Akinori Hino

Development of a continuous culture system for stable mass production of the marine rotifer Brachionus

In order to improve culture stability, as well as to reduce labor and space for mass production of the marine rotifer Brachionus, an automatic continuous culture system has been developed for practical use. The system consists of a filtration unit, a culture unit and a harvest unit. In this system, filtered water and food are continuously supplied into a rotifer culture tank at a pre-determined rate, and the same amount of culture water is transferred into a harvest tank to obtain rotifers at a significant biomass.

Resting egg formation and hatching of the S-type rotifer Brachionus plicatilis at varying salinities

The effect of salinity on resting egg formation and hatching in the marine rotifer Brachionus plicatilis rotundiformis (S-type Hawaiian stock) was examined in 1986. B. plicatilis was cultured at three salinity levels. The average number of resting eggs produced at 8, 16 and 32 ppt ranged from 22 to 615 per 10 000 rotifers, or 29 to 413 per 1.0x108Tetraselmis cells. Data on the rate of mictic female appearance, fertilization and fecundity of fertilized mictic females support these findings. Results indicated that the sexual reproduction of L- and S-type rotifers have opposing responses to salinity. After preservation for 2 mo at 5°C, simultaneous hatching of resting eggs was observed at six salinity levels at 30°C. Development time was extended and hatching rate decreased when eggs formed at 8 ppt were incubated at extremely high (40 ppt) or low (1 ppt) salinities. Hatching rates varied from 69 to 93%, and were influenced by the salinity present at the time of egg formation and incubation.

Maternal Effect by Stem Females in Brachionus plicatilis: Effect of Starvation on Mixis Induction in Offspring

We examined whether starvation during the initial period of life in stem females affected reproductive characteristics of the offspring. Starvation treatment had different effects on rotifers hatched from resting eggs and those hatched from amictic eggs. When stem females experienced starvation after hatching, this induced a higher percentage of mixis in their offspring. When the same starvation treatment was applied to rotifers hatched from amictic eggs, there was no effect on the induction of mixis. It is probable that stem females hatched from resting eggs have specific features that are vulnerable to unfavorable environmental conditions, and that these features can be inherited by their offspring through the maternal cytoplasm.

Effect of combination feeding of Nannochloropsis and freshwater Chlorella on the fatty acid composition of rotifer Brachionus plicatilis in a continuous culture

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 2000mL 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 ∑n-6/∑n-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 ∑n-6/∑n-3 ratio in continuously cultured rotifers.

Individual variations in behavior and free cortisol responses to acute stress in tiger pufferfish Takifugu rubripes

The aim of the present study was for individual variation in behavior to be characterized and related to differences in physiological stress responses in juvenile tiger pufferfish (fugu) Takifugu rubripes. A set of siblings (n=330) from wild parents was subjected to behavior tests, and some were classified into active and inactive groups (n=42 each group). The active animals consisted of those individuals that kept active swimming during 6 min after transfer from one tank to another, and the inactive animals were those that stopped swimming within 6 min. Time-course acute stress responses, which were elicited by anesthesia and subsequent removal of a pectoral fin, were compared between the active and inactive fish. The stress indicators used were free and total plasma cortisol, and plasma glucose. Although no significant difference was observed at each sampling point between groups, the inactive fish required shorter recovery time from acute stress in all three indicators than the active fish. The percentage of free to total plasma cortisol in the pre-stress condition was significantly lower in the inactive group than in the active group. Our findings indicate that fugu has behavioral variation that is associated with stress responsiveness. The inactive fish are expected to be more tolerant of stress and suitable for aquaculture.

Nitrogen-flow in the rotifer Brachionus rotundiformis and its significance in mass cultures

The nitrogen budget in the rotifer Brachionus rotundiformis wasmeasured by the stable-isotope technique. The budget was estimatedusing the difference in the turnover time between egestion andexcretion. The rotifer was fed on the algae Nannochloropsiswhich was labeled with 15N as a tracer. The turnover time ofegestion and excretion were 20 min and 2.5 hours, respectively. Where77% of the ingested nitrogen was egested, and of the assimilated23%, 18% were devoted to growth and 5% to excretion.As for the unassimilated nitrogen egested as faeces, it recycled tothe rotifer through bacteriovory. When the algae provided as foodwere almost fully consumed, bacteriovory became dominant. Thethreshold occurred when the concentration of algae in the culture wasbetween 1.5 and 0.5 million cells of Nannochloropsis per ml. Ina chemostat operated with un-limited food condition, bacterialnitrogen corresponding to 20% of algal feeding, was consumed by therotifer.In a semi-continuous mass culture where food condition was limited,bacteriovory was more effective in supporting the rotiferreproduction. It contributed to the extremely high nitrogen recoveryfrom the provided foods (algae and oil-yeast) to the harvestedrotifers. The rapid and large nitrogen outflow from rotifersaccelerated the propagation of edible bacteria and can explain thestrange paradox observed in the culture; daily supply of foods didnot cover the sum of growth and excretion.It is not too exaggerated to state that the rotifer mass culture issupported by bacteria. The future strategy for maintenance of masscultures should consider this aspect.

Establishment of shell growth analysis technique of juvenile Manila clam Ruditapes philippinarum : semidiurnal shell increment formation

The conventional acetate peel method was modified to analyze the shell growth pattern of juvenile Manila clam Ruditapes philippinarum as small as 2 mm in shell length (SL). In the outer shell layer along the axis of maximum growth, two types of growth increments were observed: distinct increments and indistinct increments, which, respectively, do and do not continue to the middle shell layer. The distinct increments were found to be formed every two days in intertidal and shallow subtidal zones by field enclosure experiments of juveniles with datum points marked with alizarin complexone. Growth patterns of juveniles (12 mm SL) collected from the Seaside Park of Yokohama in Tokyo Bay were analyzed to confirm the modified method. Mean daily shell growth rate from April to July 2005 ranged 120–142 μm/day, which was reasonable as compared with previous studies. It was impossible to backcalculate the growth to the settlement size (i.e. 0.2 mm SL) because of erosion of the outer shell surface, and the smallest backcalculated minimum shell length was 0.8 mm. Fluctuations in daily growth rate were high, ranging 29–315 μm/day, and did not show a clear two-weekly rhythm.