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Larvae of the swimming crab Portunus trituberculatus are traditionally cultured with rotifers, Artemia, and phytoplankton. However, abnormal morphology of last-stage zoeae caused by excessive nutritional intake, abnormal morphology of megalopae caused by indirect ingestion of phytoplanktons via rotifers during zoeal stages, and lack of essential nutrients due to starvation of Artemia cause mass mortality. To develop techniques addressing these issues simultaneously, we evaluated methods to prevent megalopal abnormalities while avoiding starvation of Artemia (experiment 1). Then, we evaluated the effects of Artemia feeding schedules on the occurrence of last zoeal abnormalities (experiment 2). In experiment 1, megalopal abnormality was prevented by rearing larvae with enriched Artemia supplemented with digestible Nannochloropsis, but without rotifers. However, the developmental rates were variable among individuals because of the large size of enriched Artemia for early zoeae, and last zoeal abnormality was induced because of excessive Artemia feeding, resulting in low survival. In experiment 2, newly hatched Artemia were only supplied during the first zoeal stage, and larvae were reared with low density Artemia and/or low salinity. These measures achieved high survival rate while suppressing the last zoeal abnormality. Our results suggest that P. trituberculatus larvae can be successfully cultured using only Artemia.

To elucidate possible causes of skeletal malformations in larval Japanese flounder, we reared larvae fed rotifers enriched with three types of preserved Nannochloropsis (fresh, refrigerated, and frozen). The incidence of malformations at 50 days post hatch ranged from 14.5 to 38.5 % within the three experimental groups, and elongation of the lower jaw (LJ) was the most frequently observed malformation, ranging from 68 to 89 % of total malformations. We also investigated larval parentages using microsatellite markers. Parentage analysis of the fresh Nannochloropsis group showed that one sire and a pair generated significant numbers of LJ-elongated individuals. In the refrigerated Nannochloropsis group, one dam and two sires generated significant numbers of LJ-elongated individuals. In the frozen Nannochloropsis group, no broodstocks or pairs generated significant numbers of LJ-elongated individuals. Our results suggest that LJ elongation in Japanese flounder likely results from the application of different types of preserved Nannochloropsis during rotifer feeding stage. However, there is also some level of genetic influence associated with this deformity.

We evaluated the nutrient contents of rotifers sampled from larval-rearing tanks (tank rotifers) without water exchange during the seed production of amberjack Seriola dumerili at three facilities (Kamiura, Kagoshima, and Miyazaki) and compared them with the nutrient contents of freshly enriched rotifers. Compared to the enriched rotifers, the lipid contents, especially neutral lipids and proportion of 22:6n-3, tended to decrease in the tank rotifers. These trends were clearer at Miyazaki where the tank rotifers were sampled before daily supplementation of microalgae (Nannochloropsis). Crude protein content of the tank rotifers did not decrease markedly although the proportion of lysine tended to decrease. Vitamin C and E contents of the tank rotifers decreased significantly only at Miyazaki. Calcium content of the tank rotifers increased at Kamiura and Miyazaki, and the increases in iron and manganese contents of the tank rotifers at Miyazaki and zinc content at Kagoshima were pronounced. These results suggest that the nutritional value of rotifers in larval-rearing tanks without water exchange can be maintained by appropriate supplementation of microalgae. The effect of certain minerals that became high in tank rotifers on subsequent larval development requires further investigation.

金沢大学大学院自然科学研究科 信頼性システム工学, 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 2000 mL 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 Σn-6/Σn-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 Σn-6/Σn-3 ratio in continuously cultured rotifers. © 2008 Japanese Society of Fisheries Science.

Bacterial community structures were analyzed in water used for rearing fish larvae by fluorescence in situ hybridization. In Experiment 1, red sea bream Pagrus major larvae were reared in two commercial seed production tanks. The survival rate in Tank 1 was higher than in Tank 2, even though phytoplankton, Nannochloropsis sp., was added to both tanks. In Tank 2, γ-proteobacteria became dominant (∼70% of total bacteria) on day 13, there after heavy larval mortalities occurred. In Tank 1, however, α-proteobacteria and the Cytophaga-Flavobacterium cluster were predominant from day − 1 until day 13; no significant mortality was recorded. In Experiment 2, marble goby Oxyeleotris marmoratus larvae were cultured with or without Nannochloropsis sp. At the end of the experiment, larval survival rates in aquaria with Nannochloropsis sp. were significantly (P

Phytoplankton used in fish hatcheries is mass-cultured in the open air and usually contains large numbers of bacteria. In commercial fish production, the phytoplankton cultures are usually added into the larval rearing tanks; however, the numbers and types of bacteria introduced into the rearing tanks simultaneously are unknown. In this study, the bacterial community structures in Nannochloropsis sp. cultures were analyzed by using fluorescence in situ hybridization (FISH). A direct viable count (DVC)-FISH analysis was also performed as DVC is useful for the detection of actively growing cells. Total numbers of bacteria in Nannochloropsis sp. cultures ranged from 7.72×105-2.39×106 cells/mL. High proportions of the total bacteria (31.6–53.6%) in the Nannochloropsis sp. cultures showed growth potential. DVC-FISH analysis revealed that α-proteobacteria and the Cytophaga-Flavobacterium cluster were abundant in the bacterial community of actively growing cells. Thus, the high growth potentials of the distinct bacterial communities in Nannochloropsis sp. culture must influence the bacterial communities in larval rearing tanks.

The phenomenon of mass mortality of larval mud crab Scylla serrata is described during the metamorphosis to megalops due to abnormal molting by morphologically advanced last stage zoeas (fifth stage) in the seed production. Seven seed production trials were carried out using 100 kL concrete tanks. The larvae were reared on rotifers and after the third or fourth zoeal stage with Artemia nauplii. Minced mysid was also given from the megalopal stage. The seven trials were based on the addition of different concentrations of the marine phytoplankton Nannochloropsis or of the freshwater Chlorella to the larval rearing water. The survival rate, morphological features of the fifth stage zoeas, and frequency of abnormal molting during the metamorphosis to megalops were examined. The mass mortality occurred during the metamorphosis to megalops in tanks containing high concentrations of Nannochloropsis. The fifth stage zoeas in these tanks were equipped with some advanced morphological features similar to those of megalops, such as, long endopods of the second antennae, large chelipeds and pleopods with plumose setae. The frequency of abnormal molting tended to increase depending on the degree of megalopal features in the fifth zoeal stage. We propose that zoeal morphogenesis is accelerated by the nutritional conditions of zoeas due to the supplementary effect of Nannochloropsis, which contains eicosapentaenoic acid; an essential fatty acid for larval mud crab.