Spawning induced by cubifrin in the Japanese common sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis>

The neuropeptide cubifrin-I and its derivative cubifrin-L have recently been shown to be potent substances that induce in vitro oocyte maturation and spawning in the Japanese common sea cucumber Apostichopus japonicus. We have examined the reproductive behavior of A. japonicus in vivo following the injection of cubifrin-L into the body cavity with the aim of determining the practical value of using this peptide to induce spawning in the hatchery setting. The responsiveness of ovarian fragments with oocytes >155 μm in diameter to cubifrin-L in vitro was well correlated with the spawning success induced in vivo by a cubifrin-L injection. Mature sea cucumbers injected with cubifrin-L displayed sequential reproductive behaviors, which comprised climbing up the side wall of the tank toward the water surface, waving of the head, and shedding of gametes. Gamete shedding started about 60 and 80 min after the injection of cubifrin-L in males and females, respectively, and was completed almost simultaneously in the two sexes about 2 h post-injection. Repeated injections of cubifrin-L at intervals of about 10 days successfully induced multiple spawnings in males and females. These results demonstrate that cubifrin-L can be used as an effective inducer of spawning in Japanese sea cucumber cultivation.     

Laboratory observations of habitat selection in aestivating and active adult sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis>

In order to verify which factors affect habitat selection for aestivating and in the active adult Apostichopus japonicus, animals were tested for their selection of attachment site in an experimental device (1-m pipes) in which the perceived environmental stimuli (light intensity, degree of contact with a hard surface, geotaxis) varied depending on the attachment site. During the aestivating season, the animals showed a strong selection for attachment sites during the daytime and nighttime; they also showed positive stereotaxis (thigmotaxis), negative phototaxis, and negative geotaxis. The results suggest that (1) habitats are not suitable for the aestivating adult A. japonicus unless these three environmental requirements are satisfied.     

Effect of salinity and temperature on salinity tolerance of the sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis>

Sea cucumber Apostichopus japonicus juveniles acclimated to different environmental conditions (23, 25, and 27°C combined with 25, 30, and 35 psu) were assessed for tolerance to increasing and decreasing levels of salinity at a rate of 2 psu h⁻¹. They were also tested for the LS₅₀ (median lethal salinity) when transferred directly into a series of higher salinity (32–46 psu) and lower salinity (9–25 psu). The CSMax (critical salinity maximum), CSMin (critical salinity minimum), USTL (upper salinity tolerance limit), and LSTL (lower salinity tolerance limit) were positively correlated to the acclimated salinity level but negatively correlated to temperature. The CSMax of A. japonicus was 6.2–10.0 psu higher than the USTL, and the CSMin was 5.5–8.5 psu lower than the LSTL, indicating that gradual changes in salinity resulted in the wide range of salinity tolerance that was observed, but that abrupt changes in salinity resulted in the narrow range of tolerance. Two-way analysis of variance revealed that salinity and temperature had a significant effect on 50% CSMax, 50% CSMin, USTL, and LSTL (P < 0.001). The information obtained in this study will be valuable for the further development of the sea cucumber aquaculture industry in China.     

Induced thermotolerance and expression of heat shock protein 70 in sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis>

ABSTRACT:   Thermal limits, induced thermotolerance and the expression of heat shock protein 70 (Hsp70) in an echinoderm Apostichopus japonicus were studied. The sublethal and lethal temperatures for the juveniles were 30 and 34°C, respectively; a previous sublethal heat shock exposure (30°C, 2 h) could increase the survival rates of the sea cucumbers when they were exposed to 34°C. This induced thermotolerance could last for at least 2 days. Levels of Hsp70 increased substantially after sublethal heat shock exposure and linearly decreased with time. This result indicated that a close relationship existed between the induction of thermotolerance and the levels of Hsp70 in A. japonicus.     

