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SUMMARY: A specific lot of sodium chloride (NaCl) has a contaminant toxic to the growth of Nannochloropsis oculata. This toxic effect was markedly reduced by co-culture with Flavobacterium sp., and such detoxifying ability was specific to this bacterial strain. Bioassays of the NaCl after heat treatment and ultrafiltration indicated that the NaCl was contaminated with heat-labile organic compounds of low molecular weight. Chemical analysis indicated that one contaminant in NaCl had analogous structure to a fungicide tridemorph. Commercially available tridemorph showed a toxic effect on N. oculata, and it was reduced by co-culture with Flavobacterium sp. These results indicate that the tridemorph-like substance is a causative factor for the growth inhibition of N. oculata, and that Flavobacterium sp. has an ability to detoxify the contaminant as well as tridemorph. The detoxifying ability of Flavobacterium sp. has the potential to be widely applicable for removing the trace pollutants affecting the growth of microalgae.

Mass production of the resting eggs of the marine rotifers Brachionus plicatilis (formerly called L-type) and B. rotundiformis (called S-type) were achieved using a batch culture method [reviewed by Hagiwara, A., 1994. Practical use of rotifer cysts. The Israeli Journal of Aquaculture — Bamidgeh 46, 13–21]. This paper summarizes the further advancements achieved over the last three years. The efficiency of resting egg production can be improved by employing semi-continuous culture method. Some semi-continuous cultures collapsed between the 15–20th days due to bacterial effects. However, the rotifer culture was stabilized by placing filtration mats to remove organic material. Thus harvests of 8.1 × 10 2 resting eggs/day/g (dry weight) Nannochloropsis oculata were achieved, which is 3.0 times more efficient than that from batch cultures. Resting eggs of B. rotundiformis , however, were not mass produced after the exchange of culture water. Resting eggs can be stored in sea water for more than 20 years at 5 °C under complete darkness. Hatch rates of the eggs, however, decreased when stored with abundant organic matter. Resting eggs could be canned under an atmospheric pressure of 48–61 kPa after lyophilization (− 30 °C) without reducing their hatch rates.

Scanning electron microscopy indicated that stored resting eggs of the marine rotifer Brachionus plicatilis Muller were colonized by numerous bacterial cells that affect the hatchability of resting eggs. The effectiveness of chemical disinfectants on the hatching of the resting eggs was studied using selected chemicals and antibacterial drags. The hatching success rate of the fresh and stored eggs was 26 ± 5.8% and 0%, respectively. Sodium hypochlorite (1.0 p.p.m.) increased the hatching rate to 48 ± 1.3% in freshly-collected and 28 ± 1.8% in stored eggs. Physical disintegration of the bacterial mat occurred after 1 h of treatment with sodium hypochlorite. Among antibacterial drugs, sodium nifurstyrenate (Erubaju) enhanced the hatching success rate to 40 ± 1.2% and 20 ± 1.1% for the freshly-collected eggs and stored resting eggs, respectively. However, tetracycline and oxytetracycline did not significantly influence the hatching of the stored eggs. These observations are discussed in relation to the development of an improved method for the processing and hatching of rotifer resting eggs.

The aim of this work is to control the waterenvironment for culturing larvae of the swimmingcrab, Portunus trituberculatus, using microorganisms.The bacterial strain PM-4 (Thalassobacter utilis)improved the survival rate of crab larvae andrepressed the growth of Vibrio anguillarum (bacterium)and Haliphthoros sp.(fungus) in seawater. PM-4 wascultured and added daily to seawater during the firstto third zoean growth stage of the crab with diatomsand rotifers. Numbers of PM-4 decreased in culturewater during the first 3 days, because of feeding bythe first zoean stage of larvae. The finalconcentration of PM-4 was 105 to 106 cellsml−1according to the plate count method in larval rearingwater.During 1989 to 1993, we tried seed productions of aswimming crab in 200 m3 containers at TamanoStation, Japan Sea-Farming Association. In 33trials of the biocontrol methods, average survivalrate of crab larvae was 28.3% when the bacterialstrain PM-4 was added. In 42 trials in which the strainPM-4 was not added, average survival rate of crab larvae was15.6%. We conclude that thebacterial strain PM-4 is effective as a biocontrolagent.

