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Ontogeny of tolerance to hypoxia and oxygen consumption of larval and juvenile red sea bream, Pagrus major
Aquaculture 244 (2005) 331 – 340 www.elsevier.com/locate/aqua-online
Ontogeny of tolerance to hypoxia and oxygen consumption of larval and juvenile red sea bream, Pagrus major Yasunori Ishibashia,*, Kosuke Inouea, Hiromu Nakatsukasab, Yutaka Ishitanib, Shigeru Miyashitab, Osamu Muratab a
Department of Fisheries, School of Agriculture, Kinki University, Nakamachi, Nara 631-8505, Japan b Fisheries laboratory, Kinki University, Kogaura, Shirahama, Wakayama 649-2211, Japan
Received 14 October 2004; received in revised form 17 November 2004; accepted 18 November 2004
Abstract Changes in tolerance to hypoxic stress and oxygen consumption were studied in the red sea bream, Pagrus major, from its early life stage until 42 days post-hatch. In the experiments, metamorphosis was observed mainly from days 15 to 30, and the morphological shift from larva to juvenile was completed at around 9.5 mm total length (TL). During the larval stage, lethally low dissolved oxygen (DO) levels and mass-specific metabolic rates increased with growth from 2.6 to 5 mm TL ( Pb0.01). Subsequently, the levels remained high and decreased until about 9.5 mm TL around the flexion stage and post flexion stage. Finally, beginning in the juvenile stage, lethal DO levels and mass-specific metabolic rates decreased as TL increased up to about 30 mm ( Pb0.01). The relationship between lethal DO levels and mass-specific metabolic rates was significantly linear (r=0.59, pb0.001, n=207) in fish larvae and juveniles. These results indicated that, around the stage of flexion and post flexion larvae in red sea bream, metabolic rates were highest during metamorphosis, and consequently hypoxia tolerance was lowest. It was presumed that the increasing metabolic rate during metamorphosis induced a decrease in the metabolic scope of activity and thereby induced the decrease of the tolerance to some environmental stressors in the background. D 2004 Elsevier B.V. All rights reserved. Keywords: Stress; Hypoxia; Oxygen consumption; Larvae; Development; Red sea bream
1. Introduction In teleosts, the larval stage is a period of dramatic morphological, biochemical, and physiological * Corresponding author. Tel.: +81 742 43 6305; fax: +81 742 43 1316. E-mail address: [email protected] (Y. Ishibashi). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.11.019
changes. Metamorphosis is the focus of many such changes, and some of these metamorphic changes are controlled by hormones such as thyroid hormones, cortisol, and prolactin (Inui and Miwa, 1985; Hiroi et al., 1997). Some chemical components, such as nucleic acid, proximate composition, and enzyme activity of whole body, are also altered significantly during morphological changes in the larval through
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juvenile stages (Ehrlich, 1974; Fukuda et al., 1986; Richard et al., 1991; Tanangonan et al., 1998; Gwak and Tanaka, 2002). Adaptability to various environmental changes is also altered during larval development (Ishibashi et al., 2003, 2004a). In particular, the tolerance to hypo- and hyper-salinity in some kinds of fish changes around the period of metamorphosis (Hiroi et al., 1997; Shikano and Fujio, 1999; Chona et al., 2000; Ishibashi et al., 2003). The responses of larvae to osmotic gradients have been well documented (Chona et al., 2000). Chloride cells were found in early development (Ayson et al., 1994; Bone et al., 1995). As the larvae grow into juveniles, the gills complete their development, mainly by an osmoregulation site related to salinity tolerance; this site is thought to move from the skin to the gills (Hwang, 1987; Jurss and Bastrop, 1995). Although some of the many changes that occur during larval development are extremely complicated and controversial, other kinds of adaptability to environmental changes have not been fully examined. Understanding the environmental physiology of fish is also of practical importance to industry, because cultivated fish are frequently exposed to stressors induced by fluctuations in such environmental factors as dissolved oxygen level, salinity, and water temperature (Ishibashi et al., 1992; Pihl et al., 1992; Ishibashi, 1994; Wu, 2002). In addition, disease can be a consequence of these stress factors (Chen et al., 2002; Yada and Nakanishi, 2002). For the last decade, many kinds of marine juvenile fish have been intensively cultured from eggs in fish nurseries. However, many sorts of bacterial and viral diseases have been observed in larvae and juveniles (Muroga et al., 1998), and some deformities that are thought to originate from feeding methods occur during nursery production (Matsuoka, 1987; Koumoundouros et al., 1997; Gavaia et al., 2002; Shimizu and Takeuchi, 2002; Haga et al., 2004). To protect the health of nursery-produced fish, a lot of articles have appeared about many kinds of diseases and a few protective methods in the rearing of fish larvae and juveniles (Tatner, 1996; Muroga et al., 1998; Watanabe et al., 1998). The effects of enriched rotifer and Artemia salina lipids on activities of some marine fish from larvae through juvenile stages have also been examined (Izquierdo et al., 1989; Takeuchi et al., 1990; Tago et al., 1999). However, in the seed
production of some marine cultivated fish, survival rates have not been stable enough, and further studies about larval health have been needed. Therefore, knowledge of stress tolerance and of the adaptation function in fish larvae through juvenile stages is extremely important for healthy nursery production. We previously examined the ontogenic changes in various stress tolerances of larval and juvenile red sea bream, Pagrus major, and found that all tolerances to temperature, salinity, and ammonia stresses temporarily declined at metamorphosis (Ishibashi et al., 2003). It is considered that the depression of the scope for activity based on the increased metabolic rate during metamorphosis induced the decreases in various stress tolerances in the background. Previously, we also studied the effects of hypoxia on stress response and energy metabolism in young red sea bream (Ishibashi et al., 2002a,b) as well as in the Japanese parrot fish, Oplegnathus fasciatus (Ishibashi, 1994) and the Nile tilapia, Oreochromis niloticus (Ishibashi et al., 2002c). We found that in these fishes, exposure to hypoxia was associated with cortisol release, a reduction in oxygen consumption and liver ATP levels for metabolic depression, and increased anaerobic metabolism (Ishibashi et al., 2002a,b,c). However, there has appeared no study of ontogenetic tolerance to hypoxic stress in fish from the larval through juvenile stages. In the present study, in order to obtain basic knowledge as to why some stress tolerances fall during metamorphosis, we examined ontogenic changes in tolerance to hypoxic stress and oxygen consumption among larval and juvenile red sea bream.
2. Materials and methods 2.1. Fish and feeding methods Fertilized red sea bream eggs were obtained, reared, and studied at three facilities of Kinki University, Japan. First, eggs from the university’s Fish Nursery Center were placed in several 500-l polycarbonate tanks (lot 1) at the Fisheries Laboratory, as well as in a 20,000-l concrete tank (lot 2) at the Center and in several 300-l polycarbonate tanks (lot 3) in the Fisheries Department’s laboratory. In each case, gentle aeration was provided for 2 days until the eggs hatched. The day of hatching was regarded as day 0. In the pre-
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larval stage for 2 days post-hatching, fish were held in static condition with slight aeration. In the subsequent rearing, larvae and juveniles in lots 1 and 2 were held in a flowing water system of filtered seawater with some aeration. Fish in lot 3 were reared in a recirculating water system with sterilized artificial seawater and exact temperature control. The rearing water temperature in lots 1 was at 25.7F3.3 8C; 22.8–24.1 8C at days 0–21, and 24.0–29.4 8C at days 22–42. The rearing water temperature in lots 2 and 3 was controlled at 19.8F2.1 8C and 20.1F0.2 8C. From days 3 to 42, the larvae and juveniles were fed rotifer Brachionus rotundiformis and A. salina both enriched with n-3 HUFA, and a commercially available formulated diet, in that order (Fig. 2). 2.2. Experiment 1—ontogenic changes of tolerance to hypoxic stress About 100 larvae or juveniles were selected at random in the early morning before feeding at each 7day interval during the rearing period from hatching to 42 days post-hatching. Twenty fish in lot 1 and three groups, each consisting of 10–30 fish, in lot 2 were exposed to hypoxic stress for 3–4 h. DO concentrations in seawater were adjusted using nitrogen gas. DO levels in seawater were reduced at a fixed rate by regulating the flow of nitrogen gas from about 7.0 to 0.5 mg/l for approximately 4 h. Changes in DO concentrations during hypoxic stress tests were monitored with a DO meter (YSI-58, YSI, Ohio, USA) and are shown in Fig. 1. The lethal DO level of fish was recorded just after respiratory and behavioral arrest, respectively. Median lethal DO levels, calculated by means of logit transformation of larvae and juveniles in each group, were taken as the parameter of tolerance to hypoxic stress. After the stress test, the total length, body weight, and developmental stages of each fish were measured, and these data were used to plot growth curves. 2.3. Experiment 2—relationship between lethal DO levels and oxygen consumption in larvae through juveniles 2.3.1. Measurement of oxygen consumption The water bottle method was improved and used to measure oxygen consumption of the red sea
Fig. 1. Changes in dissolved oxygen level during hypoxic tolerance tests.