Feeding activity rhythm in Japanese sea catfish <Emphasis Type="Italic">Plotosus japonicus</Emphasis>

To examine the feeding activity characteristics of Japanese sea catfish Plotosus japonicus, access to feed box (feeding activity) and the number of food pellets consumed (food intake) were recorded under laboratory conditions. When fed ad libitum under a 12:12 h light–dark (LD) cycle, all fish exhibited feeding activity and consumed food during the dark period. Feeding activity increased in the presence of food, and this increased activity level continued for several days after food removal. During restricted food availability within the light period of the LD cycle, seven of nine fish exhibited food-anticipatory activity (FAA) both before and after the 12:00 food-restricted time. This FAA persisted under constant light conditions (in nine of nine fish). The average circadian free-running period was 24 h. These results suggest that P. japonicus exhibits a nocturnal feeding activity rhythm, which may be synchronized by a food-restricted time.     

Microbial community in a multi-trophic aquaculture system of <Emphasis Type="Italic">Apostichopus japonicus</Emphasis>, <Emphasis Type="Italic">Styela clava</Emphasis> and microalgae

An integrated multi-trophic aquaculture (IMTA) system consisting of the ascidian Styela clava and the sea cucumber Apostichopus japonicus with microalgae was grown in a seawater mesocosm. Microbial populations in the water, sediment and shelters were monitored over time. The composition of the water microbial community in the IMTA system did not differ significantly from that of the traditional culture system without ascidians. Bacterial populations in the water, sediment and shelters were low in the traditional A. japonicus aquaculture system, and lower in the IMTA system. The number of Vibrios in the water, sediment and shelters in the IMTA system was significantly lower than in the traditional culture (p < 0.05), while the quantity of Bacillus was higher in the sediment and shelters. All of these results indicate that the IMTA system effectively inhibited the growth of harmful bacteria, an important positive function in the health of the culture. The bacterial diversity index in the water in the IMTA system was between those of the baited and non-baited traditional aquacultures and was slightly reduced in the shelters. Over time, the bacterial diversity index in the sediment gradually dropped, especially in summer and autumn, when it was significantly lower than in the traditional culture. In winter, the diversity index increased to some extent, approaching that of the traditional culture.     

Stage-specific distribution of Japanese sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> in Maizuru Bay,Sea of Japan,in relation to environmental factors

The Japanese sea cucumber Apostichopus japonicus plays important ecological and economic roles in the coastal areas of Japan. Here, we aim to reveal the relation between habitat characteristics and density of juvenile and adult sea cucumbers in Maizuru Bay. Density of adult sea cucumbers was estimated by underwater visual surveys and bottom trawls and that of newly settled individuals by seedling collectors distributed in the bay. The physical environment of the sea bottom was evaluated from median diameter, total organic carbon, total nitrogen, carbon-to-nitrogen ratio, carbon isotope ratio, nitrogen isotope ratio, depth, slope, and distance from the coastline. Adult sea cucumber density was higher in the east side than the west side of Maizuru Bay, the former having a no-take zone for sea cucumbers. Juvenile sea cucumbers were most abundantly collected in the central part of the bay. High organic matter from the sea, short distance from the coastline, and high seabed gradient (steepness) had a positive effect on the density of adult sea cucumbers, and depth had positive effects on their body size. We suggest that sustainable management of sea cucumbers can be achieved by a combination of the establishment of no-take zones and increased protection of nursery habitats.     

Japanese nephropid lobster <Emphasis Type="Italic">Metanephrops japonicus</Emphasis> lacks zoeal stage

ABSTRACT:   Larvae of the Japanese nephropid lobster Metanephrops japonicus hatched in the laboratory were reared at 15°C, and the development and feeding were observed. All larvae hatched at the 'prezoea stage' with no natatory setae on the exopodite of the pereiopods. Without feeding, 50% of prezoea molted into the megalopa stage, having small buds as the exopodite, within 1 h and all molted within 22 h. The megalopa fed with Artemia nauplii, shrimp meat and pelleted food molted into the first juvenile stage with no exopodite after approximately 17 days. The average carapace lengths of prezoea, megalopa and the first juvenile stage were 3.2, 3.6 and 4.4 mm, respectively. The survival rate from hatching to the first juvenile stage was high (90–100%). This lobster may be the only known nephropid species with no zoeal stage.     

