Audiogram measurement based on the auditory brainstem response for juvenile Japanese sand lance <Emphasis Type="Italic">Ammodytes personatus</Emphasis>

In this study, the auditory thresholds for juvenile Japanese sand lance Ammodytes personatus were measured based on its auditory brainstem response (ABR). The amplitude of the ABR waveforms to a sound stimulus were larger than that of the electric background noise caused by general brainwaves and myogenic signals after the averaging procedure. Japanese sand lance responded to low frequency sounds between 128 Hz and 512 Hz with a sound pressure level of 115–125 dB. As the test frequency decreased, so did the auditory threshold level, and the level was about 116 dB at 128 Hz and 181 Hz. These results indicate that Japanese sand lance can detect low frequency sound but are less sensitive than other fish species. These high thresholds are probably caused by the lack of a swim bladder.     

Reproductive potential and recruitment of sand lance <Emphasis Type="Italic">Ammodytes personatus</Emphasis> in Ise Bay,central Japan

Regular samplings of the Japanese sand lance Ammodytes personatus were carried out between 1991 and 2003 in Ise Bay in central Japan to estimate the number of adults, egg production and the number of recruits. The estimated minimum and maximum number of adults was 0.48 billion and 37.64 billion (78.4-fold variation). Age-one adults dominated in each year, and they comprised more than 80% of the total population in ten years out of the twelve years of observations. The total egg production ranged from 0.5 to 22.2 trillion (44.4-fold variation), and the total number of recruits ranged from 3.36 to 102.83 billion (30.6-fold variation). These interannual variations were both smaller than the interannual variations in the number of adults. As the total number of adults increased, the total egg production gradually became asymptotic at the saturation level, and the egg production per adult female decreased. In addition, the number of recruits per egg decreased as the total number of adults increased. It was suggested that both egg production and the initial process of mortality before recruitment exhibit density dependency.     

<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.     

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.     

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.     

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.     

Production and characterization of monoclonal antibodies to leukocytes and serum immunoglobulin in Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

Two monoclonal antibodies (MAbs: JFW1 and JFW10) were produced against peripheral blood leukocytes (PBL) in Japanese flounder. Additionally, MAbs against flounder immunoglobulin (Ig; JFW20 and JFW21) were generated for the surface marker of Ig⁺ leukocytes using purified serum Ig as an antigen. MAb JFW1 recognized the surface marker of granulocytes and monocytes and MAb JFW10 specifically bound to the surface antigen of thrombocytes. Flow cytometric analysis of PBL incubated with JFW1, JFW10, JFW20 and JFW21 revealed that 2.5–7.4, 23.7–50.1, 25.2–26.1 and 5.2–8.3% of all leukocytes were positive for these markers. Analysis of head kidney leukocytes (HKL) showed that JFW1, JFW10, JFW20 and JFW21 bound to 30.5–36.3, 1.9–2.8, 6.4–8.3 and 1.9–3.0% of all leukocytes, respectively. Western blot analysis after SDS-PAGE showed that JFW10 recognizes a protein of 115 kDa from lysed PBL. JFW20 recognized the 70 and 74 kDa proteins of the heavy chain of Ig from serum. No band was observed for either JFW1 or JFW21. These antibodies will be useful for the identification and isolation of Japanese flounder leukocyte subpopulations and will facilitate immunological studies of flounder.     

Density and sound speed contrasts of the Japanese common squid Todarodes pacificus and their influence on acoustic target strength

ABSTRACT:   In this study, density and sound speed contrasts were measured for the Japanese common squid Todarodes pacificus . Target strength (TS) data derived from an acoustic scattering model based on measurements of these two parameters were compared with TS values based on acoustic measurement data to determine whether the measured parameters are reasonable values for theoretical scattering model. Density contrast ( g ) was measured from the displacement volume and wet weight, while sound speed contrast ( h ) was measured from the acoustic measurements of travel time (time-of-flight method). The Kirchhoff ray mode model, which represents the squid body as a set of fluid-filled cylinders, was used to calculate theoretical TS. Mean g- and h -values of the squid were 1.029 and 1.041, respectively. Comparison with previous data showed that g was similar, whereas h was relatively high. The difference in the TS between the theoretical model using measured parameters and the acoustic measurement was within 3 dB. Thus, the measured g- and h -values are acceptable for theoretical models of squid.     

Mitochondrial DNA sequence variation of Japanese anchovy <Emphasis Type="Italic">Engraulis japonicus</Emphasis> from the Yellow Sea and East China Sea

Sequence variation of a fragment of the mitochondrial cytochrome b (cyt b) and cytochrome c oxidase subunit I (COI) gene was examined using polymerase chain reaction and direct sequencing among three populations of Japanese anchovy Engraulis japonicus from the Yellow Sea and East China Sea. A total of 24 and 47 nucleotide sites were detected variable defining 29 and 32 haplotypes in cyt b and COI data, respectively. All variable sites except one in COI were silent in the two sets of sequences. The Ewens-Watterson test indicated that the observed allelic configurations in both data sets were in full agreement with neutral expectations. The variation level was high, with h=0.957±0.018, π=0.644±0.387 (%) in cyt b data set and h=0.958±0.021, π=0.640±0.386 (%) in COI data set, respectively. However, at the population level, Fst values between pairs of populations were not significantly different from zero (P>0.05) in both data sets. The analysis of haplotype frequency distribution showed no significant differences among populations. Similarly, the analysis of the partitioning of molecular variance indicated that all or almost all of the genetic variation was distributed within populations. Based on the data from this study, the existence of separate genetic stocks in this area were not detected. Mixing of stocks to some extent in migration cycle and dispersal capacity of anchovy’s planktonic larvae could be the reasons for genetic homogeneity in this species in the Yellow Sea and East China Sea.     

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.     

Mitochondrial DNA variation in the East China Sea and Yellow Sea populations of Japanese Spanish mackerel <Emphasis Type="Italic">Scomberomorus niphonius</Emphasis>

Japanese Spanish mackerel Scomberomorus niphonius is a commercially important species in the East China Sea and Yellow Sea, but there is limited knowledge of its genetic population structure. In order to detect its genetic structure, sequence variation of the first hypervariable segment of the control region was analyzed among eight populations of S. niphonius from the East China Sea and Yellow Sea. A total of 119 polymorphic sites were detected in the 505-bp segment of the control region among 134 individuals of S. niphonius, defining 112 haplotypes. Mean haplotype diversity and nucleotide diversity for the eight populations were 0.9963 ± 0.0017 and 0.0236 ± 0.0119, respectively. As expected, analysis of molecular variance detected no significant differences at all hierarchical levels, and most of the conventional population Φ_ST statistics were negative, indicating that no significant population genetic structure exists in the East China Sea and Yellow Sea. Moreover, the exact test of differentiation supported the null hypothesis that S. niphonius within the East China Sea and Yellow Sea constitutes a panmictic mtDNA gene pool. Neutrality tests and mismatch distribution revealed that S. niphonius underwent population expansion in the late Pleistocene. Strong dispersal capacity of larvae and adults, long-distance migrations, and ocean currents in the studied area could be the reasons for genetic homogeneity in this species in the East China Sea and Yellow Sea. Insufficient time to accumulate genetic variation might be another explanation for the lack of genetic structure in the East China Sea and Yellow Sea.     

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.     

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.