bream, from larvae just after hatching to the juvenile stage on day 42 in lots 2 and 3. In larvae from posthatching to 14 days old, 20 to 200 normal-looking, average-sized larvae in the rearing tank were chosen and transferred, every morning before feeding, to a small tank containing filtered seawater. Some groups consisting of 2 to 10 larvae were selected from the small tank at random and carefully placed into 1- to 2-ml syringes according to the fish growth. The remaining fish in the small tank were utilized to measure the dry weight of fish. This was done because a pre-flexion larva was too small for us to determine its oxygen consumption and body weight. In the larvae older than 14 days and in juveniles, individuals were accommodated in 2- to 100-ml syringes individually, again according to size. All filled syringes were left in a water bath at 20.0F0.1 8C in a dark room. After about 1 h, while the fish appeared to be resting, the syringe was slowly revolved and the DO concentration of 100 Al seawater in the syringe was measured by a blood gas analyzer (ABL-330, Radiometer, Copenhagen, Denmark) in a dark room with slight light. The syringe and a paraffin paper seal, which were weighed in advance, were sealed at the lip and weighed to calculate the water volume. After the syringes were left in a water bath at 20.0F0.1 8C
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for 1–2 h, each syringe was slowly revolved and its DO concentration, together with exact elapsed time, was measured again. The oxygen consumption in seawater without fish was also measured in a similar way as a blank value in order to calculate the oxygen consumption of fish. The total length, body weight, and developmental stages of all fish in lot 2 were measured after the standard oxygen consumption was determined. The dry weight per fish was measured by formula method after the fish were killed by overdose of anesthetic. In larvae from post-hatching to 14 days old, 50 to 150 larvae were filtered at a time and then divided into two groups. Fish in one of the groups were weighed on the electronic scale and counted. A second group was used to determine dry weight by drying fish at 90 8C for several days. 2.3.2. Measurement of lethal DO concentrations in syringes After the measurement of oxygen consumption in lot 3, the water volume in each syringe was calculated and approximately set up to sacrifice the fish in 2–3 h in calibration with the fish’s oxygen consumption rate. The syringes were left in a mostly dark room and were occasionally revolved slowly in a water bath until the fish were dead. To determine the lethal DO level, the DO concentration in the syringe was measured a final time just after half the fish had died. Just after each lethal level was measured, the dead fish’s total length, body weight, and developmental stages were measured.
3. Results 3.1. Growth and morphological changes Fig. 2 shows the changes in the total lengths of the red sea bream, from larvae just after hatching to juveniles on day 42. The total length slowly increased before 21 days post-hatching, and then somatic growth rates were faster from the beginning of the juvenile stage about 9.5 mm TL. In particular, the growth rate of juveniles in lot 1, which were reared at a high temperature, was the fastest of all. Flexion larvae appeared at around 6.4 mm TL on days 15 to 20 in all lots. Subsequently, the morphological shift from larva to juvenile, which is based on a constant fin ray, was also observed microscopically at around 9.5 mm TL at days 24 to 30, depending on water temperature. 3.2. Experiment 1—ontogenic changes in tolerance to hypoxic stress Changes in the lethal DO levels of fish are shown in Fig. 3. In larvae, the median lethal level of low DO, which was 0.6 to 0.8 mg/l at just after hatching,
2.4. Statistics The data on lethal DO levels in lot 2 are expressed as meanFS.D., n=3. Data were analyzed by a one-way analysis of variance (ANOVA), followed by Duncan’s new multiple range test. The allometric relationships between the total length and oxygen consumption for larvae and juveniles were described by equations of linear having reflective points, depending on computed values of R 2 and significance of regression parameters. Differences between linear regressions were tested by analysis of covariance (ANCOVA). Moreover, Pearson’s correlation coefficients between mass-specific oxygen consumption and lethal dissolved oxygen were analyzed.
Fig. 2. Growth curve, feeding schedule, and developmental stages of larval and juvenile red sea bream up to day 42. (o) Lot 1 (water temperature in feeding, 25.7F3.3 8C); (4) lot 2 (19.8F2.1 8C); (5) lot 3 (20.1F0.2 8C).
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Fig. 3. Ontogenic changes in 50% lethal levels of low dissolved oxygen over a period of 3 h in larval and juvenile red sea bream. (o) Lot 1; ( ) lot 2. Each value in lot 2 represents the meanFS.D., n=3. Values with different letters in the same curves are significantly different (pb0.01).
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increased with growth until the larvae reached about 2.0 mg/l at 5 mm TL (Pb0.01). In the larval stage of about 5 mm to 9.5 mm TL, the median lethal DO levels remained high, from 2.0 to 2.3 mg/l, depending on water temperature. On the other hand, in the juvenile stage since TL reached about 9.5 mm, the lethal DO level decreased as TL increased, up to 1.2 mg/l at 32 mm TL ( Pb0.01).
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data gave three straight lines, having reflective points at about 5 mm and 9.5 mm TL. Regression lines were log M=2.762+3.968 log TL; F 1, 2 129=440.914, R =0.841, Pb0.001 in the first phase, log M=2.385+3.466 log TL; F 1, 93=322.256, R 2=0.776, Pb0.001 in the second phase, log M =2.070+3.060 log TL; F 1, 98 =5822.703, R 2=0.983, Pb0.001 in the third phase, respectively. The slopes of regression lines describing the relationship between M and TL differed, respectively ( F 2, 320=4.378, Pb0.05). Fig. 4 shows also the allometric relationships between mass-specific oxygen consumption and total length in red sea bream from the larval through juvenile stages. The allometry between mass-specific oxygen consumption and total length was triphasic. In the first phase, from 2.6 mm to 5 mm, massspecific oxygen consumption (MW) rapidly increased until reaching about 7 ml/g-dry/h (log MW=3.0379+1.2763 log TL; F 1, 130 =90.796, R 2=0.426 , Pb0.001). Subsequently, it remained high and decreased in the second phase from about 5 mm to 9.5 mm TL (log MW=4.3837–0.7061 log TL; F 1, 149=88.365, R 2=0.381, Pb0.001). Finally, in the third phase, the mass-specific metabolic rate decreased as TL increased, up to about 30 mm (log
3.3. Experiment 2—relationship between lethal DO level and oxygen consumption in larvae through juveniles The allometric equation (M=aW b) between oxygen consumption (M) and body mass (W) were calculated for ontogenic change. However, the relation between oxygen consumption and body mass was indistinct in the first phase of present experiment, because body mass barely increased until about 8 days post-hatching. Allometric relationships of oxygen consumption to total length in red sea bream from larval through juvenile stages are shown in Fig. 4. The oxygen consumption (M) increased as the fish grew, and the total length (TL)
Fig. 4. Allometric relationship of oxygen consumption and massspecific oxygen consumption to total length in larval and juvenile red sea bream. The arrows indicate inflection points. Values by the lines show the slope of the regression lines. ( ) Lot 2; (o) lot 3.
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Fig. 5. Relationship between mass-specific oxygen consumption and lethal levels of low dissolved oxygen in larval and juvenile red sea bream (lot 3).
MW=4.0314–0.3240 log TL; F 1, 55 =66.303, R 2=0.534, Pb0.001). The allometrically calculated straight-line representations of MW and TL also differed in slopes ( F 2, 334=78.259; Pb0.001). The relationship between lethal levels of low DO and mass-specific oxygen consumption in red sea bream from the larval through juvenile stages are shown in Fig. 5. Lethal DO levels increased as mass-specific oxygen consumption increased. The regression line between lethal DO levels and massspecific oxygen consumption was calculated to be y=0.1988x+0.87. Pearson’s correlation coefficient (r) was high (0.59, pb0.001, n=207), indicating a positive linear relationship.