Surface topography and layering of resting eggs of Brachionus plicatilis and B. rotundiformis are described, based on scanning and transmission electron microscope studies. In B. plicatilis, the resting eggs are spherical with smooth wavy ridges on the surface, whereas these ridges are small and condensed in B. rotundiformis. The distribution of pores on the egg surface clearly distinguishes the two species.

Water-soluble substance extracted from the coralline alga, Corallina pilulifera have the effect of induction of larval metamorphosis on the sea urchins, Pseudocentrotus depressus and Anthocidaris crassispina.The chemical cue for larval metamorphosis of P. depressus in the solution was heat stable and remained active even if its treated with organic solvent. Concentration of the potassium in an active fraction was not so high to induce the larval metamorphosis. The cue could be other inorganic ions or organic substances.

Small, tropical marine Brachionus (SS strains) are distinguished by their lorica size and shape. Their relation to other marine Brachionus species, however, remains unknown. We examined morphometry, reproductive mode, allozyme patterns and mating behavior among marine Brachionus species in an attempt to classify SS strains. Based on morphological features, reproductive mode, and allozyme patterns, SS strains were most similar to those of B. rotundiformis Tschugunoff. Observations on mating behavior between different groups indicated that there is reproductive isolation between B. plicatilis O.F. Muller and B. rotundiformis and between B. plicatilis and SS strains. In contrast, copulation was observed between B. rotundiformis and SS strains. Thus, there is no evidence that SS strains are a new marine Brachionus species, and most likely should be included in B. rotundiformis.

Previous works revealed that sexual reproduction of brachionid rotifers increases with increasing population density. It is also known that the renewal of environmental water suppresses sexual reproduction in Brachionus plicatilis O.F. Muller populations. The cause of these actions, however, remains unknown. In this study, we investigated the effects of bacterial additions to axenic rotifer B. plicatilis cultures as well as the the effect of rotifer extracts on sexual and asexual reproduction. Out of 17 bacterial strains tested, rotifer sexual reproducion (mixis) rates were 4–10 times higher than controls with addition of three Pseudomonas strains, a Moraxella strain and a Micrococcus strain. Rotifer mixis rate also increased with the addition of a water soluble extract from 1000 rotifers to 5 ml axenic rotifer cultures. Similar results were obtained for resting egg formation.

The rotifer Brachionus plicatilis requires vitamin B12. Freshwater Chlorella, which is produced by traditional culture, cannot support rotifer growth under bacteria-free conditions. However, Chlorella enriched with vitamin B12 can support rotifer growth. To attain stable mass production of Brachionus, it is desirable to develop a food that can completely support growth of the rotifers. The authors cultured rotifers at experimental and mass culture scale with concentrated Chlorella vulgaris suspension enriched with vitamin B12 in their cells. Chlorella suspensions were prepared containing different amounts of vitamin B12 in their cells, and rotifers were then cultured in 20 ml of the prepared suspensions. The highest rotifer yield was obtained from the group cultured with Chlorella containing more vitamin B12 in their cells. The suitable content of vitamin B12 in the concentrated Chlorella suspension commercially available for mass culture of the rotifer is considered to be 200 μg per 100 g dry matter of Chlorella. The amount of vitamin B12 necessary to produce one individual rotifer is calculated at 0.32 pg. The authors conducted mass production of the rotifer with baker's yeast and refrigerated concentrated Chlorella containing vitamin B12 Rotifer culture with vitamin B12 was more stable and showed 1.3 times higher production than with normal Chlorella.

