Random sequencing of genomic DNA of <Emphasis Type="Italic">Photobacterium damselae</Emphasis> subsp. <Emphasis Type="Italic">piscicida</Emphasis>

ABSTRACT:   Random sequencing of the genomic DNA of Photobacterium damselae subsp. piscicida was conducted. The sequences were assembled into 930 contigs. The total length of these contigs accounts for 37.5% of the genome of P. damselae subsp. piscicida. The contigs contain 2055 open reading frames (ORFs) that have homology with genes in the GenBank database. Furthermore, some of the ORFs have homology with reported virulence related genes, and are classified as encoding colonization factors, exotoxin, and lipopolysaccharide (LPS). Thirty-seven ORFs have homology with colonization factors. In those colonization factors, 27, three, and four ORFs have homology with polar flagellar-related genes, capsule-related genes, and others (accessory colonization factors, superoxide dismutase (Cu-Zn), MshA biogenesis protein, and heme receptor genes), respectively. Five ORFs have homology with exotoxin-related genes (2 hemolysin, phospholipase, hyaluronidase, lysophospholipase L2 genes). Further, six ORFs have homology with LPS-related genes.     

<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.     

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.     

A simple method to distinguish two commercially valuable eel species in Japan <Emphasis Type="Italic">Anguilla japonica</Emphasis> and <Emphasis Type="Italic">A. anguilla</Emphasis> using polymerase chain reaction strategy with a species-specific primer

In order to distinguish the two eel species Anguilla japonica and A. anguilla irrespective of unprocessed and processed samples, a quick, convenient method using polymerase chain reaction (PCR) with a species-specific primer was established. The comparison of partial sequences of the mitochondrial 16S ribosomal RNA gene between A. japonica and A. anguilla facilitated designing of a primer pair common to both A. japonica and A. anguilla and a primer specific to A. japonica. PCR was carried out with the three primers for total 110 specimens of A. japonica and A. anguilla. PCR products with the species-specific primer showed two bands for A. japonica and one band for A. anguilla in an agarose gel electrophoresis. This analytical procedure required only a short period and is more convenient than PCR-restriction fragment length polymorphism, one of the standard methods employed for fish species identification.     

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.     

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.     

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.     

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 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.     

Effects of potential probiotic <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> on digestive enzymatic activities of live feed <Emphasis Type="Italic">Artemia franciscana</Emphasis>

In the present study, cellulase, protease, lipase and amylase activities were performed to investigate the effects of Lactococcus lactis subsp. lactis CF4MRS bioencapsulation of Artemia franciscana. Our results show that cellulase activities (total cellulase—FPase activity, exoglucanase and endoglucanase—CMCase activity) were significantly higher (P < 0.05) in A. franciscana tissue homogenates compared to those in the control group after 8 h of L. lactic bioencapsulation. Notably, an exception case was found in β-D-glucosidase activity, whereby the cellulase activity was not significantly different (P > 0.05) compared to the control group. Administrations of L. lactis at cell concentration of 10⁸ CFU mL^?1 showed considerable improvement of other important enzymatic activities such as amylase, protease and lipase in A. franciscana. The amylase/protease ratio in probiotic-treated A. franciscana was recorded at 0.343, approximately two times higher than those without probiotic administration (0.184). In contrary, amylase/lipase ratio showed half of a reduction (0.330) in L. lactis-administrated A. franciscana compared to the control (0.614). Our study suggests that important digestive enzymes, e.g., cellulase, amylase, protease and lipase, can be enhanced through bioencapsulation of A. franciscana with L. lactis subsp. lactis, which could in turn lead to further stimulation of endogenous enzymes in the fish and shrimp larvae.     

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.     

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.     

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.     

Compensatory growth of Chinese shrimp, <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> following hypoxic exposure

Compensatory growth following stress is a strategy aquatic animals use to adjust themselves to a variable environment. Studies on the recovery growth of aquatic animals are not only of theoretical value in ecophysiology and evolution, but also important to applications in aquaculture and fisheries resource management. In this experiment, juvenile Fenneropenaeus chinensis with an initial average body weight of about 3.72 g were exposed to hypoxic water (about 2.08 and 3.11 mg/l of dissolved oxygen (DO) content) for 10 days and then switched to normoxic water (about 5.63 and 5.59 mg/l DO). Compared with juveniles in normoxia, juveniles in the hypoxia period allocated a greater proportion of energy to metabolism and exuviations but allocated less energy to daily metabolism per gram shrimp weight (J/g/day). This reduced feed conversion efficiency and feeding rate. Finally, F. chinensis suffered growth depression. The juveniles completely compensated for hypoxia-induced growth depression in 30 days after being switched into normoxic water and the compensation was achieved mainly by hyperphagia and slightly by improvement of feed conversion efficiency. During the recovery period the hypoxic-stressed shrimp showed higher daily metabolic energy (J/g/day) than controls (P < 0.05). Which means the stressed shrimp had more energy for feeding-related activities. So hyperphagia was observed. Energy analysis indicated that F. chinensis improved feed-conversion efficiency mainly by reducing the percentage of energy lost in feces and exuviations. The results showed that short-term non-lethal hypoxia would not affect the growth of juvenile F. chinensis if there were enough time for the stressed shrimp to recover. This suggested F. chinensis was capable of adapting to DO fluctuation to some extent and short-term non-lethal hypoxia would not have an obvious effect on natural, released, and cultured shrimp stock.