4. Discussion 4.1. Stress tolerance during metamorphosis and critical phase in nursery production From the results obtained in this experiment, it can be concluded that tolerance to hypoxia in red sea bream decreased from 5 mm until about 9.5 mm TL during metamorphosis (Fig. 3). In a previous study of red sea bream, we demonstrated that all tolerances to high and low temperature, hypo- and hyper-salinity, and high ammonia stressors temporarily fell during
metamorphosis at days 14 to 21 (Ishibashi et al., 2003). These results are in accordance with the critical phase, from days 13 to 20, in which red sea bream were formerly prone to die in nursery production fields (Okamoto, 1969). In other kinds of fish, hyposalinity tolerance in Japanese flounder Paralichthys olivaceus and mangrove red snapper Lutjanus argentimaculatus fell by the time of metamorphosis (Hiroi et al., 1997; Chona et al., 2002). The hyper-salinity tolerance in chum salmon Oncorhynchus keta and mangrove red snapper also declined during the larval stage (Shikano and Fujio, 1999; Chona et al., 2000). However, it has not been clear why some stress tolerances fall during metamorphosis. Although the molecular, biochemical, and morphological changes that occur during larval development are complex, the present and previous results together raise the possibility that some common factor could cause the decreases in tolerance during metamorphosis. 4.2. Metabolic activity during metamorphosis In the present experiment, mass-specific oxygen consumption was increased as larvae grew and metabolism remained high from 5 to 9.5 mm TL during metamorphosis. This experimental result is approximately in accord with the data on larval red sea bream by Oikawa et al., 1991. In red sea bream, it is known that the skeletal system is altered markedly from 6.5 to 7 mm TL and that the allometry of number and sectional area of muscle fiber have reflective points at about 6 to 10 mm TL (Matsuoka, 1987). Almost all organs were also completed development in this phase. In addition, the feeding habit of the red sea bream changed from nauplius to Copepoda or Appendicularia at around 6 mm TL (Tanaka, 1973, 1985). Because of morphogenesis, physiological and biochemical changes are dramatic in fish of this period. Itazawa and Oikawa reported that in almost all fish, the metabolic rate decreased beginning at the juvenile stage, because the weight percent of the brain and viscera, which consume more oxygen than the other parts of the body, decreased in proportion to whole body weight (Itazawa and Oikawa, 1983). It is conceivable that the metabolic rate in the whole body remains at a high level, from the stage just before flexion larvae to late metamorphic larvae, because morphogenesis
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in many organs, such as the brain and viscera, requires more oxygen. Moreover, DNA, RNA, and protein content per whole body weight are elevated from early larvae up to late metamorphic larvae in red sea bream (Takii et al., 1992). Gwak and Tanaka (2002) also indicate that cyclic phase of hyperplasia and hypertrophy occurs during metamorphosis and reduced protein synthesis during the post metamorphic phase as a result of DNA, RNA, and protein measurements in whole body Japanese flounder. The elevation of these chemical components during metamorphosis also suggests an increase in the metabolic activity of the whole body for intensive growth through cell enlargement and cell proliferation. 4.3. Relationship between stress tolerance and oxygen consumption We have previously shown the effects of hypoxia on stress response and energy metabolism in various tissues of red sea bream (Ishibashi et al., 2002a,b), the Japanese parrot fish O. fasciatus (Ishibashi, 1994), and the Nile tilapia, O. niloticus (Ishibashi et al., 2002c). We found that these three kinds of fish respond to hypoxia by first attempting to maintain oxygen uptake by increasing respiration frequency and erythrocyte pH. Subsequently, fish adapted to hypoxia by conserving energy through metabolic depression without increasing aerobic and/or anaerobic metabolism, as well as by the stress response of increasing cortisol and glucose. Finally, the fish utilized anaerobic respiration by producing lactate and creatine. Based on this rough outline, there might not be any marked differences in the basal adaptation styles of these kinds of fish. However, adaptation abilities, such as metabolic depression in tilapia, were considerably higher than in the two other species. Red sea bream were not able to sufficiently depress metabolic activity under hypoxia (Ishibashi et al., 2002a,b). Therefore, it is commonly assumed that hypoxia tolerance in fish will decrease if the basal metabolic rate increases during metamorphosis. In fact, a relationship was established between hypoxia tolerance and oxygen consumption in red sea bream from larvae through juvenile (Fig. 5). Namely, it is thought that the energy of the fish metabolic scope for activity (Fry, 1947), in which the basal metabolic rate is subtracted from the activity metabolic rate, is
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decreased during metamorphosis and consequently that hypoxic tolerance fell during metamorphosis. 4.4. Metabolic scope for activity and environmental stress tolerances It was not revealed why the tolerances to some environmental stressors fell during metamorphosis in fish. We previously reported that the tolerance to high ammonia concentrations declined in metamorphic larvae of red sea bream. Acute ammonia exposure results in a decrease of ATP levels in brain, erythrocytes, gill, liver, and plasma of teleost fish (Arillo et al., 1981; Begum, 1987; Jeney et al., 1992). It has also been reported that hepatic and brain activity of glutamine synthetase involved in converting NH 4+ to the less toxic glutamine, increased in trout subjected to a solution with a high ammonia concentration (Arillo et al., 1981; Vedel et al., 1998; Wicks and Randall, 2002). The detoxification reaction may impair the energy balance in fish due to ATP consumption by glutamine synthesis. If red sea bream larvae exposed to ammonia had a similar biodynamic, the decreased energy in the metabolic scope for activity may have been one of the reasons why ammonia tolerance decreased during metamorphosis. It has also been shown that salinity tolerances gradually decreased in larvae and increased in juveniles of chum salmon, Japanese flounder, mangrove red snapper, and red sea bream (Hiroi et al., 1997; Shikano and Fujio, 1999; Chona et al., 2000; Ishibashi et al., 2003). The development of chloride cells, which need a lot of ATP for the active transport of minerals, is very important in order for the larvae to tolerate salinity. The need for a lot of ATP for osmoregulation might be related in part to the fall in salinity tolerance during metamorphosis, because the scope for activity declines in this period. Moreover, oxygen consumption has been shown to increase in fish subjected to salinity changes (Sparks et al., 2003) and physical disturbances (Davis and Schreck, 1997). 4.5. Recovery of stress tolerance and development of cortisol stress response In red sea bream, it was found that tolerance to environmental stressors, including hypoxia, was
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developed during the juvenile stage. The results also suggested that the decrease in mass-specific oxygen consumption during juvenile was one of the reasons for the recovery of tolerance. On the other hand, the ontogeny of the cortisol stress response in larval rainbow trout Salmo gairdneri was clear beginning at 2 weeks post-hatching (Barry et al., 1995). The cortisol level in the whole body clearly responded during the metamorphosis stage in yellowtail Seriola quinqueradiata (Sakakura and Tsukamoto, 1999) and Japanese flounder P. olivaceus (Ishibashi et al., 2004a,b). In red sea bream, the cortisol stress response to hypoxia was also found to be significant beginning at late metamorphosis (Ishibashi, unpublished data). Furthermore, we demonstrated recently that survival rate, cortisol concentration, and glucocorticoid receptor activities in response to some stressors increased after larval Japanese flounder were immersed in cortisol for one hour at the stage of metamorphosis (Ishibashi et al., 2004a). This finding suggested that the development of a cortisol stress response affected the recovery of stress tolerances.
Acknowledgments The authors are grateful and indebted to Mr. Kiyokazu Hamaguchi and Mr. Kazuto Usa, both of the Department of Fisheries, School of Agriculture, Kinki University, for their help with the experiments. This study was supported in part by a Grantin-Aid for Scientific Research (15580174) and by the 21st Century COE program from the Ministry of Education, Science, and Culture of Japan. This research was also supported in part by a Grant-inAid for the encouragement of research (GS10) by Kinki University.
References Arillo, A., Margiocco, C., Melodia, F., Mesi, P., Schenone, G., 1981. Biochemical aspects of water quality criteria: the case of ammonia pollution. Environ. Technol. Lett. 2, 258 – 292. Ayson, F.G., Kaneko, T., Hasegawa, S., Hirano, T., 1994. Development of mitochondrion-rich cells in the yolk-sac membrane of embryos and larvae of tilapia, Oreochromis mossambicus, in fresh water and seawater. J. Exp. Zool. 270, 129 – 135.