The effect of initial stocking density (8-47 thousand indiv./m3) on larval mass production of kuruma prawn was studied in ten trials. The larvae were reared for 24 days from the nauplius stage to the 15-16th day of the postlarval stage. The survival rates ranged from 0.29 to 0.84, and were above 0.5 in eight trials. The daily declining pattern of survival numbers in these eight trials colud be expressed by a logarithmic linear regression throughout the rearing period, thus the population decreased in a constant proportion. By a negative correlation between initial stocking density and survival rate, 24-day rearing obtains a survival rate of more than 0.5 with an initial stocking density of less than 30 thousand indiv./m3. Relationship between initial stocking density and harvested biomass per initially stocked individual (final individual fresh body weight×survival rate) showed a more significant negative correlation. The maximum yield was estimated as approximately 80g/m3 at an initial stocking density of 30 thousand indiv./m3. The actual value of the largest yield among these trials was 87g/m3 at an initial stocking density of 25 thousand indiv./m3.

Cross-mating experiments were carried out using seven S- and L-type strains of the rotifer Brachionus plicatilis O. F. Muller, to determine the probability of sexual recombination. Rotifer rearing conditions were controlled to eliminate any effects of external factors. Of 39 crossing and selfing trials, fertilization was observed only among S-type and L-type strains. All crosses between S- and L-type strains failed. Percent fertilization obtained by cross-mating between strains of the same type was significantly lower than that of selfing. A negative correlation was found between the genetic distance of mated pairs and percent fertilization. The results suggest that whether the strains can be crossed successfully depends on the differences in their genetic constitution. Isozyme analysis on rotifers from resting eggs produced in the cross-matings indicated that classical sexual recombination occurred in S- or L-type rotifers.S- and L-type rotifers exhibit differences not only in morphological and physiological characteristics but also in genetic constitution and karyotypes. In addition, the current study has demonstrated reproductive isolation between the two types. From these facts, S- and L-type rotifers should be classified as different species.

Two trials of mass production of L-type rotifer Brachionus plicatilis resting eggs were conducted in 50m3 tanks (40m3 in water volume). Culture temperature was regulated at 18-25°C and salinity at 10-16ppt. Two replicate tanks were used for each trial. The feeding amount in Trial I (fed with Nannochloropsis oculata diet alone) was 7.81kg (dry weight), while it was 32.53kg in Trial II (fed with N. oculata and baker's yeast).The average maximum rotifer density during the 10-day culture period was 151 and 425 individ./ mlin Trials I and II, respectively. The mixis rate was 27.9 (Trial I) and 40.1% (Trial II). No significant difference was found in fertilization rates between trials. The number of resting eggs produced per tank was 1.05×109 (Trial I) and 3.53×109 (Trial II). The number of resting eggs produced from 1g dry weight diet ranged from 87×103 to 149×103, and it did not vary among trials.

The net production of eelgrass Zostera marina in tidal flats at Yanai Bay in the Seto Inland Sea, Japan, was estimated from seven field surveys using a marking method from April to July 1982. The increase of actual leaf length observed was corrected by the addition of assumed increases which had been washed away in the interval between surveys. The net production in the overground part was obtained by converting the corrected increase of leaf length into dry weight. The net production in the underground part was obtained from the weight increase of biomass during the investigation. The net production rates in overground and underground parts were 2.3 and 0.4g dry wt./m2•day, respectively. The net organic productions of the former and latter parts were 8.0 and 6.6 times greater than the biomass at the start of the investigation, respectively. The production rates of eelgrass beds in the infralittoral zone at Yanai Bay were estimated as 7.4 and 2.7g dry wt./m2•day for the former and latter parts, respectively.

The dietary value of hatched neonates from the marine rotifer Brachionus plicatilis resting eggs was investigated through larval rearing trials of red sea bream Pagrus major and chemical analysis. Rotifer resting eggs were mass-produced in 50m3 tanks by feeding Nannochloropsis oculata and baker's yeast. Eggs were preserved at 5°C under total darkness for 1-2 years and hatched at 23°C under continuous light. After nutritional enrichment with the use of N. oculata and squid oil, hatched neonates or commonly mass-cultured rotifers (control) were fed to fish larvae. Newly hatched fish larvae were stocked in 100l (80l in water volume) polycarbonate tanks at 15 or 30/l. No significant differences in larval growth (notochord length and dry weight) were found on Day 20 between groups fed on neonates from resting eggs and those fed on control rotifers. The survival rate of fish larvae fed on neonates (38%) were, however, significantly lower than the group fed on control diets (69%). This may be due to the fact that the uniform lorica size and synchronous growth of neonates during the enrichment culture after simultaneous resting egg hatching did not fit the mouth size of the fish larvae. The amount and composition of fatty acids of neonates were the same as those of the control rotifers.

