<Emphasis Type="Italic">Neoheterobothrium hirame</Emphasis> (Monogenea) alters the feeding behavior of juvenile olive flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

We monitored feeding behavior and survival of starved juvenile olive flounder experimentally infected with the gill monogenean Neoheterobothrium hirame. Infected flounder increased amount of the time spent in the water column by 117% when trying to capture live mysids, Neomysis sp. They also showed different feeding patterns from those of uninfected fish and made fewer attacks towards prey during one feeding attempt. Although the average numbers of mysids captured by individuals were similar between infected and uninfected fish, heavily infected fish tended to catch less prey. These results indicate that N. hirame reduces the feeding efficiency of the host for capturing live prey and possibly makes them more vulnerable to predation during feeding. We could not detect any obvious difference in survival rates between uninfected, lightly and heavily infected fish during 3 months of starvation. There was no evidence that starvation makes fish more susceptible to N. hirame. The present study provides first experimental evidence that N. hirame affects feeding behavior of juvenile olive flounder and supports the idea that this parasite indirectly reducing the host’s survival and may be responsible for the recent reduction of the flounder population in Japan.     

Integrated production of fish (pacu <Emphasis Type="Italic">Piaractus mesopotamicus</Emphasis> and red tilapia <Emphasis Type="Italic">Oreochromis</Emphasis> sp.) with two varieties of garnish (scallion and parsley) in aquaponics system

Aquaponics is emerging as an alternative for high-health food production. Being able to identify the technical viability of non-conventional plants and fish species would help to increase the interest and possibilities in aquaponic systems. The goal of the present study was to evaluate the aquaponics production of two garnish species: scallion (S) and parsley (P), using effluents of pacu and red tilapia culture. Two aquaponics devices were used, differing according to the fish species, generating two different effluents. Thus, for plant performance, four treatments were evaluated in a factorial design (plant species and fish effluent as main factors), as followed: Pacu-S, Tilapia-S, Pacu-P, and Tilapia-P, with three replicates each, for 35 days. Fish performance was evaluated using Student’s t test. Each experimental device included a fish tank, filters, and six experimental units for the plants (floating rafts). Results indicated that feed conversion ratio (FCR) was higher in tilapia as compared to pacu (p < 0.05); however, fish productivity and survival were similar between species. Plant performance parameters were similar with no significant differences regardless of the fish effluent (p > 0.05), except for higher number of leaves per plant in scallion cultured using pacu effluent. Plant performance comparing both plant species indicated that scallion performed better as compared to parsley in all parameters. In addition, scallion also performed better related to the plant quality index. The results indicate that pacu presented a viable alternative for the aquaponics production, and regarding to the garnish, scallion performed better results as compared to parsley.     

Repeatable immersion infection with <Emphasis Type="Italic">Photobacterium damselae</Emphasis> subsp. <Emphasis Type="Italic">piscicida</Emphasis> reproducing clinical signs and moderate mortality

Pseudotuberculosis is a bacterial septicaemia caused by Photobacterium damselae subsp. piscicida in several marine fish species. Yellowtail Seriola quinqueradiata is the most sensitive fish species to this disease. The internal organs of naturally infected yellowtail exhibit whitish spots, tubercle-like tissue structures, consisting of bacterial accumulations. There have been many trials for experimental infection, however adequate method of infection that reproduces moderate mortality and primary clinical signs has not yet established. Present investigation evaluated an immersion infection method by using logarithmic culture-phase bacteria resulting in higher mortality than that using stationary culture-phase bacteria. Typical white spots on the spleen and kidney were also observed constantly in dead fish. Transmission electron microscopy and fluorescent antibody microscopy showed bacterial clusters not only in the spleen and kidney but also in the blood channels in the secondary gill filaments. These results were confirmed repeatedly by plural experiments. The use of logarithmic-phase bacteria in immersion infection is an appropriate technique to reproduce moderate mortality and primary clinical signs, which will be a reliable infection method also for the challenge test of pseudotuberculosis vaccine.     

