimental diets were evaluated to study their effects on biodiversity of intestinal microbiota composition in gilthead seabream (Sparus aurata) juveniles. The diets were offered to duplicate groups of 15 juvenile fish (14.0 ± 0.5 g) for 70 days, and at the end of the experiment the intestinal microbiota from four specimens of each treatment was analysed by denaturing gel gradient electrophoresis. Results showed that the substitution of fish meal by algae meal induced important modifications in the intestinal microbiota community, as a big reduction of the biodiversity when the highest percentage (25 %) of U. rigida was included. On the contrary, an increase on the number of species was detected when a 15 % of algae was included. Various Lactobacillus delbrueckii subspecies were selectively stimulated when G. cornea was included in the feed, and other bacterial species, such as those included in the Vibrio genus, were reduced.     

Effects of silvering state on induced maturation and spawning in wild female Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis>

ABSTRACT:   The effects of silvering state of wild female Japanese eels Anguilla japonica on the success of induced maturation and the following spawning were examined. Thirty-eight females, collected in Mikawa Bay, were divided into four stages based on their silvering state: yellow (Y1), late-yellow (Y2), silver (S1) and late silver eels (S2). Despite injections of salmon pituitary extract (SPE) through the standard technique, Y1 and Y2 eels did not respond to the treatment with undeveloped gonad (gonad-somatic index [GSI]: 0.3–0.9), and all these females died by 5 weeks, probably due to an abnormal physiological condition. Most S1 (81%) and S2 eels (100%) matured completely (GSI: 17.8–51.4), and finally spawned successfully (69% for S1, 89% for S2). S2 eels fully matured with oocytes of over 750 μm in diameter by significantly smaller number of injections of SPE (5–6 times) than the case of S1 eels (6–8 times). The amount of eggs released by S2 eels (0.65 ± 0.11 g/fish per body weight [BW]) was significantly larger than those by S1 eels (0.54 ± 0.09 g/fish per BW). There was no difference in fertilization and hatching rates between eggs released by S1 eels and those of S2 eels. These results indicate that the success of induced maturation and spawning in wild female Japanese eels depends on their silvering state, and matured eggs can be obtained efficiently through the use of S2 eels rather than other stages.     

Characterization of <Emphasis Type="Italic">kiss2</Emphasis> and <Emphasis Type="Italic">kissr2</Emphasis> genes and the regulation of kisspeptin on the HPG axis in <Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>

Reproduction allows organisms to produce offspring. Animals shift from immature juveniles into mature adults and become capable of sexual reproduction during puberty, which culminates in the first spermiation and sperm hydration or ovulation. Reproduction is closely related to the precise control of the hypothalamic–pituitary–gonadal (HPG) axis. Kisspeptin peptides are considered as the important regulator of HPG axis in mammalian. However, the current understanding of kisspeptin in flatfish is not comprehensive. In this study, we cloned and analyzed the kiss2 and kissr2 genes in Cynoglossus semilaevis. Interesting alternative splicing in the 5′-untranslated regions (UTR) of the Cskissr2 gene was found. The expression profiles of Cskiss2 and Cskissr2 showed relative high messenger RNA (mRNA) levels at the late gastrula stage during embryonic development, at total length = 40 mm during early gonadal differentiation, and in the brains and gonads of all investigated tissues. These results suggested that the kisspeptin system participated in embryogenesis and in the regulation of gonadal differentiation and development. Considering that the control and regulatory mechanisms of kisspeptin in the central reproductive axis are still unclear, we documented that the intramuscular injection of kisspeptin caused different sGnRH and cGnRH mRNA levels in a dose- and tissue-dependent manner. The mRNA expressions of FSH and LH were stimulated in the ovary and were inhibited in the testis under the kisspeptin treatments. These results provided foundations for understanding the roles of kisspeptin in the neuroendocrine system in fish. The manipulation of the kisspeptin system may provide new opportunities to control the gonadal development and even reproduction in fish.     

