Studies on the production of reactive oxygen species (ROS) during the early development stage of various marine fish species

Devil stingers (Inimicus japonicus) and marbled rockfish (Sebastiscus marmoratus) are economically important fish in Japan. These fishes have been expected to be profitable species for marine ranching because of their migratory habits and high retail prices. Some hatcheries have tried to establish seed stock, but they have not been able to achieve stable production because of cryptogenic mass mortality during the larval rearing stage. Rearing conditions, egg quality, feeding conditions, and diseases are suspected as causes of this; however, the precise reason for this mass mortality has yet to be elucidated. There has been a report that the survival rate of the marbled rockfish larvae was notably improved by the addition of ascorbic acid into the rearing tank. Based on the above report, I speculated that the improvement in the survival rate was a result of the antioxidant effects of ascorbic acid caused by excess ROS. There has been little research about the production of ROS by fish larvae, or about the involvement of ROS in the survival rate of larvae. Herein, I tried to accumulate an elementary knowledge about ROS production of various marine fish larvae, and to elucidate the effects of the ROS level on the survival rate during the early development stage. 1. To confirm an effect of antioxidant materials, such as L-ascorbic acid phosphate magnesium (APM) and a catechin rich green tea extract, which are commercially available from Itoen(Ltd.), on the survival rate of devil stinger and marbled rockfish larvae, rearing experiments were performed. These materials dramatically decreased the mass mortality of the larvae without any negative effects on the growth. The effectual additive amounts per 1kl of rearing water were 10g of APM, 0.5g of Teaflan30A, and 0.1g of Teaflan90S, respectively. 2. I investigated the production of ROS utilizing chemiluminescence analysis on the larvae of 6 marine fish species: devil stinger; marbled rockfish; black rockfish (Sebastes inermis); sevenband grouper (Epinephelus septemfasciatus); tiger puffer (Takifugu rubripes); and, red seabream (Pagrus major). The larvae of these six marine fish species produced ROS under normal rearing conditions without extra stimuli or inducers. These results suggest that ROS generation is a common biological feature of at least the fish species tested in this study. 3. Since the addition of superoxide dismutase (SOD) resulted in a significant disappearance of chemiluminescence signals, the larvae of these six marine fish species primarily produced superoxide anions. 4. The larvae of these six marine fishes produce ROS to considerably different extents. The exact reason for these differences is unclear now, it might reflect the different biological characteristics of the ROS generation systems in the various fish species. The devil stinger, marbled rockfish, and black rockfish showed much higher levels of chemiluminescence responses than the others. Since it is known that the survival rate of these fish species are unstable because of sudden mass mortality during the larval rearing stage, the higher level of ROS production of these fish species may be somewhat linked to this incidence. 5. I examined the ROS production of the devil stinger embryo. The embryo of the devil stinger produced a certain level of ROS; furthermore, when the soluble proteins extracted from embryos were analyzed by immunoblotting, using an antibody against human neutrophil cytochrome b558 large subunit (gp91phox), a main band of approximately 91 kDa was detected in the cells of the mouse macrophage cell line RAW264.7. These results suggest that the devil stinger has an ROS generation system that is already activated at a fairly early stage of development, before the maturation of the usual immune system. 6. The ROS levels of devil stinger larvae in a rearing experiment for mass seed production were monitored utilizing chemiluminescence analysis during the initial 15 days. Although the larvae escaped extinction, outbreaks of mass mortality occurred two times out of six rearing experiments. In two experiments, the ROS levels surged for a while before increasing mortality. These results suggest that the mortality of larvae might be predictable by monitoring ROS levels during rearing. 7. The expression level of gp91phox was analyzed by immunoblotting analysis during embryogenesis of devil stingers. The variation pattern of gp91phox were coincident with the pattern of ROS generation, both the expression of gp91phox and ROS generation reached a peak at about 30 hours post fertilization. 8. The high ROS level was observed in the fish species prone to mass mortality; furthermore, increases of ROS generation coincided with mortality in the case of the devil stinger. These results demonstrate that an environmental factor, such as infection with some kind of bacteria trigger over production of ROS, and excess ROS might cause mass mortality.