Barry, T.P., Malison, J.A., Held, J.A., Parrish, J.J., 1995. Ontogeny of the cortisol stress response in larval rainbow trout. Gen. Comp. Endocrinol. 97, 57 – 65. Begum, S.J., 1987. Biochemical adaptive responses in glucose metabolism of fish (Tilapia mossambica) during ammonia toxicity. Curr. Sci. 56, 705 – 708. Bone, Q., Marshall, N.B., Blaxter, J.H.S, 1995. Biology of Fishes, 2nd edition. Chapman & Hall, London, p. 332. Chen, W.H., Sun, L.T., Tsai, C.L., Song, Y.L., Chang, C.F., 2002. Cold-stress induced the modulation of catecholamines, cortisol, immunoglobulin M, and leukocyte phagocytosis in Tilapia. Gen. Comp. Endocrinol. 126, 90 – 100. Chona, B.E., Marietta, N.D., Evelyn, T.M., Arnil, C.E., 2000. Salinity tolerance of larvae of the mangrove red snapper (Lutjanus argentimaculatus) during ontogeny. Aquaculture 190, 155 – 167. Davis, L.E., Schreck, C.B., 1997. The energetic response to handling stress in juvenile coho salmon. Trans. Am. Fish. Soc. 126, 248 – 258. Ehrlich, K.F., 1974. Chemical changes during growth and starvation of larval Pleuronectes platessa. Mar. Biol. 24, 39 – 48. Fry, F.E.J., 1947. Effects of the environment on animal activity. University of Tronto Biology Series 55, Publ. Ont. Fish. Res. Lab., vol. 68. University of Toronto Press, Toronto, pp. 1 – 62. Fukuda, M., Yano, Y., Nakano, H., Sugiyama, M., 1986. Protein and nucleic acid changes during early developmental stages of cresthead flounder. Bull. Jpn. Soc. Sci. Fish. 68, 951 – 955. Gavaia, P.J., Dinis, M.T., Cancela, M.L., 2002. Osteological development and abnormalities of the vertebral column and caudal skeleton in larval and juvenile stages of hatcheryreared Senegal sole (Solea senegalensis). Aquaculture 211, 305 – 323. Gwak, W.-S., Tanaka, M., 2002. Changes in RNA, DNA and protein contents of laboratory-reared Japanese flounder Paralichthys olivaceus during metamorphosis and settlement. Fish. Sci. 68, 27 – 33. Haga, Y., Takeuchi, T., Murayama, Y., Ohta, K., Fukunaga, T., 2004. Vitamin D3 compounds induce hypermelanosis on the blind side and vertebral deformity in juvenile Japanese flounder Paralichthys olivaceus. Fish. Sci. 70, 59 – 67. Hiroi, J., Sakakura, Y., Tagawa, M., Seikai, T., Tanaka, M., 1997. Developmental changes in low-salinity tolerance and responses of prolactin, cortisol and thyroid hormones to low-salinity environment in Japanese flounder. Zool. Sci. 14, 987 – 992. Hwang, P.P., 1987. Tolerance and ultrastructural responses of branchial chloride cells to salinity changes in the euryhaline teleost Oreochromis mossambicus. Mar. Biol. 94, 643 – 649. Inui, Y., Miwa, S., 1985. Thyroid hormone induces metamorphosis of flounder larvae. Gen. Comp. Endocrinol. 60, 450 – 454. Ishibashi, Y., 1994. Studies on the ascorbic acid requirements of cultivated marine fish. Bull. Fish. Lab. Kinki Univ. 4, 1 – 100. Ishibashi, Y., Kato, K., Ikeda, S., Murata, O., Nasu, T., Kumai, H., 1992. Effect of dietary ascorbic acid on the tolerance for intermittent hypoxic stress in Japanese parrot fish. Nippon Suisan Gakkaishi 58, 2147 – 2152. Ishibashi, Y., Hirata, H., Kumai, H., 2002a. Stress response and energy status in various tissues of red sea bream, Pagrus
Y. Ishibashi et al. / Aquaculture 244 (2005) 331–340 major, subjected to hypoxic exposure. Suisanzoshok 50, 315 – 323. Ishibashi, Y., Yonezawa, H., Miyashita, S., Kato, K., Murata, O., Hirata, H., Kumai, H., 2002b. Response of enzyme activities and metabolite concentrations in various tissues of red sea bream, Pagrus major, subjected to hypoxic exposure. Suisanzoshok 50, 325 – 331. Ishibashi, Y., Ekawa, H., Hirata, H., Kumai, H., 2002c. Stress response and energy metabolism in various tissues of Nile tilapia Oreochromis niloticus exposed to hypoxic conditions. Fish. Sci. 68, 1374 – 1383. Ishibashi, Y., Ozawa, M., Hirata, H., Kumai, H., 2003. Ontogenic changes in various stress tolerances of larval and juvenile red sea bream Pagrus major. Nippon Suisan Gakkaishi 69, 36 – 43. Ishibashi, Y., Kotaki, T., Ryu, K., 2004a. Ontogenic changes of various stress tolerances and stress response in larval and juvenile Japanese flounder Paralichthys olivaceus: role of cortisol to environmental stressors. Abstracts of the Sixth International Symposium of Fish Immunology, 24 May–29 May 2004, Turku, Finland, Nordic Soc. Fish Immun. Symp., vol. 6, p. 50. Ishibashi, Y., Kotaki, T., Kataoka, M., Yamada, Y., 2004b. Ontogeny of hypoxic tolerance and energy metabolism of larvae and juvenile in Japanese flounder Paralichthys olivaceus. Biotechnologies for Quality Short communications and extended abstracts of contributions presented at the International Conference, 20 Oct–23 Oct 2004, Barcelona, Spain. EAS Special Publication, vol. 34, pp. 436 – 437. Itazawa, Y., Oikawa, S., 1983. Metabolic rates in excised tissues of carp. Experientia 39, 160 – 161. Izquierdo, M.S., Watanabe, T., Takeuchi, T., Arakawa, T., Kitajima, C., 1989. Requirement of larval red sea bream Pagrus major for essential fatty acids. Nippon Suisan Gakkaishi 55, 859 – 867. Jeney, G., Nemeso`k, J., Jeney, Z., Ola`h, J., 1992. Acute effect of sublethal ammonia concentration on common carp (Cyprinus carpio L.): II. Effect of ammonia on blood plasma transaminase (GOT, GPT) GIDH enzyme activity and ATP value. Aquaculture 104, 149 – 156. Jurss, K., Bastrop, R., 1995. The function of mitochondria-rich cells (chloride cells) in teleost gills. Rev. Fish Biol. Fish. 5, 235 – 255. Koumoundouros, G., Oran, G., Divanach, P., Stefanakis, S., Kentouri, M., 1997. The opercular complex deformity in intensive gilthead sea bream (Sparus aurata L.) larviculture. Moment of apparition and description. Aquaculture 156, 165 – 177. Matsuoka, M., 1987. Development of the skeletal tissue and skeletal muscle in the red sea bream. Bull. Seikai Reg. Fish. Res. Lab. 65, 1 – 114. Muroga, K., Furusawa, T., Furusawa, I., 1998. Viral nervous necrosis in striped jack, Pseudocaranx dentex. Suisanzoshok 46, 473 – 480. Oikawa, S., Itazawa, Y., Gotoh, M., 1991. Ontogenetic change in the relationship between metabolic rate and body mass in red sea bream. J. Fish Biol. 38, 483 – 496.
339
Okamoto, R., 1969. Rearing of red sea bream larva. Nippon Suisan Gakkaishi 35, 563 – 566. Pihl, L., Baden, S.P., Diaz, R.J., Schaffner, L.C., 1992. Hypoxiainduced structural changes in the diet of bottom-feeding fish and Crustacea. Mar. Biol. 112, 349 – 361. Richard, P., Bergeron, J.P., Boulhic, M., Galois, R., Person-Le Ruyet, J., 1991. Effect of starvation on RNA, DNA and protein content of laboratory-reared larvae and juveniles of Solea solea. Mar. Ecol., Prog. Ser. 72, 69 – 77. Sakakura, Y., Tsukamoto, K., 1999. Ontogeny of aggressive behavior in schools of yellowtail, Seriola quinqueradiata. Environ. Biol. Fishes 56, 231 – 242. Shikano, T., Fujio, Y., 1999. Decline in salinity tolerance during larval stage of chum salmon Oncorhynchus keta. Fish. Sci. 65, 661 – 662. Shimizu, H., Takeuchi, H., 2002. Bone abnormrity of hatcheryreared bluefin tuna Thunnus orientalis. Suisanzoshok 50, 71 – 78. Sparks, R.T., Shepherd, B.S., Ron, B., Richman, N.H., Riley, L.G., Iwama, G.K., Hirano, T., Grau, G., 2003. Effects of environmental salinity and 17a-methyltestosterone on growth and oxygen consumption in the tilapia, Oreochromis mossambicus. Comp. Biochem. Physiol. 136A, 657 – 665. Tago, A., Yamamoto, Y., Teshima, S., Kanazawa, A., 1999. Effects of 1,2-di-20:5-phosphatidylcholine (PC) and 1,2-di22:6-PC on growth and stress tolerance of Japanese flounder (Paralichthys olivaceus) larvae. Aquaculture 179, 231 – 239. Takeuchi, T., Toyota, M., Satoh, S., Watanabe, T., 1990. Requirement of juvenile red sea bream Pagrus major for eicosapentaenoic and docosahexaenoic acids. Nippon Suisan Gakkaishi 56, 1263 – 1269. Takii, K., Nakamura, M., Takaoka, O., Furuta, S., Kumai, H., 1992. Nucleic acid content and chemical composition of red sea bream, from larvae just after hatching to juveniles. Suisanzoshok 40, 285 – 290. Tanaka, M., 1973. Studies on the structure and function of digestive system of teleost larvae. Ph.D thesis, Kyoto University, Kyoto. Tanaka, M., 1985. Factors affecting the inshore migration of pelagic larval and demersal juvenile red sea bream Pagrus major to a nursery ground. Trans. Am. Fish. Soc. 114, 471 – 477. Tanangonan, J.B., Nakano, H., Tanaka, M., 1998. Changes in DNA, RNA, and protein content during early growth and development of Japanese flounder Paralichthys olivaceus. Suisanzoshok 46, 243 – 252. Tatner, M.F., 1996. Natural changes in the immune system of fish. In: Iwama, G.K., Nakanishi, T. (Eds.), The Fish Immune System, Organism, Pathogen, and Environment. Academic Press, New York, pp. 255 – 287. Vedel, N.E., Korsgaard, B., Jensen, F.B., 1998. Isolated and combined exposure to ammonia and nitrite in rainbow trout (Oncorhynchus mykiss): effects on electrolyte status, blood respiratory properties and brain glutamine/glutamate concentrations. Aquat. Toxicol. 41, 325 – 342.
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Y. Ishibashi et al. / Aquaculture 244 (2005) 331–340
Watanabe, K., Suzuki, S., Nishizawa, T., Suzuki, K., Yoshimizu, M., Ezura, Y., 1998. Control strategy for viral nervous necrosis of barfin flounder. Fish Pathol. 33, 445 – 446. Wicks, B.J., Randall, D.J., 2002. The effect of sub-lethal ammonia exposure on fed and unfed rainbow trout: the role of glutamine in regulation of ammonia. Comp. Biochem. Physiol. 132A, 275 – 285.
Wu, R.S.S., 2002. Hypoxia: from molecular responses to ecosystem responses. Mar. Pollut. Bull. 45, 35 – 45. Yada, T., Nakanishi, T., 2002. Interaction between endocrine and immune systems in fish. Int. Rev. Cytol. 220, 35 – 92.