Corn gluten meal in pacu <Emphasis Type="Italic">Piaractus mesopotamicus</Emphasis> diets: effects on growth,haematology, and meat quality

Corn gluten meal (CGM) has a high protein content and absence of antinutrients. However, it has high levels of carotenoids that can cause a yellowing of fish fillets and impair further commercialisation. Graded levels of CGM were incorporated in pacu diets to replace soybean meal (SM) protein. The experimental diets were formulated to be isonitrogenous (220 g kg^?1 digestible protein) and isoenergetic (13.4 MJ kg^?1 digestible energy) with 0, 25, 50, 75, and 100 % replacement of SM protein by CGM protein over a 60-day period. One hundred fish (32.79 ± 3.43 g) were randomly distributed in 20 experimental cages (70 L) placed within five (1000 L) aquaria with continuous water renewal. The experimental design was completely randomised with five treatments and four replicates. A quadratic effect (P < 0.05) was observed for fillet and carcass yields, feed conversion ratio, weight gain, and specific growth rate, with the optimum values for replacement of SM protein by CGM protein estimated as 21.95, 29.13, 30.94, 37.71, and 38.75 %, respectively. There were no differences (P > 0.05) for blood parameters, proximal composition, water-holding capacity, pH, lipid oxidation, and a* value of fillets. The L* and b* values of fillets showed differences (P < 0.05). Replacement between 21.95 and 38.75 % of SM protein by CGM protein (5.37 and 9.48 % of inclusion in the diet) improves the growth performance and body yield of pacu juveniles without altering blood parameters or affecting the fillet quality.     

Induction of diploid gynogenesis in turbot <Emphasis Type="Italic">Scophthalmus maximus</Emphasis> with left-eyed flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> sperm

Turbot Scophthalmus maximus exhibits sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. In this paper, gynogenetic diploids of turbot were induced by activating egg development with ultraviolet (UV)-irradiated left-eyed flounder Paralichthys olivaceus sperm combined with cold shock to prevent extrusion of the second polar body. The results of UV irradiation experiments showed that survival, motility, and duration of activity of P. olivaceus sperm generally decreased with increase in UV dose. The typical Hertwig’s effect was observed after fertilized turbot eggs with UV-irradiated P. olivaceus sperm and the optimal UV dose for gynogenetic haploid production was 36,000 erg mm⁻². At 15°C, appropriate timing of cold shock for retention of the second polar body in turbot eggs was at 6 min after fertilization. Results of different combinations of two shock temperatures (1 or 3°C) and four shock durations (15, 25, 35 or 45 min) at 6 min after fertilization demonstrated that shock of 25 min at 1°C gave the highest production of diploid gynogens (39.58% relative to its diploid control). The results of this study reveal that the use of UV-irradiated P. olivaceus sperm for activation of turbot eggs and cold shock for polar body retention is an effective method to produce gynogenetic offspring.     

Effects of soy sauce <Emphasis Type="Italic">koji</Emphasis> and lactic acid bacteria on the fermentation of fish sauce from freshwater silver carp <Emphasis Type="Italic">Hypophthalmichthys molitrix</Emphasis>

The effects of soy sauce koji and the lactic acid bacterium, Tetragenococcus halophilus, were studied on the fermentation of fish sauce prepared from Chinese silver carp. The fish sauce prepared without koji and the lactic acid bacterium contained low levels of organic materials, total nitrogen, and organic acids. The use of koji was effective in increasing these qualitative parameters and further improved the amino acid score of the fish sauce. Addition of T. halophilus had an effect on lowering the pH value during the initial period of fermentation when the soy sauce koji was also supplemented. In contrast, T. halophilus-like bacteria were found to be predominant for all tanks fermented under the different starting conditions. Although it was not examined whether the T. halophilus-like bacteria observed after fermentation were the same as the starter-bacterium or not, it was suggested that T. halophilus plays an important role in the successful fermentation of silver carp fish sauce. Sensory evaluation conducted with Japanese and Chinese panelists also suggested the superiority of the use of koji for fermentation of silver carp fish sauce.     