The growth of eelgrass Zostera marina in tidal flats was investigated in Yanai Bay, Seto Inland Sea, Japan, from late April to late July 1982. Field surveys were conducted seven times with a marking method at about 15 day intervals. In May and June, the fastest growth of vegetative shoots of eelgrass was observed. The average number of leaves on a vegetative shoot was 4.5 and the plastochrone interval was about 10 days. The average life of a leaf on a vegetative shoot was about 40 days. The growth rates of leaves 10 and 30 days after formation, were 2.5 cm/day and 0.5 cm/day, respectively. The growth rate of rhizomes was about 2cm/day in May. The formation of nodes was synchronized with a loss of leaves by washing away. In generative shoots, neither growth of rhizome nor branching was observed in the period of investigation. Spadices were fromed from late April and became dominant in June. In July, all spadices were washed away and subsequently all generative shoots withered. Our observation suggests that seeds were released together with the spadix when it was washed away, but not scattered one by one.

The reduction of environmental water exchange and the supply of an ample amount of diet are required for inducing rotifer resting egg formation. The use of a preserved diet is essential for conducting trials of mass formation of resting eggs in a large scale tank. The potential for applying preserved diets for L-and S-type rotifer Brachionus plicatilis resting egg production was investigated in the laboratory. Dietary values of frozen or refrigerated diets (baker's yeast, Chlorella vulgaris, Euglena gracilis, and Nannochloropsis oculata) in condensed forms were compared with fresh N. oculata diet (control).Frequencies of rotifer mastax movement when fed most of the preserved diets were equal with the control. In the individual culture experiment, growth and mixis rates obtained by feeding preserved diets were much less than the control. Growth rates increased to the level of the control, however, when tested diets were fed to batch-cultured rotifers in a 20l medium. The remarkable increase of mixis rates in batch cultures was observed when preserved diets were fed to S-type rotifers. The number of resting eggs formed from 1g dry weight preserved diet was 7.8-112.0 (×103) and 2.1-5.0 (×103) for L-and S-type rotifers, respectively. Meanwhile, it was 138.0 for L-type and 15.4 for S-type with the control diet.

A comparative study on the karyotypes of S and L types of the rotifer Brachionus plicatilis O.F. Muller was carried out to clarify their cytological differences. Embryos, stained with alcoholic hydrochloric acid-carmine, were made into squash chromosome preparations. Mitotic chromosomes of S type prometaphase cells and L type metaphase cells were studied. The two karyotypes differed in chromosome number and morphology. The female diploid embryo of the S type had 25 chromosomes (2n = 25), whereas that of L type had 22 chromosomes (2n = 22). The chromosome numbers of male haploid embryos of S and L types were n = 12 and 11, respectively. Differences in karyotypes suggest that S and L types are so differentiated they should be considered different species. The possible evolutionary steps of chromosomes changes leading to the two types are discussed.

The rotifer Brachionus plicatilis O.F. Muller has been used extensively for aquaculture in Japan; two morphotypes, S (small) and L (large), are distinguished, based on morphological and physiological differences. Morphological differences between these two types were studied biometrically. 67 strains of the rotifer collected from all over the world were classified by eye into 37 S and 30 L type strains by differences in the shape of anterior spines of lorica. For each of the 67 strains, seven characters of the lorica were measured on each of 20 individuals derived parthenogenetically from one individual. Classifications based on both cluster and stepwise discriminant analyses yielded equivalent results. Based on cluster analysis using the seven measurements, the 67 strains were classified into two groups containing the predesignated 37 S and 30 L type strains. Lorica length, lorica shape and shape of anterior spine were considered as the distinguishing morphological characters between S and L types. Two linear discriminant functions associated with S-L types were also derived, which can be used to classify new strains that may be observed in the future into S or L type. Accordingly, the rotifer was clearly divided morphologically into the two types, S and L.