Ontogeny of tolerance to hypoxia and oxygen consumption of larval and juvenile red sea bream, Pagrus major Yasunori Ishibashia,*, Kosuke Inouea, Hiromu Nakatsukasab, Yutaka Ishitanib, Shigeru Miyashitab, Osamu Muratab a
Department of Fisheries, School of Agriculture, Kinki University, Nakamachi, Nara 631-8505, Japan b Fisheries laboratory, Kinki University, Kogaura, Shirahama, Wakayama 649-2211, Japan
Received 14 October 2004; received in revised form 17 November 2004; accepted 18 November 2004
Abstract Changes in tolerance to hypoxic stress and oxygen consumption were studied in the red sea bream, Pagrus major, from its early life stage until 42 days post-hatch. In the experiments, metamorphosis was observed mainly from days 15 to 30, and the morphological shift from larva to juvenile was completed at around 9.5 mm total length (TL). During the larval stage, lethally low dissolved oxygen (DO) levels and mass-specific metabolic rates increased with growth from 2.6 to 5 mm TL ( Pb0.01). Subsequently, the levels remained high and decreased until about 9.5 mm TL around the flexion stage and post flexion stage. Finally, beginning in the juvenile stage, lethal DO levels and mass-specific metabolic rates decreased as TL increased up to about 30 mm ( Pb0.01). The relationship between lethal DO levels and mass-specific metabolic rates was significantly linear (r=0.59, pb0.001, n=207) in fish larvae and juveniles. These results indicated that, around the stage of flexion and post flexion larvae in red sea bream, metabolic rates were highest during metamorphosis, and consequently hypoxia tolerance was lowest. It was presumed that the increasing metabolic rate during metamorphosis induced a decrease in the metabolic scope of activity and thereby induced the decrease of the tolerance to some environmental stressors in the background. D 2004 Elsevier B.V. All rights reserved. Keywords: Stress; Hypoxia; Oxygen consumption; Larvae; Development; Red sea bream
1. Introduction In teleosts, the larval stage is a period of dramatic morphological, biochemical, and physiological * Corresponding author. Tel.: +81 742 43 6305; fax: +81 742 43 1316. E-mail address: [email protected] (Y. Ishibashi). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.11.019
changes. Metamorphosis is the focus of many such changes, and some of these metamorphic changes are controlled by hormones such as thyroid hormones, cortisol, and prolactin (Inui and Miwa, 1985; Hiroi et al., 1997). Some chemical components, such as nucleic acid, proximate composition, and enzyme activity of whole body, are also altered significantly during morphological changes in the larval through
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juvenile stages (Ehrlich, 1974; Fukuda et al., 1986; Richard et al., 1991; Tanangonan et al., 1998; Gwak and Tanaka, 2002). Adaptability to various environmental changes is also altered during larval development (Ishibashi et al., 2003, 2004a). In particular, the tolerance to hypo- and hyper-salinity in some kinds of fish changes around the period of metamorphosis (Hiroi et al., 1997; Shikano and Fujio, 1999; Chona et al., 2000; Ishibashi et al., 2003). The responses of larvae to osmotic gradients have been well documented (Chona et al., 2000). Chloride cells were found in early development (Ayson et al., 1994; Bone et al., 1995). As the larvae grow into juveniles, the gills complete their development, mainly by an osmoregulation site related to salinity tolerance; this site is thought to move from the skin to the gills (Hwang, 1987; Jurss and Bastrop, 1995). Although some of the many changes that occur during larval development are extremely complicated and controversial, other kinds of adaptability to environmental changes have not been fully examined. Understanding the environmental physiology of fish is also of practical importance to industry, because cultivated fish are frequently exposed to stressors induced by fluctuations in such environmental factors as dissolved oxygen level, salinity, and water temperature (Ishibashi et al., 1992; Pihl et al., 1992; Ishibashi, 1994; Wu, 2002). In addition, disease can be a consequence of these stress factors (Chen et al., 2002; Yada and Nakanishi, 2002). For the last decade, many kinds of marine juvenile fish have been intensively cultured from eggs in fish nurseries. However, many sorts of bacterial and viral diseases have been observed in larvae and juveniles (Muroga et al., 1998), and some deformities that are thought to originate from feeding methods occur during nursery production (Matsuoka, 1987; Koumoundouros et al., 1997; Gavaia et al., 2002; Shimizu and Takeuchi, 2002; Haga et al., 2004). To protect the health of nursery-produced fish, a lot of articles have appeared about many kinds of diseases and a few protective methods in the rearing of fish larvae and juveniles (Tatner, 1996; Muroga et al., 1998; Watanabe et al., 1998). The effects of enriched rotifer and Artemia salina lipids on activities of some marine fish from larvae through juvenile stages have also been examined (Izquierdo et al., 1989; Takeuchi et al., 1990; Tago et al., 1999). However, in the seed
production of some marine cultivated fish, survival rates have not been stable enough, and further studies about larval health have been needed. Therefore, knowledge of stress tolerance and of the adaptation function in fish larvae through juvenile stages is extremely important for healthy nursery production. We previously examined the ontogenic changes in various stress tolerances of larval and juvenile red sea bream, Pagrus major, and found that all tolerances to temperature, salinity, and ammonia stresses temporarily declined at metamorphosis (Ishibashi et al., 2003). It is considered that the depression of the scope for activity based on the increased metabolic rate during metamorphosis induced the decreases in various stress tolerances in the background. Previously, we also studied the effects of hypoxia on stress response and energy metabolism in young red sea bream (Ishibashi et al., 2002a,b) as well as in the Japanese parrot fish, Oplegnathus fasciatus (Ishibashi, 1994) and the Nile tilapia, Oreochromis niloticus (Ishibashi et al., 2002c). We found that in these fishes, exposure to hypoxia was associated with cortisol release, a reduction in oxygen consumption and liver ATP levels for metabolic depression, and increased anaerobic metabolism (Ishibashi et al., 2002a,b,c). However, there has appeared no study of ontogenetic tolerance to hypoxic stress in fish from the larval through juvenile stages. In the present study, in order to obtain basic knowledge as to why some stress tolerances fall during metamorphosis, we examined ontogenic changes in tolerance to hypoxic stress and oxygen consumption among larval and juvenile red sea bream.
2. Materials and methods 2.1. Fish and feeding methods Fertilized red sea bream eggs were obtained, reared, and studied at three facilities of Kinki University, Japan. First, eggs from the university’s Fish Nursery Center were placed in several 500-l polycarbonate tanks (lot 1) at the Fisheries Laboratory, as well as in a 20,000-l concrete tank (lot 2) at the Center and in several 300-l polycarbonate tanks (lot 3) in the Fisheries Department’s laboratory. In each case, gentle aeration was provided for 2 days until the eggs hatched. The day of hatching was regarded as day 0. In the pre-
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larval stage for 2 days post-hatching, fish were held in static condition with slight aeration. In the subsequent rearing, larvae and juveniles in lots 1 and 2 were held in a flowing water system of filtered seawater with some aeration. Fish in lot 3 were reared in a recirculating water system with sterilized artificial seawater and exact temperature control. The rearing water temperature in lots 1 was at 25.7F3.3 8C; 22.8–24.1 8C at days 0–21, and 24.0–29.4 8C at days 22–42. The rearing water temperature in lots 2 and 3 was controlled at 19.8F2.1 8C and 20.1F0.2 8C. From days 3 to 42, the larvae and juveniles were fed rotifer Brachionus rotundiformis and A. salina both enriched with n-3 HUFA, and a commercially available formulated diet, in that order (Fig. 2). 2.2. Experiment 1—ontogenic changes of tolerance to hypoxic stress About 100 larvae or juveniles were selected at random in the early morning before feeding at each 7day interval during the rearing period from hatching to 42 days post-hatching. Twenty fish in lot 1 and three groups, each consisting of 10–30 fish, in lot 2 were exposed to hypoxic stress for 3–4 h. DO concentrations in seawater were adjusted using nitrogen gas. DO levels in seawater were reduced at a fixed rate by regulating the flow of nitrogen gas from about 7.0 to 0.5 mg/l for approximately 4 h. Changes in DO concentrations during hypoxic stress tests were monitored with a DO meter (YSI-58, YSI, Ohio, USA) and are shown in Fig. 1. The lethal DO level of fish was recorded just after respiratory and behavioral arrest, respectively. Median lethal DO levels, calculated by means of logit transformation of larvae and juveniles in each group, were taken as the parameter of tolerance to hypoxic stress. After the stress test, the total length, body weight, and developmental stages of each fish were measured, and these data were used to plot growth curves. 2.3. Experiment 2—relationship between lethal DO levels and oxygen consumption in larvae through juveniles 2.3.1. Measurement of oxygen consumption The water bottle method was improved and used to measure oxygen consumption of the red sea
Fig. 1. Changes in dissolved oxygen level during hypoxic tolerance tests.