Effects of heat-shock selection during pelagic stages on thermal sensitivity of juvenile sea cucumber, <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> Selenka

The effects of heat-shock during pelagic stages on growth and survival of juvenile sea cucumber Apostichopus japonicus were investigated to test the possibility to acquire high thermal tolerance individuals after the heat-shock selection. Larvae at the stages of blastula, gastrula, and auricularia were heat-shocked at selected temperatures (21.5, control; 26, 28, and 30°C) for 45 min and returned to 21.5°C for continuous rearing. There was a significant difference in thermal sensitivity among different developmental stages, and thermal tolerance of the larvae was correlated with the expression of heat shock protein 70 (Hsp70). Juveniles after the heat-shock selection at pelagic stages showed higher induced thermotolerance than those without heat-shock. Therefore, heat-shock application at early pelagic development stages is potentially a useful way to select high-thermotolerance variety of the sea cucumbers.     

Effect of yeast polysaccharide on the immune function of juvenile sea cucumber, <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> Selenka under pH stress

In the present study, we evaluated the effect of yeast polysaccharide (YSP) on the immune function of Apostichopus japonicus at different pH levels. Juvenile sea cucumbers were fed artificial feed containing 0.5% YSP. Phagocytic activity and activity of lysozyme (LSZ), acid phosphatase (ACP) and alkaline phosphatase (ALP) were measured under pH stress after 24 h. The results showed that the phagocytic index, the activity of LSZ and ACP in the intestines increased at pH 10.0, and ACP activity in the body wall decreased. The phagocytic rate and ALP activity in the intestines were not different from the control group. The phagocytic index was also not different from the control group at pH 5.5. The activity of LSZ and ACP in the intestines and the activity of ALP increased, while the activity of ACP in the body wall decreased. The phagocytic rate of YSP group increased and the phagocytic rate of control group decreased. Thus, this study indicated that YSP can modulate the non-specific immunity of A. japonicus, and the reasonable dose of YSP might be used as the immunostimulants in sea cucumbers farming under pH stress.     

Release size and stocking density for grow-out of <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> in the sea with raft-cultured macroalgae

Sea cucumber, Apostichopus japonicus, is the main cultured species in China. The main culture style for this species is the sea ranching model. Field trials were conducted in bottom-cages to preliminary reveal optimal releasing size, as well as maximum density of A. japonicus in an integrated multi-trophic aquaculture sea ranching area. Different sizes (4–30 g ind^?1) and densities (336–1342 g m^?2) of sea cucumbers were cultured for 13 months in Rongcheng Bay, Shandong Province, China. The size experiment showed that sea cucumber of all sizes grew throughout the experimental period. Sea cucumbers <15 g had high mortality in summer and low SGR in winter, while larger individual (>20 g ind^?1) had no advantage of growth. Sea cucumber sizes of 15–20 g may be suitable for release, considering their higher survival rate and SGR. The density experiment showed that the high biomass group had a low SGR and that the maximum release biomass was 793 g m^?2 based on a regression analysis. The optimal practical release season for sea cucumber was spring based on the results of two field experiments.     

Effects of dietary rhubarb, <Emphasis Type="Italic">Bacillus cereus</Emphasis>, yeast polysaccharide,and florfenicol supplementation on growth,intestinal morphology,and immune responses of sea cucumber (<Emphasis Type="Italic">Apostichopus japonicus</Emphasis>)