Allozyme variation was determined for 67 strains of the rotifer Brachionus plicatilis O.F. Muller including 37 strains of S type and 30 strains of L type collected from all over the world, using horizontal starch gel electrophoresis. Six of the 18 enzymes assayed were variable, viz., lactate dehydrogenase (LDH), malate dehydrogenase (MDH), 6-phosphogluconate dehydrogenase (6PGD), superoxide dismutase (SOD), phosphoglucomutase (PGM) and glucose phosphate isomerase (GPI). 33 of the 46 alleles detected for the six loci were specific for either S or L type strains. On the basis of genetic distances calculated from allele frequencies of the 46 alleles, a dendrogram of the similarity among 67 strains was drawn. The strains were divided into two groups at a large genetic distance. One group consisted of 37 strains which coincided with the strains classified as S type in morphometric analysis. The other group consisted of 30 strains which coincided with ones classified as L type in morphometric analysis. The result indicates that the two groups of the rotifer, S and L, are genetically divergent.

Recent research has indicated that the hatchability of mass-produced rotifer (Brachionus plicatilis) resting eggs (stored in sea water in the dark at 4°C) gradually decreases due to bacterial infection of the porous egg surface. We investigated whether lyophilization as well as a canning treatment could solve the problem of bacterial infection of resting eggs. The canning method preserves eggs in a dry form, after lyophilization at −40°C. The resting eggs were packed in cans under various pressures: 21, 34, 48, 61, 75, 88 and 101 kPa. The viability of the canned resting eggs was tested by hatching experiments done 6 and 12 months after canning and storage at 5°C. When partial pressures were less than 88 kPa, percent hatching after 6 months ranged between 16 and 20%, similar to that of uncanned eggs (19% hatching), but significantly lower (9 and 11%, P

The practical mass culture of marine microalgae, faces occasionally unexpected problems or collapse. The effect of a marine bacterium, Flavobacterium sp., which was found to promote growth of a marine diatom Chaetoceros gracilis in the axenic culture condition, was examined on the mass cultures of three marine microalgae.

Condensed suspension of Chlorellavulgaris was used for the food of the rotifer Brachionus plicatilis and B. rotundiformis inplace of Nannochloropsis oculata. Thisreport describes the characteristics of C. vulgaris as arotifer food in comparison with N. oculata and thepresent status of this field.The cell components of C. vulgarissuch as protein content, amino acids, minerals andvitamins are generally similar to those of N. oculata. However, the taxonomic status of thesealgal species are different. Based on thesimilarity of cell components, the dietary value ofC. vulgaris is equal in value to that of N. oculata for rotifer growth. Dietary value ofC. vulgaris can be improved by addition ofvitamin B12. This improved C. vulgaris is currently widely used as an indispensable food organism for rotifer culture. Recent investigationshave shown that the use of the condensed suspensionof C. vulgaris makes it possible tosignificantly increase the rotifer density atharvest. Application of condensed C. vulgaris has made rotifer culture quite easy because theculture of N. oculata is no longer required,and intensive rotifer production in aquaculture cannow be realized.

Copepods and protozoans often co-exist in marine rotifer mass cultures. Interspecific interaction between the rotifer Brachionus rotundiformis Tschugunoff and eight other zooplankton species, namely Brachionus plicatilis O. F. Muller (rotifer), Diaphanosoma celebensis Stingelin (cladoceran), Tigriopus japonicus Mori, Acartia sp. (copepod), Euplotes sp., Vorticella sp., an unidentified protozoan species (P1 strain) (protozoan) and Artemia sp. (anostracan) at two developmental stages (nauplii — 0.95 mm, 0 days old; adults — 3.3 mm, 19 days old) were investigated in the laboratory.

