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Prevention of surface death of marine fish larvae by the addition of egg white into rearing water
Aquaculture 224 (2003) 313 – 322 www.elsevier.com/locate/aqua-online
Prevention of surface death of marine fish larvae by the addition of egg white into rearing water Tatsuya Kaji a,*, Masaaki Kodama b, Hiroshi Arai b, Masaru Tanaka a, Masatomo Tagawa a a
Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto 606-8502, Japan b Tokyo Sea Life Park, 6-2-3, Rinkai, Edogawa, Tokyo 134-8587, Japan
Received 22 August 2002; received in revised form 20 February 2003; accepted 25 February 2003
Abstract In the seed production of several marine teleosts, large numbers of dead larvae are frequently observed on the water surface around the time of first feeding. We tried to prevent such ‘‘surface death’’ by the addition of chicken egg white (EW) into the rearing water. In the first experiment, larvae of striped bonito, Sarda orientalis, at the first feeding stage (2 and 3 days after hatching) were transferred to plastic beakers using a 5-ml pipette at a density of 10 larvae/l, in the absence or presence of EW (80 Al/l). No diet was supplied, the water was not renewed throughout the experiment, and dead larvae were removed daily. Numbers of surviving larvae of the EW-treated group were significantly higher than those of the control group. In particular, survival at the first observation (next day after transfer) was less than 30% in the control, while 100% in the EW-treated group. In a second experiment, mortality at 2 h after transfer was compared with special attention to the location of the dead larvae. Significantly ( P < 0.01) higher mortality was found in the control group, and more than 70% of the dead larvae were located on the water surface. Upward swimming toward the water surface was observed in both groups, but larvae ‘‘trapped’’ at the surface were only observed in the control group. In a third experiment, an oil film that was artificially created on the water surface prevented larval death after the transfer as well as the dissolved EW. Since the oil film is known to prevent ‘‘surface trap’’ of larvae and decrease ‘‘surface death’’ in a grouper, the addition of EW was considered to have a similar effect in reducing mortality. In a fourth experiment using 30-l tanks, survival of larval pez cochero, Dules auriga, was also higher in the presence of EW, especially during a period when dead larvae were frequently found on the water surface in the control. Thus, the addition of EW to the rearing water is suggested to prevent surface death in a
* Corresponding author. Tel.: +81-75-753-6222; fax: +81-75-753-6229. E-mail address: [email protected] (T. Kaji). 0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0044-8486(03)00243-6
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variety of marine fish larvae, and the possibility of applying the method in practical rearing is discussed. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Striped bonito; Sarda orientalis; Survival; Stress; Mortality; Pez cochero; Dules auriga
1. Introduction In the aquaculture of marine teleosts, one of the most serious problems associated with mass production of larval fish is the high mortality during the early larval period. Mortality during the yolk-sac to first feeding stages is caused by poor quality of eggs (Kjørsvik et al., 1990) and/or various abiotic conditions, since larvae do not require any external nutrition before and around the time of first feeding. ‘‘Surface death’’ is a heavy mortality factor in which most of the dead larvae are found on the water surface, typically reported for prelarvae (during yolk absorption) of red spotted grouper, Epinephelus akaara (Yamaoka et al., 2000). The authors also reported the complete prevention of death by artificially forming an oil film on the water surface, perhaps due to the reduction of surface tension (Yamaoka et al., 2000). Reduction of mortality after handling has been demonstrated by the addition of protein, such as egg white (EW), into rearing water in yolk-sac larvae of the Japanese flounder, Paralichthys olivaceus (Tagawa et al., submitted for publication). This phenomenon could be due to the protective effect of dissolved protein on the injured larvae, however further study is needed for application of the method to practical and mass rearing of marine fish larvae. The present study attempts to demonstrate the prevention of surface death by the addition of EW into rearing water in larval striped bonito, Sarda orientalis. The application of this method was also tested on a practical rearing scale using larvae of pez cochero, Dules auriga.
2. Materials and methods 2.1. Fish All experiments were conducted at the Tokyo Sea Life Park from November to December 2001. Striped bonito broodstock were maintained in an exhibition tank (2200 tons) at 23 jC and pez cochero broodstock were in an exhibition tank (5 tons) at 18 jC. Fertilized eggs from both species were collected in the morning after spontaneous spawning by water flow into a net installed to each tank. Floating eggs of the striped bonito were transferred to a 30-l rearing tank (stock tank), aerated by glass tube and incubated at 23 F 1 jC. Hatched larvae were kept in the tank without diet or renewal of the rearing water until used in experiments. The pez cochero eggs were used directly in the experiments described below.