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.     

Intraspecific diversity of <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> (Harvey) Suringar (Laminariales,Phaeophyta) from Japan,China and Korea,based on the <Emphasis Type="Italic">cox1</Emphasis> gene and ITS2 sequences

As a trial to develop a method of authenticating the place of origin of circulated Undaria pinnatifida products, we investigated their intraspecific genetic diversity using the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) and the internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal DNA (rDNA) sequence. Four dried U. pinnatifida products labeled with their origins (one from Japan, one from China and two from Korea), natural plants collected from three locations (two from Japan and one from China), and cultivated plants collected from two locations (one from Japan and one from China) were used in the present study. The amplified fragments of cox1 were 664 bp in length, and the aligned sequences were highly homologous. Among the nine sequences, no insertions or deletions were found and six substitution positions were detected, and they were classified into five haplotypes. In contrast, multiple highly variable regions were found in ITS2, and some of them carried a restriction site for Mbo II. Polymerase chain reaction-restriction fragment length polymorphism analysis showed different restricted profiles among the tested samples. The availability of molecular markers for authenticating food products of U. pinnatifida is discussed.     

Plasma biochemical responses of the omnivorous crucian carp (<Emphasis Type="Italic">Carassius auratus</Emphasis>) to crude cyanobacterial extracts

Healthy crucian carp (Carassius auratus) were treated by intraperitoneal (i.p.) injection of crude cyanobacterial extracts at two doses, 50 and 200 μg MC-LR equiv kg⁻¹ BW. High mortality (100%) was observed within 60 h post injection in the high-dose group. In the treated fish, activities of four plasma enzymes, alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), all showed substantial increases, with both dose and time-dependent effects. These increases of enzyme activity indicate severe impairment occurred in the liver of crucian carp over time. Plasma concentrations of energy-related biomolecules including glucose (GLU), cholesterol (CHO), triglyceride (TG), and total protein (TP) showed marked changes in the high-dose group, possibly a nutritional imbalance correlated with the liver injury caused by intraperitoneal exposure to crude cyanobacterial extracts.     

Circadian melatonin release <Emphasis Type="Italic">in vitro</Emphasis> by European sea bass pineal

Melatonin production by the fish pineal is under the control of photoperiod. In addition, in some fish species pineal melatonin production is self-sustained by an intrapineal circadian pacemaker, not in salmonids, though. The aim of this research was to test melatonin release in vitro by the pineal of sea bass held under LD 12:12 and DD conditions to determine the endogenous nature of melatonin rhythm. Circadian rhythmicity ( = 28.3 h) was found since rhythmic melatonin release persisted on DD.     

Dietary supplementation of curcumin augments heat stress tolerance through upregulation of <Emphasis Type="Italic">nrf-2</Emphasis>-mediated antioxidative enzymes and <Emphasis Type="Italic">hsps</Emphasis> in <Emphasis Type="Italic">Puntius sophore</Emphasis>