Microbial techniques of biocontrol using the interaction of microorganisms to repress the growth of deleterious bacteria and viruses were developed. The bacterial strain used in this work also improved the growth of fishes and crustaceans. Using the concept and procedures of the biocontrol method described here, the aquaculture production became stable and even increased.

A technique was developed for the semi-mass culture of the unarmored dinoflagellate, Gymnodinium splendens under laboratory conditions. A maximum cell density of 4600 to 6800 cells m1−1 was observed within 8 to 11 days of culture. An initial feeding test for 8 days with three important marine finfish larvae showed that red spotted grouper, Epinephelus akaara preferred G. splendens fed 200 cells m1−1 with 44% survival. The Japanese stripe knife jaw, Oplegnathus fasciatus, attained 22% survival fed a combination of G. splendens and rotifers (200 cells m1−1 and 5 ind. m1−1, respectively). Red sea bream, Pagrus major larvae did not respond well to the initial feeding of G. splendens alone. Red sea bream were observed to be solely dependent on rotifers (5 ind. m1−1) as initial food. Gymnodinium splendens may be used as a live food in the initial feeding of red spotted grouper larvae (E. akaara) to reduce mortality and to further enhance growth during the critical first few days of rearing.

Diapause and hatching of Brachionus plicatilis Muller resting eggs were examined through histological and optical approaches. Compound microscope observations on 1% toluidine blue-stained embryo sections suggests that the total number of nuclei in an embryo during the internal diapause period increased from 22 on Day 2 to 39 (each n = 1) on Day 6. The outer layer of embryo membrane gradually thickens from 1.2 (Day 0) to 4.0 μm (Day 8) (each n = 10).

Fecundity patterns of S and L type rotifers Brachionus plicatilis, which were previously found to have distinct growth ability, were analyzed using data of age-specific fecundity. The data were obtained by individual cultures of S and L type strains at 17, 24 and 34 °C. The pattern was analyzed by using normal probability functions. When the age was transformed into logarithmic value, the S and L types had an identical pattern of fecundity at every temperature. This fact indicates that the difference of the growth response to the temperature between S and L type strains stemmed only from the differences in net reproduction rate, not from the pattern.

The response of Bugula neritina in the early growth stage of a colony to the environmental water temperature was studied. The larvae attached on slide glasses were immersed 1 m below the surface in the sea at five experimental sites. In the colonial growth, the increase of the zooid number had a parallel relation with the increase of branch number and of the dry and wet weights. The growth speed of a colony could be expressed as the function of the number of zooid (N):dN/dt=rN(t, day: r, intrinsic rate of natural increase of zooid). The experiment on colonial growth conducted in four seasons at various temperatures revealed that the lowest temperature for survival was about 9°C, the lower limit of temperature for the exponential growth was about 13°C. The relation between the intrinsic rate (r) and water temperature (T) for a temperature range from 13 to 28°C could be represented by the following equation: logr=0.04T-1.3. B. neritina was supposed to tolerate without any abnormal symptom even a high temperature of 30°C.

Colonies attached to glass slides were suspended in the sea. They were also immersed in low salinity waters once for one or two hours or repeatedly for one, two or six hours once a day to examine the effects of salinity changes on growth. The effect was also examined with a culture method in beakers using water of various salinity (33.7, 31.0, 27.9, 24.1 and 18.1‰), with colonies on glass slides with Rhodomonas as food.Exposure to fresh water for even a short time caused severe damage to the animal, but salinities higher than 9‰ were not injurious for their further growth. Colonies repeatedly exposed to seawater lower than 20‰ will not grow to be a mature colony. If the seawater has a salinity higher than 25‰ they might grow to a mature stage and in salinities higher than 30‰ they grow well.