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2.2. Procedure for dissolving the EW and forming oil film The EW was separated from the egg yolk, mixed 1 part EW to 49 parts of seawater (SW), and added to each experimental container for a final concentration of 80 Al EW/l rearing water. This concentration of EW (1/12,500) is about three times higher than the effective concentration (1/30,000) reported in Japanese flounder (Tagawa et al., submitted for publication). An equivalent volume of SW (without EW) was added to control containers. An oil film was formed using Super Selco (INVE Aquaculture NV, Belgium), a product originally used for nutritional enrichment of rotifer and brine shrimp (Artemia spp.). Two drops of Super Selco were added and mixed well in 2 l of SW in a plastic beaker. The surface layer of floating oil was collected by a pipette 10 min later and 5 ml of the surface layer were transferred into each experimental beaker with 2 l SW. 2.3. Time course of larval death in the presence or absence of EW Two sets of three plastic beakers (2-l in volume) were filled with 1 l of seawater with or without EW. Ten striped bonito larvae 2 and 3 days after hatching (DAH) were individually transferred from the stock tank to the beaker using a glass pipette (5 ml in volume) and kept at 23 F 1 jC without aeration. Dead larvae were counted and removed by pipette every 24 h. Water was not renewed and no food was supplied during the experiment. 2.4. Location of dead larvae in the presence or absence of EW or oil film To determine the location of dead larvae in the presence or absence of EW, 20 striped bonito larvae (0 – 3 DAH) were transferred to the experimental beakers containing 2 l seawater, with or without EW, and maintained as described above. Dead larvae were counted and removed 2 h after the transfer in order to clarify the location of dead larvae as ‘‘surface’’ or ‘‘other’’ (middle or bottom). Swimming behavior of the larvae was also observed by the naked eye during the experiment. Experiments were carried out in six replicates. To examine the effect of oil film, three sets of six beakers containing 2 l of seawater were assigned as control, EW, and oil film. Twenty striped bonito larvae were transferred to each of the beakers on 3 DAH, and larval death was observed 2 h after the transfer as described above. 2.5. Effect of EW in a large scale experiment To test the practical application of EW for rearing larvae on a large scale, fertilized eggs of pez cochero were collected and placed into two tanks (30-l) with and without EW. The initial stocking density was 222 eggs/l (6000 eggs in 27 l SW). A glass tube provided gentle aeration and the water temperature was 20 jC. No food was supplied throughout the experiment. On 1 and 2 DAH, one third of the seawater was exchanged and an appropriate amount of EW was added to keep the EW concentration constant. The number of surviving larvae was estimated by counting the larvae in 300-ml samples of well-mixed rearing water in triplicate.
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2.6. Histological observation on mucous cells To observe the mucous cells on the body surface, 2 DAH pez cochero larvae were sampled from the experimental tank, fixed in Bouin’s solution, and preserved in 80% ethanol. Later, the larvae were dehydrated and embedded in paraffin (Parahisto, Nakarai tesque, Japan). Serial sections (sagittal plane) were made by a microtome at a thickness of 5 Am. Sections were stained with Mayer’s hematoxylin and eosin. For the body surface of striped bonito, histological sections of 2 DAH larvae were examined using the previously prepared sections (Kaji et al., 2002). 2.7. Statistics Fisher’s exact test was employed to compare the number of dead larvae between the treatments in each experiment, except for large scale experiment where no replication was made. In the experiment to examine the location of dead larvae (Experiments 2 and 3), the numbers of dead larvae at different locations in each treatment were compared by exact test of goodness of fitness.
3. Results When larvae of striped bonito were transferred to beakers with or without EW on 2 and 3 DAH, the number of surviving larvae was constantly and significantly higher ( P < 0.01 by Fisher’s exact test) in the EW group than in the control (Fig. 1). In particular, on the next day after the transfer, the survival rates of control groups were about 30%, while those of the EW group were 100%.
Fig. 1. Effect of egg white (EW) on the larval survival of unfed striped bonito. Ten larvae were transferred to 2-l beakers on 2 and 3 days after hatching (DAH). Each point and vertical line represent the mean and S.E.M., respectively (n = 3). **Significantly different ( P < 0.01) from the control group.
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Stage specificity of larval death was examined at 2 h after the transfer in the presence or absence of EW (Fig. 2). Significantly higher mortality was observed in the control groups on 0, 2 and 3 DAH, than in the EW groups ( P < 0.01 by Fisher’s exact test). When the position of dead larvae were examined in the control group, more than 70% of the dead larvae were found on the water surface and were significantly higher than those at the middle and bottom ( P < 0.01 on 0 and 3 DAH, and P < 0.05 on 2 DAH by exact test of goodness of fitness). However, on 1 DAH, very low mortality was recorded in both the control and EW groups. The swimming behavior was different between 1 DAH and other ages. On 0, 2 and 3 DAH, it was observed that larvae in both groups swam upward to the water
Fig. 2. Number of dead larvae in the presence or absence of egg white. Twenty larvae of striped bonito were transferred to 2-l beakers on 0 – 3 days after hatching (DAH). Dead larvae at the water surface and those at the middle and bottom were separately counted 2 h after transfer. Each column and vertical bars represents the mean and S.E.M., respectively (n = 6). **P < 0.01, *P < 0.05.