bream, from larvae just after hatching to the juvenile stage on day 42 in lots 2 and 3. In larvae from posthatching to 14 days old, 20 to 200 normal-looking, average-sized larvae in the rearing tank were chosen and transferred, every morning before feeding, to a small tank containing filtered seawater. Some groups consisting of 2 to 10 larvae were selected from the small tank at random and carefully placed into 1- to 2-ml syringes according to the fish growth. The remaining fish in the small tank were utilized to measure the dry weight of fish. This was done because a pre-flexion larva was too small for us to determine its oxygen consumption and body weight. In the larvae older than 14 days and in juveniles, individuals were accommodated in 2- to 100-ml syringes individually, again according to size. All filled syringes were left in a water bath at 20.0F0.1 8C in a dark room. After about 1 h, while the fish appeared to be resting, the syringe was slowly revolved and the DO concentration of 100 Al seawater in the syringe was measured by a blood gas analyzer (ABL-330, Radiometer, Copenhagen, Denmark) in a dark room with slight light. The syringe and a paraffin paper seal, which were weighed in advance, were sealed at the lip and weighed to calculate the water volume. After the syringes were left in a water bath at 20.0F0.1 8C
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for 1–2 h, each syringe was slowly revolved and its DO concentration, together with exact elapsed time, was measured again. The oxygen consumption in seawater without fish was also measured in a similar way as a blank value in order to calculate the oxygen consumption of fish. The total length, body weight, and developmental stages of all fish in lot 2 were measured after the standard oxygen consumption was determined. The dry weight per fish was measured by formula method after the fish were killed by overdose of anesthetic. In larvae from post-hatching to 14 days old, 50 to 150 larvae were filtered at a time and then divided into two groups. Fish in one of the groups were weighed on the electronic scale and counted. A second group was used to determine dry weight by drying fish at 90 8C for several days. 2.3.2. Measurement of lethal DO concentrations in syringes After the measurement of oxygen consumption in lot 3, the water volume in each syringe was calculated and approximately set up to sacrifice the fish in 2–3 h in calibration with the fish’s oxygen consumption rate. The syringes were left in a mostly dark room and were occasionally revolved slowly in a water bath until the fish were dead. To determine the lethal DO level, the DO concentration in the syringe was measured a final time just after half the fish had died. Just after each lethal level was measured, the dead fish’s total length, body weight, and developmental stages were measured.
3. Results 3.1. Growth and morphological changes Fig. 2 shows the changes in the total lengths of the red sea bream, from larvae just after hatching to juveniles on day 42. The total length slowly increased before 21 days post-hatching, and then somatic growth rates were faster from the beginning of the juvenile stage about 9.5 mm TL. In particular, the growth rate of juveniles in lot 1, which were reared at a high temperature, was the fastest of all. Flexion larvae appeared at around 6.4 mm TL on days 15 to 20 in all lots. Subsequently, the morphological shift from larva to juvenile, which is based on a constant fin ray, was also observed microscopically at around 9.5 mm TL at days 24 to 30, depending on water temperature. 3.2. Experiment 1—ontogenic changes in tolerance to hypoxic stress Changes in the lethal DO levels of fish are shown in Fig. 3. In larvae, the median lethal level of low DO, which was 0.6 to 0.8 mg/l at just after hatching,
2.4. Statistics The data on lethal DO levels in lot 2 are expressed as meanFS.D., n=3. Data were analyzed by a one-way analysis of variance (ANOVA), followed by Duncan’s new multiple range test. The allometric relationships between the total length and oxygen consumption for larvae and juveniles were described by equations of linear having reflective points, depending on computed values of R 2 and significance of regression parameters. Differences between linear regressions were tested by analysis of covariance (ANCOVA). Moreover, Pearson’s correlation coefficients between mass-specific oxygen consumption and lethal dissolved oxygen were analyzed.
Fig. 2. Growth curve, feeding schedule, and developmental stages of larval and juvenile red sea bream up to day 42. (o) Lot 1 (water temperature in feeding, 25.7F3.3 8C); (4) lot 2 (19.8F2.1 8C); (5) lot 3 (20.1F0.2 8C).
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Fig. 3. Ontogenic changes in 50% lethal levels of low dissolved oxygen over a period of 3 h in larval and juvenile red sea bream. (o) Lot 1; ( ) lot 2. Each value in lot 2 represents the meanFS.D., n=3. Values with different letters in the same curves are significantly different (pb0.01).
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increased with growth until the larvae reached about 2.0 mg/l at 5 mm TL (Pb0.01). In the larval stage of about 5 mm to 9.5 mm TL, the median lethal DO levels remained high, from 2.0 to 2.3 mg/l, depending on water temperature. On the other hand, in the juvenile stage since TL reached about 9.5 mm, the lethal DO level decreased as TL increased, up to 1.2 mg/l at 32 mm TL ( Pb0.01).
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data gave three straight lines, having reflective points at about 5 mm and 9.5 mm TL. Regression lines were log M=2.762+3.968 log TL; F 1, 2 129=440.914, R =0.841, Pb0.001 in the first phase, log M=2.385+3.466 log TL; F 1, 93=322.256, R 2=0.776, Pb0.001 in the second phase, log M =2.070+3.060 log TL; F 1, 98 =5822.703, R 2=0.983, Pb0.001 in the third phase, respectively. The slopes of regression lines describing the relationship between M and TL differed, respectively ( F 2, 320=4.378, Pb0.05). Fig. 4 shows also the allometric relationships between mass-specific oxygen consumption and total length in red sea bream from the larval through juvenile stages. The allometry between mass-specific oxygen consumption and total length was triphasic. In the first phase, from 2.6 mm to 5 mm, massspecific oxygen consumption (MW) rapidly increased until reaching about 7 ml/g-dry/h (log MW=3.0379+1.2763 log TL; F 1, 130 =90.796, R 2=0.426 , Pb0.001). Subsequently, it remained high and decreased in the second phase from about 5 mm to 9.5 mm TL (log MW=4.3837–0.7061 log TL; F 1, 149=88.365, R 2=0.381, Pb0.001). Finally, in the third phase, the mass-specific metabolic rate decreased as TL increased, up to about 30 mm (log
3.3. Experiment 2—relationship between lethal DO level and oxygen consumption in larvae through juveniles The allometric equation (M=aW b) between oxygen consumption (M) and body mass (W) were calculated for ontogenic change. However, the relation between oxygen consumption and body mass was indistinct in the first phase of present experiment, because body mass barely increased until about 8 days post-hatching. Allometric relationships of oxygen consumption to total length in red sea bream from larval through juvenile stages are shown in Fig. 4. The oxygen consumption (M) increased as the fish grew, and the total length (TL)
Fig. 4. Allometric relationship of oxygen consumption and massspecific oxygen consumption to total length in larval and juvenile red sea bream. The arrows indicate inflection points. Values by the lines show the slope of the regression lines. ( ) Lot 2; (o) lot 3.
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Fig. 5. Relationship between mass-specific oxygen consumption and lethal levels of low dissolved oxygen in larval and juvenile red sea bream (lot 3).
MW=4.0314–0.3240 log TL; F 1, 55 =66.303, R 2=0.534, Pb0.001). The allometrically calculated straight-line representations of MW and TL also differed in slopes ( F 2, 334=78.259; Pb0.001). The relationship between lethal levels of low DO and mass-specific oxygen consumption in red sea bream from the larval through juvenile stages are shown in Fig. 5. Lethal DO levels increased as mass-specific oxygen consumption increased. The regression line between lethal DO levels and massspecific oxygen consumption was calculated to be y=0.1988x+0.87. Pearson’s correlation coefficient (r) was high (0.59, pb0.001, n=207), indicating a positive linear relationship.