A 60-day feeding trial was conducted to investigate the effects of dietary Chinese herbal medicine (rhubarb, Rheum officinale Bail), probiotic (Bacillus cereus BC-01), prebiotic (yeast polysaccharide), and antibiotic (florfenicol) supplementation on the growth, intestinal morphology, and immune responses of juvenile sea cucumbers (Apostichopus japonicus Selenka; initial weight, 4.67 ± 0.06 g). The control treatment was fed only the basal diet (CT treatment), whereas four other treatments were fed the basal diet supplemented with rhubarb (RH treatment), B. cereus BC-01 (BC treatment), yeast polysaccharide (PO treatment), or florfenicol (FL treatment). Groups of ten sea cucumbers were housed in separate tanks, and each diet was fed to five randomly selected groups. The specific growth rates of the individuals in the RH and BC treatments were significantly higher than those in the CT, PO, and FL treatments. The apparent digestibility coefficient of crude protein in the RH and BC treatments was significantly higher than that of crude protein in the CT and PO treatments. The alkaline phosphatase activity of the coelomocytes in the RH and BC treatments was significantly higher than that in the CT and FL treatments. Coelomocytes in the PO treatment had the highest acid phosphatase activity. Higher than average lysozyme activity was observed in the PO and FL treatments. Fold and enterocyte heights in the mid-intestinal tract of sea cucumbers in the CT and RH treatments were lower than those in the BC, PO, and FL treatments. Microvillus height was greatest in the RH treatment and lowest in the FL treatment. Scanning electron microscopy revealed remarkable atrophy of microvilli and apoptosis of intestinal epithelial cells in the FL treatment. These results suggest that dietary rhubarb, B. cereus, and yeast polysaccharide supplementation can improve the health of sea cucumbers, whereas long-term florfenicol supplementation may have a negative impact on their intestinal healthy and immune response of sea cucumbers.     

Seasonal distribution pattern of adult sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> (Stichopodidae) in Yoshimi Bay,western Yamaguchi Prefecture,Japan

To examine the distribution pattern of adult Apostichopus japonicus, approximately monthly surveys were conducted during a 2-year period in the subtidal zone of a harbor area in Yoshimi Bay, western Yamaguchi Prefecture. Results showed a clear seasonal change in distribution pattern: many animals were distributed on the seabed and the lower part of a jetty during winter to spring, whereas most animals occurred on the upper part of a jetty during summer to autumn, namely, the aestivation season. Such change is considered to be due to the migration of animals within the present study site. Y. Yamana is a Research Fellow of the Japan Society for the Promotion of Science.     

Spatial variation of quantitative color traits in green and black types of sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> (Stichopodidae) using image processing

It has been suggested that the Japanese sea cucumber, Apostichopus japonicus, has three color types (red, green, and black), although the qualitative difference between the color types, particularly between the green and black types, is unclear because of continuous color variation among color types. This study elucidated the color variation between green and black types using image processing (RGB, red–green–blue system) and multivariate analysis to demonstrate whether or not the black and green types can be quantitatively classified. Moreover, spatial variation of the RGB value among various local sites was clarified to estimate potential environmental factors that may affect the color variation. The series of analyses revealed that a quantitative boundary between green and black types could be provisionally established, and also that spatial variability in the intermediate (continuous) color trait between green and black types was significant. Potential environmental factors (depth and industrial activity index) were correlated with the color traits in both color types. These results suggest that the green and black types cannot be regarded as independent color traits and that the color variation between green and black types may be influenced by local environmental factors.     

Immune response of sea bass <Emphasis Type="Italic">Dicentrarchus labrax</Emphasis> to <Emphasis Type="Italic">Tenacibaculum maritimum</Emphasis> antigens

ABSTRACT:   Seawater fishes are affected by a pathology commonly called 'myxobacteriosis', caused by Tenacibaculum maritimum (formerly Flexibacter maritimus ). The disease is characterized by fin erosion and necrotic ulcers of skin and muscle, and by low but constant mortality in cultured marine fish; in Italy is one of the most important and widely spread diseases affecting sea bass Dicentrarchus labrax , gilthead seabream Sparus aurata , sharp-snouted bream Diplodus puntazzo , white bream Diplodus sargus , and six-tooted bream Dentex dentex . In order to obtain an effective vaccine against the disease, formalin killed cells (FKC), extracellular products (ECPs) and crude lipopolysaccharide (LPS) preparations were obtained from the T. maritimum strain SPVId and injected intraperitoneally twice into the sea bass. The fish immune response to the preparations was studied: agglutinating antibody titer and in vitro phagocytosis were determined after the first and second injection in order to evaluate whether the preparations are immunogenic or not and if the booster effect took place. The results show that FKC and LPS preparations increased the antibody titer after the first injection when compared to the control sea bass. Moreover, all the preparations stimulated a secondary (booster) response. In vitro phagocytosis of the total blood was significantly higher for all the preparations when compared to the controls, but the crude LPS immunized sea bass showed the highest activity.     

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.     

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.