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Fig. 3. Effect of egg white and oil film on the number of dead larvae of striped bonito. Twenty larvae were transferred to 2-l beakers on 3 days after hatching. Dead larvae at the water surface and those at the middle and bottom were separately counted 2 h after the transfer. Each column and vertical bars represents the mean and S.E.M., respectively (n = 6). **P < 0.01, *P < 0.05.
surface after the transfer, but such an upward swimming was not observed on 1 DAH. When the larvae of 0, 2 and 3 DAH touched the water surface, it was observed that some larvae were ‘‘trapped’’ by the water surface and died in the
Fig. 4. Effect of egg white on the larval survival of pez cochero under starvation. Six thousands eggs were stocked in a 30-l tank and number of live larvae was estimated by water column sampling, expressed as means of three measurements.
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control group, while no larvae were ‘‘trapped’’ by the water surface in the EW group. Since the mode of larval death observed in a second experiment was similar to the ‘‘surface death’’, the effect of an oil film was examined using the striped bonito larvae on 3 DAH (Fig. 3). In the control group, more than 60% of the larvae died within 2 h after the transfer, with 80% of the dead larvae located on the water surface. The mortality was significantly reduced by the EW ( P < 0.01 by Fisher’s exact test) as well as by the oil film ( P < 0.01 for the mortality at the surface and P < 0.05 for the mortality at the middle and bottom by Fisher’s exact test). There was no statistical difference ( P>0.05) in the mortality and the location of dead larvae between the EW group and the oil film group. In a large-scale experiment using 6000 eggs of pez cochero in a 30-l tank, the number of surviving larvae was always higher in the tank with EW than those in control throughout the experiment (Fig. 4). On 1 and 2 DAH, more dead larvae appeared to be present at the water surface in the control tank. All the larvae died on 3 DAH both in the EW treated and control tanks. The body surfaces of striped bonito and pez cochero larvae were covered with thin skin at a thickness of 4.47 and 7.10 Am, respectively. Mucous cells were scattered at a density of 0.0023 and 0.013 cells/Am body surface, respectively.
4. Discussion ‘‘Surface death’’ is recognized as one of the most serious obstacles to mass seed production of groupers around the first feeding stage (Yamaoka et al., 2000; Sawada et al., 1999). In addition, similar larval death has been noted in other marine fishes such as striped bonito and kawakawa, Euthynus affinis (Kaji et al., unpublished). This paper describes a novel method for preventing the surface death of marine fish larvae by the addition of EW into the rearing water. After transfer to the experimental container, larval mortality of striped bonito was significantly less in the group with EW added to the rearing water (Fig. 1), similar to that reported in the Japanese flounder larvae (Tagawa et al., submitted for publication). However, the mode of death in the present study may be fundamentally different from that of the Japanese flounder for the following reasons. Since the larval mortality of Japanese flounder occurred just after the transfer through the narrow tip of a Pasteur pipette, injury to the body surface is the most probable cause of death. On the other hand, larvae in the present study were transferred through a broad tip of a 5-ml pipette, suggesting less injury to the body. This idea is strongly supported by the absence of significant mortality in the control group on 1 DAH (Fig. 2). Even though they received similar handling by being transferred with a 5-ml pipette as on the other days, there was no transfer-related mortality. A significant difference in swimming behavior between 1 DAH and other ages (0, 2 and 3 DAH) was noted. Particularly, there was an absence of upward swimming on 1 DAH. Moreover, it was observed that the water surface was the major site of larval death in other ages (Fig. 2). Consequently, the main reason for the death of bonito larvae is entrapment in the water surface tension, and not due to handling injury. In the presence of EW, although the larvae swam upward as in the control group, no larvae were ‘‘trapped’’ at the water
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surface, suggesting that the addition of EW is effective at preventing the surface entrapment. In red spotted grouper, prelarvae swim upward attracted by light, and were ‘‘trapped’’ at the surface by water surface tension (Yamaoka et al., 2000). The formation of an oil film on the water surface prevented surface death by reducing the water surface tension (Yamaoka et al., 2000). As shown in Fig. 3, the mortality reduced by EW was also reduced by an oil film, suggesting the similarity between EW and the oil film in the mechanism of ‘‘surface death’’ prevention by reduction of water surface tension. In order to examine the effect of EW, pez cochero was employed in large scale experiment (Fig. 4). Because striped bonito larvae around the first feeding stage are much larger than those of most marine fish (Kaji et al., in press), generalization is difficult. In addition, pez cochero died frequently at the water surface during the early larval period (Arai, H., personal communications). The number of larvae surviving in the EW-treated tank was always higher than in the control tank (Fig. 4). Although some other benefit of EW addition may be present, the results suggest that the addition of EW is effective in preventing surface death in a wide variety of teleosts and that the method would be applicable to larval rearing on a practical scale. Surface death has been regarded as a problem peculiar to larval groupers. In