Predation on glass eels of Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis> in the Tone River Estuary,Japan

Aquaculture of Japanese eel Anguilla japonica relies upon the natural recruitment of their glass eels (juveniles); however, predation that could influence glass eel recruitment remains unknown. In the present study, we aimed to elucidate the proportion of predation on A. japonica glass eels through stomach content analysis of predatory fishes collected in the estuary region of the Tone River system and its vicinity in Japan. Species of the predated glass eels were identified by DNA barcoding. A total of 270 predatory fishes of 15 taxa was collected over 2 years. The overall proportion of predation on glass eels, genetically identified as Japanese eel, was 0.7%, but this rose to 2.0% when data were limited to fishes caught during the peak months of glass eel recruitment. A glass eel was found in the stomach contents of a channel catfish Ictalurus punctatus, an invasive species in this river system, and a blackfin sea bass Lateolabrax latus. These fishes are therefore considered potential predators of A. japonica glass eels. However, as the proportion of predation was low, and the glass eels represented only small proportions of predator stomach contents, further investigation is needed for a better understanding of predation on A. japonica glass eels, and its effects on the early life stages of this endangered species.     

Monitoring of <Emphasis Type="Italic">Francisella noatunensis</Emphasis> subsp. <Emphasis Type="Italic">orientalis</Emphasis> in farmed Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>) in Brazil

Francisella noatunensis orientalis is a bacterium that causes emerging bacteriosis in Nile tilapia (Oreochromis niloticus) in many parts of the world, including Brazil. It is a non-motile, Gram-negative, strictly aerobic, facultative intracellular coccobacillus. This species of bacteria is responsible for low to high mortality in fish farms, causing economic losses for fish farmers. This study aimed to detect the presence of F. noatunensis orientalis using qPCR (real-time polymerase chain reaction) and to describe lesions caused by the bacterium in O. niloticus in Brazilian aquaculture. For this purpose, 360 fish from six fish farms (30 per farm) were sampled at two time points (n = 180 per sampling). Necropsies and histopathology were performed for lesion observation, in addition to qPCR and sequencing for detection and identification of Francisella species. Environmental data were collected using a multiparameter sonde YSI EXO2. All measured limnological variables were within the optimum range for cultivation of Nile tilapia. The major lesions present were melanization of the skin, splenomegaly, granulomas, and inflammatory cell responses. The prevalence of francisellosis varied from 0 to 86.66% between time periods and fish farms analyzed, and an outbreak was observed during the second sampling period. This study describes the prevalence of francisellosis in O. niloticus and reports that the lesions found are not exclusively associated with this bacterial disease.     

Influence of <Emphasis Type="Italic">Tribulus terrestris</Emphasis> on testicular enzyme in fresh water ornamental fish <Emphasis Type="Italic">Poecilia latipinna</Emphasis>

The influence of Tribulus terrestris on the activities of testicular enzyme in Poecilia latipinna was assessed in lieu of reproductive manipulation. Different concentrations of (100, 150, 200, 250, and 300 mg) Tribulus terrestris extract and of a control were tested for testicular activity of enzymes in Poecilia latipinna for 2 months. The testis and liver were homogenized separately in 0.1 mol/l potassium phosphate buffer (0.1 mol/l, pH 7.2). The crude homogenate was centrifuged, and supernatant obtained was used as an enzyme extract for determination of activities. The activities of testicular functional enzyme ALP, ACP, SDH, LDH, and G6PDH levels were changed to different extent in treated groups compared with that of the control. The total body weight and testis weight were increased with the Tribulus terrestris-treated fish (Poecilia latipinna). These results suggest that Tribulus terrestris induced the testicular enzyme activity that may aid in the male reproductive functions. It is discernible from the present study that Tribulus terrestris has the inducing effect on reproductive system of Poecilia latipinna.     

Effect of <Emphasis Type="Italic">Cratoxylum formosum</Emphasis> on innate immune response and disease resistance against <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> in tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

The effect of aqueous extract of Cratoxylum formosum on innate immune response and disease resistance in tilapia Oreochromis niloticus was investigated. The fish were fed diets containing 0% (control), 0.5% (diet 1), 1% (diet 2), and 1.5% (diet 3) of C. formosum aqueous extract for 30 days. At the end of the experimental period, parameters of innate immune response including phagocytosis of blood leukocytes, lysozyme activity in plasma, and respiratory bust activity were examined. Feeding the fish with diet 2 and diet 3 for 20 days enhanced phagocytic activity and for 30 days stimulated lysozyme and respiratory burst activities. Diet 1 increased the phagocytic activity at 30 days, but did not affect the other measured parameters. All parameters were not significantly changed (P > 0.05) in the control group throughout the experiment. Following 30 days of feeding, fish were infected with S. agalactiae. The cumulative mortalities of bacterial-infected tilapia that were fed diet 1, diet 2, and diet 3 were 56, 12, and 10%, respectively, compared with 85% in the control group. These results indicate that the aqueous extract of C. formosum may elevate the innate immune response and enhance disease resistance in tilapia.