doi:10.1016/j.fsi.2005.11.006 
Copyright © 2005 Elsevier Ltd All rights reserved.
Short communication
Production of reactive oxygen species (ROS) by devil stinger (Inimicus japonicus) during embryogenesis
Kazushi Kadomuraa, Takuji Nakashimab, c, Maki Kurachib, Kenichi Yamaguchib and Tatsuya Odab, 
, 
aNagasaki Prefectural Institute of Fisheries, Nagasaki, Japan
bDivision of Biochemistry, Faculty of Fisheries, Nagasaki University, Nagasaki 852-8521, Japan
cNagasaki Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, JST, Nagasaki, Japan
Received 16 September 2005;
revised 9 November 2005;
accepted 10 November 2005.
Available online 27 December 2005.
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Abstract
In aquacultural industry, the seed production of devil stinger, a valuable fish in Japan, has not succeeded yet due to the cryptogenic mass mortality. We found that survival rate of the larvae of devil stinger increased by the addition of green tea extract rich in catechin into rearing tank. Generation of reactive oxygen species (ROS) was detected in the embryo of devil stinger by chemiluminescence analysis under the normal growth conditions without addition of specific stimulants. Even in the unfertilized egg, certain level of ROS was detected. ROS were continuously detected during the development from fertilized egg to larva and tended to increase gradually. Observation of embryos and post-hatching larvae with hypersensitive photon-counting microscopy indicated that ROS were produced on the surface of embryo and the head region of larva especially peripheries of eyes. When the embryo proteins were analyzed by immunoblotting using antibody against the human neutrophil cytochrome b558 large subunit (gp91 phox), a main band of approximately 91 kDa was detected, suggesting the presence of NADPH oxidase-like ROS generating system in the embryo of devil stinger. After treatment with streptomycin and penicillin G for 1 day, the level of ROS production in larvae decreased with increase in the survival rate of larvae. Our results suggest that devil stinger has ROS generation system that is already activated at fairly early stage of development before the maturation of usual immune system.
Keywords: Devil stinger; Fish embryo; Fish larva; Reactive oxygen species; NADPH oxidase
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Fig. 1. The generation of ROS by devil stinger during early development. (A) Effect of green tea extract on the survival rate of larvae of devil stinger after hatching. Green tea extract (final concentration 1 μg ml−1) was added to one tank (test group), and the viability of the larvae in the tank was observed together with two other control tanks. This experiment was repeated several times, and the representative results are shown (Expt. 1; ○ +catechin, ●, control; Expt. 2: □, +catechin, ▪, control). Statistical analysis was performed using the Mann–Whitney U-test for comparison of two groups. *P < 0.01, Significant difference between the control group and green tea extract-treated group. (B) Time course of L012-mediated chemiluminescence response of the embryo of devil stinger. Day 1 embryo after fertilization was put into a well of a white 96-well plate and chemiluminescence response was measured after addition of L012 (final 10 μM). Each value represents the mean of 10 embryos. (C) Relationship between the number of embryos and chemiluminescence response intensity. From 1 to 20 embryos were put into a well, and the relative intensity of integrated emission of each well during initial 30 min was measured. (D) Time course of chemiluminescence response levels during the development from the fertilized egg to aged larvae. Ten specimens withdrawn from each of four separate tanks were put into a well and the chemiluminescence responses were measured every day. ○ (black), tank A; ○ (red), tank B; □ (blue), tank C; □ (green), tank D; ●, unfertilized eggs. (E) Location of ROS generation in embryo and larva of devil stinger. After addition of L012 (final 10 μM), each specimen was observed with a hypersensitive photon-counting microscope equipped with a real-time digital CCD camera. (A) embryo; (B) larva 0 days after hatching; (C) larva 2 days after hatching; (D) larva 3 days after hatching. Blue color indicates L012-mediated chemiluminescence emission. Each bar indicates 500 μm.
Fig. 2. (A) Effects of various agents on L012-mediated chemiluminescence response of the embryo of devil stinger. In the presence of each agent at the indicated final concentration, relative intensity of integrated chemiluminescence emission of each well containing 10 embryos during the initial 30 min was measured at 26 °C. All these reagents were purchased from Sigma (St. Louis, MO). Statistical analysis was performed using the Student t-test for independent samples. *P < 0.01, significant difference between the control group and reagent-treated group. (B) Immunoblotting analysis of the embryo proteins with the primary antibody against human neutrophil cytochrome b558 large subunit (gp91phox) of NADPH oxidase. Proteins were extracted from 10 embryos with SDS sample buffer. After electrophoretic separation, the usual immunoblot analysis was conducted using the antibody mentioned above. Lane A, embryo; lane B, RAW264.7 cells (mouse macrophage cell line).
Fig. 3. Effects of antibiotics on the number of bacteria in rearing seawater, hatching rate, and chemiluminescence response of the embryos of devil stinger. After 24 h treatment with 100 μg ml−1 penicillin G and streptomycin, the number of viable bacteria in the rearing seawater, chemiluminescence response levels of the larvae, and hatching rate were measured. Statistical analysis was performed using the Student t-test for independent samples. Significant differences between the control group and antibiotics-treated group: *P < 0.05, **P < 0.01.
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