the larvae of the red spotted grouper, well-developed mucous cells covered almost all the body surface (Kaji et al., 1995). When the density of mucous cells was measured from the figure in the paper (Kaji et al., 1995), the density was 0.081 cells/Am body surface. Moreover, secreted mucus has been suggested to act as glue when larvae swim up and touch the water surface (Yamaoka et al., 2000). Similar mucous cells and related mortality at the water surface have been reported in another grouper, Epinephelus bruneus (Sawada et al., 1999). In contrast, the striped bonito and pez cochero do not have such highly developed mucous cells on the body surface at the first feeding stage and the density of mucous cells on their body surface is much lower than that of the red spotted grouper (0.0023 cells/Am body surface for striped bonito and 0.013 cells/Am body surface for pez cochero). Thus, ‘‘surface death’’ can occur in a wide variety of marine fish larvae, including larvae without well-developed mucous cells. In addition, the larval size at yolk-sac and first feeding stages of striped bonito (Kaji et al., in press) was much larger than that of the pez cochero or other groupers (Sawada et al., 1999; Ukawa et al., 1966). Therefore, the addition of EW should prevent surface death in larvae of various sizes. Addition of EW decreases water quality due to the fermentation of the dissolved EW. In fact, the bad smell of the rearing water was noticed 1 day after the addition of EW in experiments 1 and 4. However, the time when all the larvae died was not different between the control and the EW treated group in both experiments (Figs. 1 and 4). Since no diet was given in these experiments, starvation is probably the main reason for the death and the poor water quality in the EW group will likely have a minor effect on larval death up to 4 days. In addition, surface death is known to mainly occur at the mouth opening stage in red spotted grouper (Yamaoka et al., 2000) and on 2 and 3 DAH in striped bonito (Figs. 2 and 3) when larvae are in the first feeding stage (Kaji et al., in press). This suggests a stage- or agespecificity of the surface death. Thus, the addition of EW may only be required at a specific and short period during larval rearing. It is necessary to determine the timing and duration of the occurrence of surface death. Then, the addition of EW should only be necessary when
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the peak of mortality is expected. The short duration of EW addition would be important also for successful swim bladder inflation in some species, because initial swim bladder inflation is inhibited by oil film blocking the larval access to water surface (Kitajima et al., 1994). The mechanism for prevention of surface death by addition of EW is unclear so far, but surfactant effect of EW is a strong possibility. Further studies on physical and chemical changes in the rearing water by the addition of EW, such as strength of water surface tension, will help us to understand the underlying mechanism. In addition, experiments using practical rearing protocols are also essential for future application of this method to mass production of larvae, since all of the experiments in the present study were conducted using small containers without food and with minimal water exchange. Successful feeding in EW dissolved water has been observed in striped bonito in our preliminary experiment (Kaji et al., unpublished). In the use of oil film to prevent surface death, the effect seemed to differ among hatcheries, and the removal of oil film from the rearing tank was difficult, and therefore an alternative substance was desired (Yamaoka, 2001). Although more research needs to be conducted before practical use, we believe that the addition of EW is the strongest candidate for an alternative to oil film to prevent surface death, as well as to protect from the physical damage reported by Tagawa et al. (submitted for publication).
Acknowledgements This study was conducted under a cooperative program between the Tokyo Sea Life Park and Kyoto University. We wish to express our sincere thanks to the staff of Tokyo Sea Life Park for their support during our studies. Thanks are also due to Dr. G. Joan Holt of University of Texas Marine Science Institute for critical reviewing of this article. This study was supported by grants-in-aid from the Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists to T.K. This study was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports and Science, and from the Ministry of Agriculture, Forestry, and Fisheries to M.T. and M.T.
References Kaji, T., Yamaoka, K., Isshiki, T., Yamada, T., 1995. Mucous cell development on the body surface in larvae of the red-spotted grouper, Epinephelus akaara. Bull. Mar. Sci. Fish., Kochi Univ. 15, 117 – 120. Kaji, T., Kodama, M., Arai, H., Tagawa, M., Tanaka, M., 2002. Precocious development of the digestive system in relation to early appearance of piscivory in striped bonito Sarda orientalis larvae. Fish. Sci. 68, 1212 – 1218. Kitajima, C., Watanebe, T., Tsukashima, Y., Fujita, S., 1994. Lordotic deformation and abnormal development of swim bladders in some hatchery-bred marine physoclistous fish in Japan. J. World Aquac. Soc. 25, 64 – 77. Kjørsvik, E., Mangor-Jensen, A., Holmefjord, I., 1990. Egg quality in fishes. Adv. Mar. Biol. 26, 71 – 113. Sawada, Y., Kato, K., Okada, T., Kurata, M., Mukai, Y., Miyashita, S., Murata, O., Kumai, H., 1999. Growth and morphological development of larval and juvenile Epinephelus bruneus (Perciformes: Serranidae). Ichthyol. Res. 46, 245 – 257. Tagawa, M., Kaji, T., Kinoshita, M., Tanaka, M., 2002. Effect of stocking density and addition of proteins on larval survival in Japanese flounder, Paralichthys olivaceus. Aquaculture (submitted for publication).