Heat stress is one of the major environmental concerns in global warming regime and rising temperature has resulted in mass mortalities of animals including fishes. Therefore, strategies for high temperature stress tolerance and ameliorating the effects of heat stress are being looked for. In an earlier study, we reported that Nrf-2 (nuclear factor E2-related factor 2) mediated upregulation of antioxidative enzymes and heat shock proteins (Hsps) provide survivability to fish under heat stress. In this study, we have evaluated the ameliorative potential of dietary curcumin, a potential Nrf-2 inducer in heat stressed cyprinid Puntius sophore. Fishes were fed with diet supplemented with 0.5, 1.0, and 1.5% curcumin at the rate 2% of body weight daily in three separate groups (n = 40 in each group) for 60 days. Fishes fed with basal diet (without curcumin) served as the control (n = 40). Critical thermal maxima (CTmax) was determined for all the groups (n = 10, in duplicates) after the feeding trial. Significant increase in the CTmax was observed in the group fed with 1.5% curcumin- supplemented fishes whereas it remained similar in groups fed with 0.5%, and 1% curcumin-supplemented diet, as compared to control. To understand the molecular mechanism of elevated thermotolerance in the 1.5% curcumin supplemented group, fishes were given a sub-lethal heat shock treatment (36 °C) for 6 h and expression analysis of nrf-2, keap-1, sod, catalase, gpx, and hsp27, hsp60, hsp70, hsp90, and hsp110 was carried out using RT-PCR. In the gill, expression of nrf-2, sod, catalase, gpx, and hsp60, hsp70, hsp90, and hsp110 was found to be elevated in the 1.5% curcumin-fed heat-shocked group compared to control and the basal diet-fed, heat-shocked fishes. Similarly, in the liver, upregulation in expression of nrf-2, sod, catalase, and hsp70 and hsp110 was observed in 1.5% curcumin supplemented and heat shocked group. Thus, this study showed that supplementation of curcumin augments tolerance to high temperature stress in P. sophore that could be attributed to nrf-2-induced upregulation of antioxidative enzymes sod, catalase, gpx, and the hsps.     

Weaning of greater amberjack (<Emphasis Type="Italic">Seriola dumerilii</Emphasis> Risso 1810) juveniles from moist to dry pellet

The weaning of greater amberjack (Seriola dumerilii Risso 1810) to a dry diet was studied in two consecutive experiments. The experimental fish were hatchery-produced and grown to their initial size on a soft pellet containing 50% trash fish and 50% commercial dry pellet. In the first experiment, three homogenous groups of greater amberjacks (n = 153, mean weight: 374 ± 43 g), initially adapted for a period of 30 days to a diet with a 40% moisture content, were fed on commercial pellets containing three different levels of dietary moisture (7, 20 and 40%, respectively) for 126 days. We found that fish needed a month to adapt to the 7%- and 20%-moisture diets and that moisture content of the diet had a significant effect on fish growth in terms of increasing feed consumption and lowering feed conversion ratio. The group fed on the 20%-moisture diet not only compensated for the initial adaptation period but presented the higher growth rate and best feed conversion ratio. In the second experiment, all groups of experiment 1 were fed the same commercial diet with a 7% moisture content for 72 days. The group that had been previously fed on the 20%-moisture diet showed the best growth performance. The implication of the above results for the weaning of greater amberjack to dry diets is discussed in terms of the digestion process and physiology.     

Circadian locomotor activity in Japanese sea catfish <Emphasis Type="Italic">Plotosus lineatus</Emphasis>

To find out whether or not the locomotor activity of the Japanese sea catfish, Plotosus lineatus, shows a circadian rhythmicity, the locomotor activity of this catfish was recorded in a laboratory. All of the catfish recorded (n = 20) showed a nocturnal locomotor activity rhythm under a light–dark (LD) cycle (LD 12:12). The locomotor activity started after the light was turned off, and declined during the period of darkness. In five of the six catfish we examined, locomotor activity shifted gradually over the course of 3–4 days in order to synchronize to the LD cycle after it was advanced by 6 h. Locomotor activity persisted within a constant darkness (DD) in all eight of the catfish. The average free-running period of locomotor activity under DD lasted for 24.2 ± 0.4 h (mean ± SD). Thus, the present results demonstrate that the Japanese sea catfish has an endogenous circadian oscillator entrained by an LD cycle.     