4. Discussion 4.1. Stress tolerance during metamorphosis and critical phase in nursery production From the results obtained in this experiment, it can be concluded that tolerance to hypoxia in red sea bream decreased from 5 mm until about 9.5 mm TL during metamorphosis (Fig. 3). In a previous study of red sea bream, we demonstrated that all tolerances to high and low temperature, hypo- and hyper-salinity, and high ammonia stressors temporarily fell during
metamorphosis at days 14 to 21 (Ishibashi et al., 2003). These results are in accordance with the critical phase, from days 13 to 20, in which red sea bream were formerly prone to die in nursery production fields (Okamoto, 1969). In other kinds of fish, hyposalinity tolerance in Japanese flounder Paralichthys olivaceus and mangrove red snapper Lutjanus argentimaculatus fell by the time of metamorphosis (Hiroi et al., 1997; Chona et al., 2002). The hyper-salinity tolerance in chum salmon Oncorhynchus keta and mangrove red snapper also declined during the larval stage (Shikano and Fujio, 1999; Chona et al., 2000). However, it has not been clear why some stress tolerances fall during metamorphosis. Although the molecular, biochemical, and morphological changes that occur during larval development are complex, the present and previous results together raise the possibility that some common factor could cause the decreases in tolerance during metamorphosis. 4.2. Metabolic activity during metamorphosis In the present experiment, mass-specific oxygen consumption was increased as larvae grew and metabolism remained high from 5 to 9.5 mm TL during metamorphosis. This experimental result is approximately in accord with the data on larval red sea bream by Oikawa et al., 1991. In red sea bream, it is known that the skeletal system is altered markedly from 6.5 to 7 mm TL and that the allometry of number and sectional area of muscle fiber have reflective points at about 6 to 10 mm TL (Matsuoka, 1987). Almost all organs were also completed development in this phase. In addition, the feeding habit of the red sea bream changed from nauplius to Copepoda or Appendicularia at around 6 mm TL (Tanaka, 1973, 1985). Because of morphogenesis, physiological and biochemical changes are dramatic in fish of this period. Itazawa and Oikawa reported that in almost all fish, the metabolic rate decreased beginning at the juvenile stage, because the weight percent of the brain and viscera, which consume more oxygen than the other parts of the body, decreased in proportion to whole body weight (Itazawa and Oikawa, 1983). It is conceivable that the metabolic rate in the whole body remains at a high level, from the stage just before flexion larvae to late metamorphic larvae, because morphogenesis
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in many organs, such as the brain and viscera, requires more oxygen. Moreover, DNA, RNA, and protein content per whole body weight are elevated from early larvae up to late metamorphic larvae in red sea bream (Takii et al., 1992). Gwak and Tanaka (2002) also indicate that cyclic phase of hyperplasia and hypertrophy occurs during metamorphosis and reduced protein synthesis during the post metamorphic phase as a result of DNA, RNA, and protein measurements in whole body Japanese flounder. The elevation of these chemical components during metamorphosis also suggests an increase in the metabolic activity of the whole body for intensive growth through cell enlargement and cell proliferation. 4.3. Relationship between stress tolerance and oxygen consumption We have previously shown the effects of hypoxia on stress response and energy metabolism in various tissues of red sea bream (Ishibashi et al., 2002a,b), the Japanese parrot fish O. fasciatus (Ishibashi, 1994), and the Nile tilapia, O. niloticus (Ishibashi et al., 2002c). We found that these three kinds of fish respond to hypoxia by first attempting to maintain oxygen uptake by increasing respiration frequency and erythrocyte pH. Subsequently, fish adapted to hypoxia by conserving energy through metabolic depression without increasing aerobic and/or anaerobic metabolism, as well as by the stress response of increasing cortisol and glucose. Finally, the fish utilized anaerobic respiration by producing lactate and creatine. Based on this rough outline, there might not be any marked differences in the basal adaptation styles of these kinds of fish. However, adaptation abilities, such as metabolic depression in tilapia, were considerably higher than in the two other species. Red sea bream were not able to sufficiently depress metabolic activity under hypoxia (Ishibashi et al., 2002a,b). Therefore, it is commonly assumed that hypoxia tolerance in fish will decrease if the basal metabolic rate increases during metamorphosis. In fact, a relationship was established between hypoxia tolerance and oxygen consumption in red sea bream from larvae through juvenile (Fig. 5). Namely, it is thought that the energy of the fish metabolic scope for activity (Fry, 1947), in which the basal metabolic rate is subtracted from the activity metabolic rate, is
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decreased during metamorphosis and consequently that hypoxic tolerance fell during metamorphosis. 4.4. Metabolic scope for activity and environmental stress tolerances It was not revealed why the tolerances to some environmental stressors fell during metamorphosis in fish. We previously reported that the tolerance to high ammonia concentrations declined in metamorphic larvae of red sea bream. Acute ammonia exposure results in a decrease of ATP levels in brain, erythrocytes, gill, liver, and plasma of teleost fish (Arillo et al., 1981; Begum, 1987; Jeney et al., 1992). It has also been reported that hepatic and brain activity of glutamine synthetase involved in converting NH 4+ to the less toxic glutamine, increased in trout subjected to a solution with a high ammonia concentration (Arillo et al., 1981; Vedel et al., 1998; Wicks and Randall, 2002). The detoxification reaction may impair the energy balance in fish due to ATP consumption by glutamine synthesis. If red sea bream larvae exposed to ammonia had a similar biodynamic, the decreased energy in the metabolic scope for activity may have been one of the reasons why ammonia tolerance decreased during metamorphosis. It has also been shown that salinity tolerances gradually decreased in larvae and increased in juveniles of chum salmon, Japanese flounder, mangrove red snapper, and red sea bream (Hiroi et al., 1997; Shikano and Fujio, 1999; Chona et al., 2000; Ishibashi et al., 2003). The development of chloride cells, which need a lot of ATP for the active transport of minerals, is very important in order for the larvae to tolerate salinity. The need for a lot of ATP for osmoregulation might be related in part to the fall in salinity tolerance during metamorphosis, because the scope for activity declines in this period. Moreover, oxygen consumption has been shown to increase in fish subjected to salinity changes (Sparks et al., 2003) and physical disturbances (Davis and Schreck, 1997). 4.5. Recovery of stress tolerance and development of cortisol stress response In red sea bream, it was found that tolerance to environmental stressors, including hypoxia, was
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developed during the juvenile stage. The results also suggested that the decrease in mass-specific oxygen consumption during juvenile was one of the reasons for the recovery of tolerance. On the other hand, the ontogeny of the cortisol stress response in larval rainbow trout Salmo gairdneri was clear beginning at 2 weeks post-hatching (Barry et al., 1995). The cortisol level in the whole body clearly responded during the metamorphosis stage in yellowtail Seriola quinqueradiata (Sakakura and Tsukamoto, 1999) and Japanese flounder P. olivaceus (Ishibashi et al., 2004a,b). In red sea bream, the cortisol stress response to hypoxia was also found to be significant beginning at late metamorphosis (Ishibashi, unpublished data). Furthermore, we demonstrated recently that survival rate, cortisol concentration, and glucocorticoid receptor activities in response to some stressors increased after larval Japanese flounder were immersed in cortisol for one hour at the stage of metamorphosis (Ishibashi et al., 2004a). This finding suggested that the development of a cortisol stress response affected the recovery of stress tolerances.
Acknowledgments The authors are grateful and indebted to Mr. Kiyokazu Hamaguchi and Mr. Kazuto Usa, both of the Department of Fisheries, School of Agriculture, Kinki University, for their help with the experiments. This study was supported in part by a Grantin-Aid for Scientific Research (15580174) and by the 21st Century COE program from the Ministry of Education, Science, and Culture of Japan. This research was also supported in part by a Grant-inAid for the encouragement of research (GS10) by Kinki University.
References Arillo, A., Margiocco, C., Melodia, F., Mesi, P., Schenone, G., 1981. Biochemical aspects of water quality criteria: the case of ammonia pollution. Environ. Technol. Lett. 2, 258 – 292. Ayson, F.G., Kaneko, T., Hasegawa, S., Hirano, T., 1994. Development of mitochondrion-rich cells in the yolk-sac membrane of embryos and larvae of tilapia, Oreochromis mossambicus, in fresh water and seawater. J. Exp. Zool. 270, 129 – 135.