The larvae attached on slide glasses were immersed two meters below the surface at two stations in Omura Bay and Nomo Inlet located near Nagasaki City. Colonial growth shown by the increase of zooid number, sexual maturation and the time span of the colony from attachment on to falling off from the slide glass (life span of the colony) were investigated in four seasons from 1982 to 1983.There was a linear relation between the number of zooids (N) and the height (Hmm) of the colony, which was represented as follows: ln N=2.46 ln H-0.22 (r=0.96).The lowest temperature for survival of the colony and the lower limit of temperature for the exponential growth were estimated as about 9°C and 13°C, respectively. The life span of the colony varied from 1.5-2 months in spring and summer to more than 3 months in fall and winter, depending on water temperature.We could observe the first brood chamber in zooids when the colony had grown up at zooid number of 200-1, 000. Our observation suggested that the colony could release the larvae after growing up at the zooid number of 1, 000 to 5, 000 (height, 2-4cm). The minimal size of the colony in sexual maturation showed little fluctuation seasonally in zooid number.The maximal size of a colony was estimated as 20, 000-50, 000 in zooid number (height of the colony, 6-10cm).

Two slide glasses each bearing three to five larvae of Bugula neritina attached had been placed in a one-liter beaker holding 800m/ of filtered seawater. After the daily renewal, adequate volume (10-60m/) of the cultured plankton was supplied into the beaker to make the food suspension at a definite density. The geometric means of the zooid number per colony after ten-day's culture with various food planktons were determined to compare the dietary effect. The salinity and temperature of culture water were maintained from 33.4 to 34.3‰ and from 21 to 23°, respectively. Of 24 species examined, Rhodomonas sp. supported the best growth; all young colonies exponentially increased the zooid number, from one to 72 in ten days. The growth was almost equal to that under the best natural conditions. Oxyrrhis marina cultured in the medium enriched with yeast extract was the most suitable food next to Rhodomonas (48 zooids in ten days). When fed on Dunaliella tertiolecta and Prorocentrum obtusidens, the colonies increased the zooid number only to 18 and 12, respectively. Diatoms and armored planktons had no dietary value for the animal.

The larval preference of Bugula neritina for settlement to each constituent of primary film, such as detrital particles, attaching bacteria and attaching diatoms which had been coated on a glass slide by immersing in the sea and in a closed circulating aquarium was studied through a choice test. Two hundred larvae were put in a chamber where six not film-coated and six film-coated glass slides were stuck on the inner side of the Iid of the chamber. The settlement index on film-coated glass slides was defined as the difference between the numbers of larvae which settled on the film-coated and not film-coated surfaces. The glass slide coated with the primary film was preferable for the settlement of the larvae to the not film-coated glass slide. The attaching diatom showed a quite similar pattern of increase to that of the settlement index. The attaching diatoms on the surface of the glass slide was the most attractive for the settlement of the larva.

The culture of the sterilized rotifer Brachionus plicatilis with washed cells of a baker's yeast revealed that the yeast had no nutritive effect on the pupulation growth of the rotifer. Nutritive quality of experimental suspensions was evaluated by two kinds of culture, one of which was individual culture in which the rotifers were cultured separately in test tubes and observed daily to obtain the survival rate and fecundity while food suspension was renewed daily. From daily counts, intrinsic rate of population increase and net reproduction rate were calculated as the indices of nutritive quality of the suspension. The other was batch culture in which teh rotifers were maintained in the food suspension without any daily treatment. After 6 or 7 days culture, the increase in individual number was counted. Out of the nutrients tested on the supplementary effect, vitamin B12 was most effective. Pollack liver oil (feed oil) and cystine were also effective in strengthening the nutritive quality of the yeast cell suspension. None of the oil spluble vitamins could substitute for the supplementary role of the feed oil.

The dietary value of dried, commercial Chlorella was compared with that of living marine Chlorella, and yeast, in relation to growth of the rotifer Brachionus plicatilis raised individually and by batch culture methods. A concentration of 50 μg/ml of dried Chlorella powder is near an optimal density for rotifer growth. The dried material in suspension is less effective for growth than living marine Chlorella, although it is much more effective than a suspension of yeast at the same density (50 μg/ml). In batch culture (12-l glass vessel), the rotifers grew from an initial inoculation of 13.2 individuals/ml to a density of 434 individuals/ml by the 16th day. About 107 rotifers could be removed from one batch culture in five harvests in the 41-day experimental period. The results indicate that dried Chlorella powder is an effective food for the rotifer Brachionus plicatilis.