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Ukawa, M., Higuchi, M., Mito, S., 1966. Spawning habits and early life history of a serranid fish, Epinephelus akaara (Temminck et Schlegel). Jpn. J. Ichthyol. 13, 156 – 161 (in Japanese, with English summary). Yamaoka, K., 2001. Mechanism of surface tension-related deaths of prelarval red spotted grouper. Aquac. Mag., Feb., 76 – 80 (in Japanese). Yamaoka, K., Nanbu, T., Miyagawa, M., Isshiki, T., Kusaka, A., 2000. Water surface tension-related deaths in prelarval red-spotted grouper. Aquaculture 189, 165 – 176.
Prevention of surface death of marine fish larvae by the addition of egg white into rearing water Tatsuya Kaji a,*, Masaaki Kodama b, Hiroshi Arai b, Masaru Tanaka a, Masatomo Tagawa a a
Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto 606-8502, Japan b Tokyo Sea Life Park, 6-2-3, Rinkai, Edogawa, Tokyo 134-8587, Japan
Received 22 August 2002; received in revised form 20 February 2003; accepted 25 February 2003
Abstract In the seed production of several marine teleosts, large numbers of dead larvae are frequently observed on the water surface around the time of first feeding. We tried to prevent such ‘‘surface death’’ by the addition of chicken egg white (EW) into the rearing water. In the first experiment, larvae of striped bonito, Sarda orientalis, at the first feeding stage (2 and 3 days after hatching) were transferred to plastic beakers using a 5-ml pipette at a density of 10 larvae/l, in the absence or presence of EW (80 Al/l). No diet was supplied, the water was not renewed throughout the experiment, and dead larvae were removed daily. Numbers of surviving larvae of the EW-treated group were significantly higher than those of the control group. In particular, survival at the first observation (next day after transfer) was less than 30% in the control, while 100% in the EW-treated group. In a second experiment, mortality at 2 h after transfer was compared with special attention to the location of the dead larvae. Significantly ( P < 0.01) higher mortality was found in the control group, and more than 70% of the dead larvae were located on the water surface. Upward swimming toward the water surface was observed in both groups, but larvae ‘‘trapped’’ at the surface were only observed in the control group. In a third experiment, an oil film that was artificially created on the water surface prevented larval death after the transfer as well as the dissolved EW. Since the oil film is known to prevent ‘‘surface trap’’ of larvae and decrease ‘‘surface death’’ in a grouper, the addition of EW was considered to have a similar effect in reducing mortality. In a fourth experiment using 30-l tanks, survival of larval pez cochero, Dules auriga, was also higher in the presence of EW, especially during a period when dead larvae were frequently found on the water surface in the control. Thus, the addition of EW to the rearing water is suggested to prevent surface death in a
* Corresponding author. Tel.: +81-75-753-6222; fax: +81-75-753-6229. E-mail address: [email protected] (T. Kaji). 0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0044-8486(03)00243-6
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variety of marine fish larvae, and the possibility of applying the method in practical rearing is discussed. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Striped bonito; Sarda orientalis; Survival; Stress; Mortality; Pez cochero; Dules auriga
1. Introduction In the aquaculture of marine teleosts, one of the most serious problems associated with mass production of larval fish is the high mortality during the early larval period. Mortality during the yolk-sac to first feeding stages is caused by poor quality of eggs (Kjørsvik et al., 1990) and/or various abiotic conditions, since larvae do not require any external nutrition before and around the time of first feeding. ‘‘Surface death’’ is a heavy mortality factor in which most of the dead larvae are found on the water surface, typically reported for prelarvae (during yolk absorption) of red spotted grouper, Epinephelus akaara (Yamaoka et al., 2000). The authors also reported the complete prevention of death by artificially forming an oil film on the water surface, perhaps due to the reduction of surface tension (Yamaoka et al., 2000). Reduction of mortality after handling has been demonstrated by the addition of protein, such as egg white (EW), into rearing water in yolk-sac larvae of the Japanese flounder, Paralichthys olivaceus (Tagawa et al., submitted for publication). This phenomenon could be due to the protective effect of dissolved protein on the injured larvae, however further study is needed for application of the method to practical and mass rearing of marine fish larvae. The present study attempts to demonstrate the prevention of surface death by the addition of EW into rearing water in larval striped bonito, Sarda orientalis. The application of this method was also tested on a practical rearing scale using larvae of pez cochero, Dules auriga.