Determination of hormones inducing oocyte maturation in <Emphasis Type="Italic">Chalcalburnus tarichi</Emphasis> (Pallas, 1811)

Chalcalburnus tarichi is an endemic cyprinid species living in the Lake Van basin, in eastern Anatolia, Turkey. The present study was undertaken to determine which hormones induce oocyte maturation in C. tarichi. The levels of 17α,20β,21-trihydroxyprogesterone (20β-S), progesterone (P), 17α-hydroxyprogesterone (17α-HOP), 11-deoxycortisol (11-DOC), and 17α-hydroxy-20β-dihydroprogesterone (17,20β-P) were measured in fish caught from Lake Van and the Karasu River, and injected with human chorionic hormone (hCG) (1,000 and 1,500 IU/kg). Oocytes of fish caught from the lake were also incubated in vitro with different doses (50, 200, and 1,000 ng/ml) of 20β-S, 17α-HOP, 11-DOC, and 17,20β-P. 11-DOC was found to be the most effective hormone among those measured for inducing oocyte maturation in vivo and in vitro. 17,20β-P could not be determined in the plasma of any fish in vivo (P < 0.05). 1,000 IU/kg dose of hCG given by injection caused a statistically significant increase in all plasma hormone levels (P < 0.05). It was found that there was a significant decrease in the P level only at 1,500 IU/kg dose of hCG injected (P < 0.05), while the level of other hormones increased at this dose (P < 0.05). It was also determined that all the hormones were effective in germinal vesicle breakdown (GVBD) in in vitro oocyte culture (P < 0.05). However, 11-DOC was found to be the most effective hormone in GVBD at a dose of 200 ng/ml (70% GVBD). In conclusion, 11-DOC synthesized during final oocyte maturation in C. tarichi was found to be a potent inducer of GVBD, which shows that 11-DOC may be described as an oocyte maturation steroid in this species.     

Motility Parameters of Rainbow Trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) Spermatozoa in Relation to Sequential Collection of Milt,Time of <Emphasis Type="Italic">Post-mortem</Emphasis> Storage and Anesthesia

The present study investigated the effects of sequential collection of milt, time of post-mortem storage and anesthesia on rainbow trout (Oncorhynchus mykiss) sperm motility parameters (using computer-assisted sperm analysis – CASA) as well as seminal plasma osmolality and sperm concentration. The post-mortem storage and time of anesthesia altered motility characteristics of rainbow trout sperm to different extents. The moderate impact of time of anesthesia was manifested in a shortened duration of sperm motility after 10 min exposure of fish to anesthetic. The prolonged post-mortem storage (≥40–60 min), in addition to lowering sperm motility duration, also significantly influenced sperm motility parameters, such as sperm velocities, percentage of motile sperm and sperm trajectory parameters. These results clearly demonstrate that when milt from sacrificed fish is used for sperm motility studies, the time of post-mortem storage significantly alters sperm motility characteristics. Since sperm motility rate and swimming velocity could predict fertilizing ability, detrimental effects of prolonged post-mortem storage may lead to reduced fertilization success. Sperm concentration and seminal plasma osmolality were lower in the first fractions and increased with successive collections of milt. It suggests the presence of urine contamination of the first milt fractions which were collected by stripping. Therefore, testing of sperm concentration and/or seminal plasma osmolality should be mandatory while handling stored milt.     

Cryopreservation diluents for spermatozoa of Sakhalin taimen <Emphasis Type="Italic">Hucho perryi</Emphasis>

To develop a suitable cryopreservation diluent for spermatozoa of the endangered Sakhalin taimen Hucho perryi, all possible combinations of cryoprotectants (glycerol, dimethyl sulfoxide [DMSO], methanol) and extenders (fetal bovine serum [FBS], 300 mM glucose solution [GS], artificial seminal plasma for masu salmon) were examined by observing sperm motility 10 s after thawing. Spermatozoa cryopreserved with diluents such as mixtures of 10% glycerol plus 90% FBS, 10% DMSO plus 90% FBS, and 10% methanol plus 90% GS showed the highest motility. The maximal post-thaw motility was observed at 10% among all concentrations (0, 5, 10, 15 and 20%) of these three cryoprotectants. No significant difference among three diluents was observed in motility at 10 s. Mixtures of 10% glycerol plus 90% FBS, 10% DMSO plus 90% FBS, and 10% methanol plus 90% GS are suitable cryopreservation diluents for Sakhalin taimen spermatozoa.