Barry, T.P., Malison, J.A., Held, J.A., Parrish, J.J., 1995. Ontogeny of the cortisol stress response in larval rainbow trout. Gen. Comp. Endocrinol. 97, 57 – 65. Begum, S.J., 1987. Biochemical adaptive responses in glucose metabolism of fish (Tilapia mossambica) during ammonia toxicity. Curr. Sci. 56, 705 – 708. Bone, Q., Marshall, N.B., Blaxter, J.H.S, 1995. Biology of Fishes, 2nd edition. Chapman & Hall, London, p. 332. Chen, W.H., Sun, L.T., Tsai, C.L., Song, Y.L., Chang, C.F., 2002. Cold-stress induced the modulation of catecholamines, cortisol, immunoglobulin M, and leukocyte phagocytosis in Tilapia. Gen. Comp. Endocrinol. 126, 90 – 100. Chona, B.E., Marietta, N.D., Evelyn, T.M., Arnil, C.E., 2000. Salinity tolerance of larvae of the mangrove red snapper (Lutjanus argentimaculatus) during ontogeny. Aquaculture 190, 155 – 167. Davis, L.E., Schreck, C.B., 1997. The energetic response to handling stress in juvenile coho salmon. Trans. Am. Fish. Soc. 126, 248 – 258. Ehrlich, K.F., 1974. Chemical changes during growth and starvation of larval Pleuronectes platessa. Mar. Biol. 24, 39 – 48. Fry, F.E.J., 1947. Effects of the environment on animal activity. University of Tronto Biology Series 55, Publ. Ont. Fish. Res. Lab., vol. 68. University of Toronto Press, Toronto, pp. 1 – 62. Fukuda, M., Yano, Y., Nakano, H., Sugiyama, M., 1986. Protein and nucleic acid changes during early developmental stages of cresthead flounder. Bull. Jpn. Soc. Sci. Fish. 68, 951 – 955. Gavaia, P.J., Dinis, M.T., Cancela, M.L., 2002. Osteological development and abnormalities of the vertebral column and caudal skeleton in larval and juvenile stages of hatcheryreared Senegal sole (Solea senegalensis). Aquaculture 211, 305 – 323. Gwak, W.-S., Tanaka, M., 2002. Changes in RNA, DNA and protein contents of laboratory-reared Japanese flounder Paralichthys olivaceus during metamorphosis and settlement. Fish. Sci. 68, 27 – 33. Haga, Y., Takeuchi, T., Murayama, Y., Ohta, K., Fukunaga, T., 2004. Vitamin D3 compounds induce hypermelanosis on the blind side and vertebral deformity in juvenile Japanese flounder Paralichthys olivaceus. Fish. Sci. 70, 59 – 67. Hiroi, J., Sakakura, Y., Tagawa, M., Seikai, T., Tanaka, M., 1997. Developmental changes in low-salinity tolerance and responses of prolactin, cortisol and thyroid hormones to low-salinity environment in Japanese flounder. Zool. Sci. 14, 987 – 992. Hwang, P.P., 1987. Tolerance and ultrastructural responses of branchial chloride cells to salinity changes in the euryhaline teleost Oreochromis mossambicus. Mar. Biol. 94, 643 – 649. Inui, Y., Miwa, S., 1985. Thyroid hormone induces metamorphosis of flounder larvae. Gen. Comp. Endocrinol. 60, 450 – 454. Ishibashi, Y., 1994. Studies on the ascorbic acid requirements of cultivated marine fish. Bull. Fish. Lab. Kinki Univ. 4, 1 – 100. Ishibashi, Y., Kato, K., Ikeda, S., Murata, O., Nasu, T., Kumai, H., 1992. Effect of dietary ascorbic acid on the tolerance for intermittent hypoxic stress in Japanese parrot fish. Nippon Suisan Gakkaishi 58, 2147 – 2152. Ishibashi, Y., Hirata, H., Kumai, H., 2002a. Stress response and energy status in various tissues of red sea bream, Pagrus
Y. Ishibashi et al. / Aquaculture 244 (2005) 331–340 major, subjected to hypoxic exposure. Suisanzoshok 50, 315 – 323. Ishibashi, Y., Yonezawa, H., Miyashita, S., Kato, K., Murata, O., Hirata, H., Kumai, H., 2002b. Response of enzyme activities and metabolite concentrations in various tissues of red sea bream, Pagrus major, subjected to hypoxic exposure. Suisanzoshok 50, 325 – 331. Ishibashi, Y., Ekawa, H., Hirata, H., Kumai, H., 2002c. Stress response and energy metabolism in various tissues of Nile tilapia Oreochromis niloticus exposed to hypoxic conditions. Fish. Sci. 68, 1374 – 1383. Ishibashi, Y., Ozawa, M., Hirata, H., Kumai, H., 2003. Ontogenic changes in various stress tolerances of larval and juvenile red sea bream Pagrus major. Nippon Suisan Gakkaishi 69, 36 – 43. Ishibashi, Y., Kotaki, T., Ryu, K., 2004a. Ontogenic changes of various stress tolerances and stress response in larval and juvenile Japanese flounder Paralichthys olivaceus: role of cortisol to environmental stressors. Abstracts of the Sixth International Symposium of Fish Immunology, 24 May–29 May 2004, Turku, Finland, Nordic Soc. Fish Immun. Symp., vol. 6, p. 50. Ishibashi, Y., Kotaki, T., Kataoka, M., Yamada, Y., 2004b. Ontogeny of hypoxic tolerance and energy metabolism of larvae and juvenile in Japanese flounder Paralichthys olivaceus. Biotechnologies for Quality Short communications and extended abstracts of contributions presented at the International Conference, 20 Oct–23 Oct 2004, Barcelona, Spain. EAS Special Publication, vol. 34, pp. 436 – 437. Itazawa, Y., Oikawa, S., 1983. Metabolic rates in excised tissues of carp. Experientia 39, 160 – 161. Izquierdo, M.S., Watanabe, T., Takeuchi, T., Arakawa, T., Kitajima, C., 1989. Requirement of larval red sea bream Pagrus major for essential fatty acids. Nippon Suisan Gakkaishi 55, 859 – 867. Jeney, G., Nemeso`k, J., Jeney, Z., Ola`h, J., 1992. Acute effect of sublethal ammonia concentration on common carp (Cyprinus carpio L.): II. Effect of ammonia on blood plasma transaminase (GOT, GPT) GIDH enzyme activity and ATP value. Aquaculture 104, 149 – 156. Jurss, K., Bastrop, R., 1995. The function of mitochondria-rich cells (chloride cells) in teleost gills. Rev. Fish Biol. Fish. 5, 235 – 255. Koumoundouros, G., Oran, G., Divanach, P., Stefanakis, S., Kentouri, M., 1997. The opercular complex deformity in intensive gilthead sea bream (Sparus aurata L.) larviculture. Moment of apparition and description. Aquaculture 156, 165 – 177. Matsuoka, M., 1987. Development of the skeletal tissue and skeletal muscle in the red sea bream. Bull. Seikai Reg. Fish. Res. Lab. 65, 1 – 114. Muroga, K., Furusawa, T., Furusawa, I., 1998. Viral nervous necrosis in striped jack, Pseudocaranx dentex. Suisanzoshok 46, 473 – 480. Oikawa, S., Itazawa, Y., Gotoh, M., 1991. Ontogenetic change in the relationship between metabolic rate and body mass in red sea bream. J. Fish Biol. 38, 483 – 496.
339
Okamoto, R., 1969. Rearing of red sea bream larva. Nippon Suisan Gakkaishi 35, 563 – 566. Pihl, L., Baden, S.P., Diaz, R.J., Schaffner, L.C., 1992. Hypoxiainduced structural changes in the diet of bottom-feeding fish and Crustacea. Mar. Biol. 112, 349 – 361. Richard, P., Bergeron, J.P., Boulhic, M., Galois, R., Person-Le Ruyet, J., 1991. Effect of starvation on RNA, DNA and protein content of laboratory-reared larvae and juveniles of Solea solea. Mar. Ecol., Prog. Ser. 72, 69 – 77. Sakakura, Y., Tsukamoto, K., 1999. Ontogeny of aggressive behavior in schools of yellowtail, Seriola quinqueradiata. Environ. Biol. Fishes 56, 231 – 242. Shikano, T., Fujio, Y., 1999. Decline in salinity tolerance during larval stage of chum salmon Oncorhynchus keta. Fish. Sci. 65, 661 – 662. Shimizu, H., Takeuchi, H., 2002. Bone abnormrity of hatcheryreared bluefin tuna Thunnus orientalis. Suisanzoshok 50, 71 – 78. Sparks, R.T., Shepherd, B.S., Ron, B., Richman, N.H., Riley, L.G., Iwama, G.K., Hirano, T., Grau, G., 2003. Effects of environmental salinity and 17a-methyltestosterone on growth and oxygen consumption in the tilapia, Oreochromis mossambicus. Comp. Biochem. Physiol. 136A, 657 – 665. Tago, A., Yamamoto, Y., Teshima, S., Kanazawa, A., 1999. Effects of 1,2-di-20:5-phosphatidylcholine (PC) and 1,2-di22:6-PC on growth and stress tolerance of Japanese flounder (Paralichthys olivaceus) larvae. Aquaculture 179, 231 – 239. Takeuchi, T., Toyota, M., Satoh, S., Watanabe, T., 1990. Requirement of juvenile red sea bream Pagrus major for eicosapentaenoic and docosahexaenoic acids. Nippon Suisan Gakkaishi 56, 1263 – 1269. Takii, K., Nakamura, M., Takaoka, O., Furuta, S., Kumai, H., 1992. Nucleic acid content and chemical composition of red sea bream, from larvae just after hatching to juveniles. Suisanzoshok 40, 285 – 290. Tanaka, M., 1973. Studies on the structure and function of digestive system of teleost larvae. Ph.D thesis, Kyoto University, Kyoto. Tanaka, M., 1985. Factors affecting the inshore migration of pelagic larval and demersal juvenile red sea bream Pagrus major to a nursery ground. Trans. Am. Fish. Soc. 114, 471 – 477. Tanangonan, J.B., Nakano, H., Tanaka, M., 1998. Changes in DNA, RNA, and protein content during early growth and development of Japanese flounder Paralichthys olivaceus. Suisanzoshok 46, 243 – 252. Tatner, M.F., 1996. Natural changes in the immune system of fish. In: Iwama, G.K., Nakanishi, T. (Eds.), The Fish Immune System, Organism, Pathogen, and Environment. Academic Press, New York, pp. 255 – 287. Vedel, N.E., Korsgaard, B., Jensen, F.B., 1998. Isolated and combined exposure to ammonia and nitrite in rainbow trout (Oncorhynchus mykiss): effects on electrolyte status, blood respiratory properties and brain glutamine/glutamate concentrations. Aquat. Toxicol. 41, 325 – 342.
340
Y. Ishibashi et al. / Aquaculture 244 (2005) 331–340
Watanabe, K., Suzuki, S., Nishizawa, T., Suzuki, K., Yoshimizu, M., Ezura, Y., 1998. Control strategy for viral nervous necrosis of barfin flounder. Fish Pathol. 33, 445 – 446. Wicks, B.J., Randall, D.J., 2002. The effect of sub-lethal ammonia exposure on fed and unfed rainbow trout: the role of glutamine in regulation of ammonia. Comp. Biochem. Physiol. 132A, 275 – 285.
Wu, R.S.S., 2002. Hypoxia: from molecular responses to ecosystem responses. Mar. Pollut. Bull. 45, 35 – 45. Yada, T., Nakanishi, T., 2002. Interaction between endocrine and immune systems in fish. Int. Rev. Cytol. 220, 35 – 92.