2. Materials and methods 2.1. Fish All experiments were conducted at the Tokyo Sea Life Park from November to December 2001. Striped bonito broodstock were maintained in an exhibition tank (2200 tons) at 23 jC and pez cochero broodstock were in an exhibition tank (5 tons) at 18 jC. Fertilized eggs from both species were collected in the morning after spontaneous spawning by water flow into a net installed to each tank. Floating eggs of the striped bonito were transferred to a 30-l rearing tank (stock tank), aerated by glass tube and incubated at 23 F 1 jC. Hatched larvae were kept in the tank without diet or renewal of the rearing water until used in experiments. The pez cochero eggs were used directly in the experiments described below.
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2.2. Procedure for dissolving the EW and forming oil film The EW was separated from the egg yolk, mixed 1 part EW to 49 parts of seawater (SW), and added to each experimental container for a final concentration of 80 Al EW/l rearing water. This concentration of EW (1/12,500) is about three times higher than the effective concentration (1/30,000) reported in Japanese flounder (Tagawa et al., submitted for publication). An equivalent volume of SW (without EW) was added to control containers. An oil film was formed using Super Selco (INVE Aquaculture NV, Belgium), a product originally used for nutritional enrichment of rotifer and brine shrimp (Artemia spp.). Two drops of Super Selco were added and mixed well in 2 l of SW in a plastic beaker. The surface layer of floating oil was collected by a pipette 10 min later and 5 ml of the surface layer were transferred into each experimental beaker with 2 l SW. 2.3. Time course of larval death in the presence or absence of EW Two sets of three plastic beakers (2-l in volume) were filled with 1 l of seawater with or without EW. Ten striped bonito larvae 2 and 3 days after hatching (DAH) were individually transferred from the stock tank to the beaker using a glass pipette (5 ml in volume) and kept at 23 F 1 jC without aeration. Dead larvae were counted and removed by pipette every 24 h. Water was not renewed and no food was supplied during the experiment. 2.4. Location of dead larvae in the presence or absence of EW or oil film To determine the location of dead larvae in the presence or absence of EW, 20 striped bonito larvae (0 – 3 DAH) were transferred to the experimental beakers containing 2 l seawater, with or without EW, and maintained as described above. Dead larvae were counted and removed 2 h after the transfer in order to clarify the location of dead larvae as ‘‘surface’’ or ‘‘other’’ (middle or bottom). Swimming behavior of the larvae was also observed by the naked eye during the experiment. Experiments were carried out in six replicates. To examine the effect of oil film, three sets of six beakers containing 2 l of seawater were assigned as control, EW, and oil film. Twenty striped bonito larvae were transferred to each of the beakers on 3 DAH, and larval death was observed 2 h after the transfer as described above. 2.5. Effect of EW in a large scale experiment To test the practical application of EW for rearing larvae on a large scale, fertilized eggs of pez cochero were collected and placed into two tanks (30-l) with and without EW. The initial stocking density was 222 eggs/l (6000 eggs in 27 l SW). A glass tube provided gentle aeration and the water temperature was 20 jC. No food was supplied throughout the experiment. On 1 and 2 DAH, one third of the seawater was exchanged and an appropriate amount of EW was added to keep the EW concentration constant. The number of surviving larvae was estimated by counting the larvae in 300-ml samples of well-mixed rearing water in triplicate.
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2.6. Histological observation on mucous cells To observe the mucous cells on the body surface, 2 DAH pez cochero larvae were sampled from the experimental tank, fixed in Bouin’s solution, and preserved in 80% ethanol. Later, the larvae were dehydrated and embedded in paraffin (Parahisto, Nakarai tesque, Japan). Serial sections (sagittal plane) were made by a microtome at a thickness of 5 Am. Sections were stained with Mayer’s hematoxylin and eosin. For the body surface of striped bonito, histological sections of 2 DAH larvae were examined using the previously prepared sections (Kaji et al., 2002). 2.7. Statistics Fisher’s exact test was employed to compare the number of dead larvae between the treatments in each experiment, except for large scale experiment where no replication was made. In the experiment to examine the location of dead larvae (Experiments 2 and 3), the numbers of dead larvae at different locations in each treatment were compared by exact test of goodness of fitness.
3. Results When larvae of striped bonito were transferred to beakers with or without EW on 2 and 3 DAH, the number of surviving larvae was constantly and significantly higher ( P < 0.01 by Fisher’s exact test) in the EW group than in the control (Fig. 1). In particular, on the next day after the transfer, the survival rates of control groups were about 30%, while those of the EW group were 100%.
Fig. 1. Effect of egg white (EW) on the larval survival of unfed striped bonito. Ten larvae were transferred to 2-l beakers on 2 and 3 days after hatching (DAH). Each point and vertical line represent the mean and S.E.M., respectively (n = 3). **Significantly different ( P < 0.01) from the control group.