In mass culture tanks for the rotifer Brachionus plicatilis, the concentrations of inorganic. nitrogen compounds, pH, water temperature and specific gravity were measured daily at the Aqua-culture Research Laboratory of Nagasaki Prefectural Institute of Fisheries from April to July, 1984. The rotifer density decreased with an increased concentration of un-ionized ammonia nitrogen (NH3-N). The highest concentration of NH3-N observed was 3.6ppm. The rotifer densities above 150 rods./ml were observed, only when the pH ranged from 7.3-7.8. Acute toxicity test of NH3-N to rotifers showed that 24h LC50 value was 17.0ppm at 23°C. Chronic toxicity test showed that concentrations of NH3-N at which the population growth and the reproduction reduced by 50% of the controls were 13.2 and 7.8ppm, respectively. The concentrations at which the intrinsic rate of population growth and the net reproduction rate were reduced by l% of those of the control, were adopted as the “no-effect” concentration of NH3-N. They were estimated as 2.1 and 2.4ppm, respectively. The results from mass culture and toxicity tests showed that higher concentration of NH3-Nmight be one of restrictive factors affecting the increase of the rotifer.

The development of the capacity to store vitamin B12 is necessary for improvement in the dietary value of Chlorella as a food for mass production of the rotifer Brachionus plicatilis. In the present study, the uptake of vitamin B12 by fresh-water Chlorella was investigated under controlled culture conditions.The vitamin B12 content of nine Chlorella strains grown in a medium without enrichment of vitamin B12 were at less than 0.26μg/100g, regardless of the culture methods of autotrophy, mixotrophy and heterotrophy. The test of vitamin B12 uptake from the medium clarified that the strains were divided into two groups. The first group was composed of 5 strains and has the ability to take up more than 81% of the vitamin B12 which had been added to the medium. The other group of 4 strains took up less than 19% of the vitamin B12 supplied. The former group has no ability to produce secondary carotenoids, which are related to the structure and composition of cell walls, while the latter group has the ability.The addition of vitamin B12 at various concentrations into the culture medium makes it possible for Chlorella vulgaris K-22 to store vitamin B12 in the cells at various levels from 0.2 to 1100μg/100g. The vitamin B12 content of cells remained virtually unchanged during a thirty-day cold storage and during a three-day suspension in artificial sea water.

Nutritional effects of marine phytoplankton on population growth of the rotifer Brachionus plicatilis were investigated under sterile conditions. First-laid eggs of the rotifers were divided into two groups, and one was cultured with a species of phytoplankton and the other was cultured with Chlorella sp. (marine type) as control. They were cultured separately in many test tubes, each containing two individuals and were observed daily to obtain the survival rate and the fecundity, while food phytoplankton suspension was renewed daily. The phytoplankton suspension was adjusted at the concentration which had been decided previously as the desirable cell concentration for population growth of the rotifers. From these daily counts, intrinsic rate of population growth and net reproduction rate were calculated as the indices for evaluating the nutritional effect of phytoplankton on population growth of the rotifers. Eight species of phytoplankton were evaluated in terms of nutritional effect on population growth of the rotifers as follows. Two species of Synechococcus elongatus and Chlorella sp. were evaluated as excellent food plankton, four species of Chlamydomonas sp, , Monochrysis lutheri, Dunaliella tertiolecta and Cyclotella cryptica as average and two species of Eutreptiella sp. and Nitzschia clostelium as deficient.

This paper describes the chemical changes in culture water in closed culture systems with sand filter. Stabilization of inorganic nitrogen after sequential changes of ammonium-N and nitrite-N in a culture system with new filters takes from 40 to 60 days. The characteristic changes in chemical composition of aged culture water, namely an accumulation of nitrate and phosphate and lowering of pH and alkalinity, are discussed. The carrying capacity in a closed culture system is determined by using oxygen consumption during filtration as an index to indicate the degree of purification by filtration.