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Stage specificity of larval death was examined at 2 h after the transfer in the presence or absence of EW (Fig. 2). Significantly higher mortality was observed in the control groups on 0, 2 and 3 DAH, than in the EW groups ( P < 0.01 by Fisher’s exact test). When the position of dead larvae were examined in the control group, more than 70% of the dead larvae were found on the water surface and were significantly higher than those at the middle and bottom ( P < 0.01 on 0 and 3 DAH, and P < 0.05 on 2 DAH by exact test of goodness of fitness). However, on 1 DAH, very low mortality was recorded in both the control and EW groups. The swimming behavior was different between 1 DAH and other ages. On 0, 2 and 3 DAH, it was observed that larvae in both groups swam upward to the water
Fig. 2. Number of dead larvae in the presence or absence of egg white. Twenty larvae of striped bonito were transferred to 2-l beakers on 0 – 3 days after hatching (DAH). Dead larvae at the water surface and those at the middle and bottom were separately counted 2 h after transfer. Each column and vertical bars represents the mean and S.E.M., respectively (n = 6). **P < 0.01, *P < 0.05.
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Fig. 3. Effect of egg white and oil film on the number of dead larvae of striped bonito. Twenty larvae were transferred to 2-l beakers on 3 days after hatching. Dead larvae at the water surface and those at the middle and bottom were separately counted 2 h after the transfer. Each column and vertical bars represents the mean and S.E.M., respectively (n = 6). **P < 0.01, *P < 0.05.
surface after the transfer, but such an upward swimming was not observed on 1 DAH. When the larvae of 0, 2 and 3 DAH touched the water surface, it was observed that some larvae were ‘‘trapped’’ by the water surface and died in the
Fig. 4. Effect of egg white on the larval survival of pez cochero under starvation. Six thousands eggs were stocked in a 30-l tank and number of live larvae was estimated by water column sampling, expressed as means of three measurements.
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control group, while no larvae were ‘‘trapped’’ by the water surface in the EW group. Since the mode of larval death observed in a second experiment was similar to the ‘‘surface death’’, the effect of an oil film was examined using the striped bonito larvae on 3 DAH (Fig. 3). In the control group, more than 60% of the larvae died within 2 h after the transfer, with 80% of the dead larvae located on the water surface. The mortality was significantly reduced by the EW ( P < 0.01 by Fisher’s exact test) as well as by the oil film ( P < 0.01 for the mortality at the surface and P < 0.05 for the mortality at the middle and bottom by Fisher’s exact test). There was no statistical difference ( P>0.05) in the mortality and the location of dead larvae between the EW group and the oil film group. In a large-scale experiment using 6000 eggs of pez cochero in a 30-l tank, the number of surviving larvae was always higher in the tank with EW than those in control throughout the experiment (Fig. 4). On 1 and 2 DAH, more dead larvae appeared to be present at the water surface in the control tank. All the larvae died on 3 DAH both in the EW treated and control tanks. The body surfaces of striped bonito and pez cochero larvae were covered with thin skin at a thickness of 4.47 and 7.10 Am, respectively. Mucous cells were scattered at a density of 0.0023 and 0.013 cells/Am body surface, respectively.
4. Discussion ‘‘Surface death’’ is recognized as one of the most serious obstacles to mass seed production of groupers around the first feeding stage (Yamaoka et al., 2000; Sawada et al., 1999). In addition, similar larval death has been noted in other marine fishes such as striped bonito and kawakawa, Euthynus affinis (Kaji et al., unpublished). This paper describes a novel method for preventing the surface death of marine fish larvae by the addition of EW into the rearing water. After transfer to the experimental container, larval mortality of striped bonito was significantly less in the group with EW added to the rearing water (Fig. 1), similar to that reported in the Japanese flounder larvae (Tagawa et al., submitted for publication). However, the mode of death in the present study may be fundamentally different from that of the Japanese flounder for the following reasons. Since the larval mortality of Japanese flounder occurred just after the transfer through the narrow tip of a Pasteur pipette, injury to the body surface is the most probable cause of death. On the other hand, larvae in the present study were transferred through a broad tip of a 5-ml pipette, suggesting less injury to the body. This idea is strongly supported by the absence of significant mortality in the control group on 1 DAH (Fig. 2). Even though they received similar handling by being transferred with a 5-ml pipette as on the other days, there was no transfer-related mortality. A significant difference in swimming behavior between 1 DAH and other ages (0, 2 and 3 DAH) was noted. Particularly, there was an absence of upward swimming on 1 DAH. Moreover, it was observed that the water surface was the major site of larval death in other ages (Fig. 2). Consequently, the main reason for the death of bonito larvae is entrapment in the water surface tension, and not due to handling injury. In the presence of EW, although the larvae swam upward as in the control group, no larvae were ‘‘trapped’’ at the water
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surface, suggesting that the addition of EW is effective at preventing the surface entrapment. In red spotted grouper, prelarvae swim upward attracted by light, and were ‘‘trapped’’ at the surface by water surface tension (Yamaoka et al., 2000). The formation of an oil film on the water surface prevented surface death by reducing the water surface tension (Yamaoka et al., 2000). As shown in Fig. 3, the mortality reduced by EW was also reduced by an oil film, suggesting the similarity between EW and the oil film in the mechanism of ‘‘surface death’’ prevention by reduction of water surface tension. In order to examine the effect of EW, pez cochero was employed in large scale experiment (Fig. 4). Because striped bonito larvae around the first feeding stage are much larger than those of most marine fish (Kaji et al., in press), generalization is difficult. In addition, pez cochero died frequently at the water surface during the early larval period (Arai, H., personal communications). The number of larvae surviving in the EW-treated tank was always higher than in the control tank (Fig. 4). Although some other benefit of EW addition may be present, the results suggest that the addition of EW is effective in preventing surface death in a wide variety of teleosts and that the method would be applicable to larval rearing on a practical scale. Surface death has been regarded as a problem peculiar to larval groupers. In the larvae of the red spotted grouper, well-developed mucous cells covered almost all the body surface (Kaji et al., 1995). When the density of mucous cells was measured from the figure in the paper (Kaji et al., 1995), the density was 0.081 cells/Am body surface. Moreover, secreted mucus has been suggested to act as glue when larvae swim up and touch the water surface (Yamaoka et al., 2000). Similar mucous cells and related mortality at the water surface have been reported in another grouper, Epinephelus bruneus (Sawada et al., 1999). In contrast, the striped bonito and pez cochero do not have such highly developed mucous cells on the body surface at the first feeding stage and the density of mucous cells on their body surface is much lower than that of the red spotted grouper (0.0023 cells/Am body surface for striped bonito and 0.013 cells/Am body surface for pez cochero). Thus, ‘‘surface death’’ can occur in a wide variety of marine fish larvae, including larvae without well-developed mucous cells. In addition, the larval size at yolk-sac and first feeding stages of striped bonito (Kaji et al., in press) was much larger than that of the pez cochero or other groupers (Sawada et al., 1999; Ukawa et al., 1966). Therefore, the addition of EW should prevent surface death in larvae of various sizes. Addition of EW decreases water quality due to the fermentation of the dissolved EW. In fact, the bad smell of the rearing water was noticed 1 day after the addition of EW in experiments 1 and 4. However, the time when all the larvae died was not different between the control and the EW treated group in both experiments (Figs. 1 and 4). Since no diet was given in these experiments, starvation is probably the main reason for the death and the poor water quality in the EW group will likely have a minor effect on larval death up to 4 days. In addition, surface death is known to mainly occur at the mouth opening stage in red spotted grouper (Yamaoka et al., 2000) and on 2 and 3 DAH in striped bonito (Figs. 2 and 3) when larvae are in the first feeding stage (Kaji et al., in press). This suggests a stage- or agespecificity of the surface death. Thus, the addition of EW may only be required at a specific and short period during larval rearing. It is necessary to determine the timing and duration of the occurrence of surface death. Then, the addition of EW should only be necessary when
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the peak of mortality is expected. The short duration of EW addition would be important also for successful swim bladder inflation in some species, because initial swim bladder inflation is inhibited by oil film blocking the larval access to water surface (Kitajima et al., 1994). The mechanism for prevention of surface death by addition of EW is unclear so far, but surfactant effect of EW is a strong possibility. Further studies on physical and chemical changes in the rearing water by the addition of EW, such as strength of water surface tension, will help us to understand the underlying mechanism. In addition, experiments using practical rearing protocols are also essential for future application of this method to mass production of larvae, since all of the experiments in the present study were conducted using small containers without food and with minimal water exchange. Successful feeding in EW dissolved water has been observed in striped bonito in our preliminary experiment (Kaji et al., unpublished). In the use of oil film to prevent surface death, the effect seemed to differ among hatcheries, and the removal of oil film from the rearing tank was difficult, and therefore an alternative substance was desired (Yamaoka, 2001). Although more research needs to be conducted before practical use, we believe that the addition of EW is the strongest candidate for an alternative to oil film to prevent surface death, as well as to protect from the physical damage reported by Tagawa et al. (submitted for publication).
Acknowledgements This study was conducted under a cooperative program between the Tokyo Sea Life Park and Kyoto University. We wish to express our sincere thanks to the staff of Tokyo Sea Life Park for their support during our studies. Thanks are also due to Dr. G. Joan Holt of University of Texas Marine Science Institute for critical reviewing of this article. This study was supported by grants-in-aid from the Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists to T.K. This study was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports and Science, and from the Ministry of Agriculture, Forestry, and Fisheries to M.T. and M.T.
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