Process and Timing of Initial Swim Bladder Inflation Inlongtooth

Aquacult. Sci. 64（4），349－358（2016）

Process and timing of initial swim bladder inflation in longtooth grouper Epinephelus bruneus and red spotted grouper Epinephelus akaara

1 2 3 Takashi IWASAKI , Sho MIZUTA , Takayuki KOGANE , 4 5 2,＊ Jun SATOH , Shigeki DAN and Katsuyuki HAMASAKI

Abstract: To understand the process of initial swim bladder inflation, swim bladder development was histologically examined in larvae of longtooth grouper, Epinephelus bruneus, and red spotted grouper, Epinephelus akaara. The posterodorsal wall of the larval digestive tract of both species protruded, the swim bladder formed, and the pneumatic duct connected the swim bladder to the digestive tract during 6-10 days after hatching (DAH) (3.6-5.0 mm standard length, SL) and 5-8 DAH (3.0-3.3 mm SL) in longtooth and red spotted grouper larvae, respectively. Larvae of both species were reared in tanks with or without treatments to remove oil film from the water surface. In both species, more larvae developed inflated swim bladders in the tank with the oil-film-removal treatment than in the tank without, and the numbers of larvae with inflated swim bladders increased from 6 to 9 DAH (3.2-3.9 mm SL) and from 5 to 8 DAH (2.6-3.6 mm SL) in longtooth and red spotted grouper, respectively. Our results indicated that larvae inflated their swim bladders by gulping air from the water surface, and that timing of initial swim bladder inflation was 4-5 days in the early life stage by 10 DAH.

Key words: Grouper; Larvae; Seed production; Swim bladder

Grouper is economically important species However, high mortality due to water surface in the East and Southeast Asian countries tension-related death and bottoming death syn- (Fukuhara 1989; Liao et al. 2001; Marte 2003). dromes of larvae up to 10 days after hatching Mass seed production techniques for groupers (DAH) have been reported. These syndromes have been developed recently, and grouper have caused serious problems for mass seed species are expected to be new aquaculture production of finfishes, including grouper spe- commodities because of their high market cies (Yamaoka et al. 2000; Tsuchihashi et al. prices (e.g., at least 1,500 yen/kg in Japan) 2003; Miyashita 2006; Teruya and Yoseda 2006; (Tsuchihashi et al. 2003; Teruya and Yoseda Tanaka et al. 2009; Takebe et al. 2011). 2006; Iwasaki et al. 2011a; Takebe et al. 2011). Water surface tension-related death occurs

Received 6 July 2016; Accepted 21 September 2016. 1 Yaeyama Laboratory, Seikai National Fisheries Research Institute, Japan Fisheries Research and Education Agency, Ishigaki, Okinawa 907-0451, Japan. 2 Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Minato, Tokyo 108-8477, Japan. 3 Yashima Laboratory, National Research Institute of Fisheries and Environment of Inland Sea, Japan Fisheries Research and Education Agency, Takamatsu, Kagawa 761-0111, Japan. 4 Kamiura Laboratory, National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, Saiki, Oita 872-2602, Japan. 5 Momoshima Laboratory, National Research Institute of Fisheries and Environment of Inland Sea, Japan Fisheries Research and Education Agency, Momoshima, Onomichi, Hiroshima 722-0061, Japan. ＊Corresponding author: Tel., +81-3-5463-0538; Fax, +81-3-5463-0538; Email, [email protected] (K. Hamasaki). 350 T. Iwasaki, S. Mizuta, T. Kogane, J. Satoh, S. Dan and K. Hamasaki when larvae approach the water surface layer and are trapped by surface tension, and thus Materials and Methods an oil film is often spread on the water surface to reduce surface tension and prevent this type Larviculture and ontogenetic development of the of mortality (Yamaoka et al. 2000; Tsuchihashi swim bladder et al. 2003; Miyashita 2006). However, larvae The longtooth grouper (LG) larvae were of many fish species gulp air from the water cultured during May-June 2012 at the Kamiura surface to inflate their swim bladders, and an oil Laboratory, National Research Institute of film on the water surface prevents air gulping Aquaculture, Japan Fisheries Research and and proper swim bladder inflation (Kitajima et Education Agency (FRA), Saiki, Oita, Japan. al. 1981; Chatain and Ounais-Guschemann 1990; Fertilized eggs of LG were obtained by artificial Trotter et al. 2005; Kawabe and Kimura 2008; fertilization according to the methods of Woolley and Qin 2010; Imai et al. 2011; Kurata Iwasaki et al. (2016). The fertilized eggs were et al. 2012; Tsuji et al. 2016). Fish larvae without placed in a 2-l measuring cylinder containing 2 l an inflated swim bladder have a higher body seawater, and were separated into floating eggs density and sink to the tank bottom, resulting in and sinking eggs. About 5 minutes after adding a high risk of bottoming death (Miyashita 2006; the eggs to the measuring cylinder, the floating Hirata et al. 2009; Tanaka et al. 2009; Teruya et eggs were transferred to a 1 kl cylindrical al. 2009). Furthermore, in several finfish species tank and stocked for 20 h at 22°C with water including grouper species such as longtooth exchanged at a rate of 5 l/min. About 50,000 grouper Epinephelus bruneus, and sevenband floating fertilized eggs were stocked into a 3 kl grouper Hyporthodus septemfasciatus (synonym: square tank and the larvae were reared until 25 E. septemfasciatus), the failure of larvae to initially DAH. The oil film was removed from the water inflate the swim bladder leads to abnormal mor- surface using two surface skimmers made of phological development, particularly lordosis PVC pipe (13 mm outer diameter and 25 cm (Kitajima et al. 1981; Chatain 1994; Kitajima et square) with an air jet system (Shiozawa et al. al. 1994, Goolish and Okutake 1999; Tsuji et al. 2003; Kawabe and Kimura 2008) were posi- 2014; Uji 2014). Therefore, it is very important tioned at the water surface at opposing corners to understand the mechanism of initial swim of the tank, and the oil film was concentrated bladder inflation so that appropriate larval in the skimmer and removed from the rearing management strategies can be developed. This tank. The larvae were fed rotifers, Brachionus information will contribute to stable mass seed plicatilis species complex (S- and L-types) production in finfish. enriched with a commercial enrichment At present, little is known about the frequency material (Super Fresh Chlorella V-12; SV-12, of initial swim bladder inflation in grouper larvae Chlorella Industry, Tokyo, Japan) for 20-28 h (Kawabe and Kimura 2008; Tsuji et al. 2016). and were fed at a density of 10-15 individuals/ Moreover, to our knowledge, no information is ml from 3 DAH until the end of experiment available on the morphological development (S-type, 3-10 DAH and L-type, 10-25 DAH). of the swim bladder in grouper larvae. In the The tank water was not exchanged during the present study, we focused on LG and red spot- experiment. The water temperature was main- ted grouper, E. akaara, which are important tained at 22°C until hatching and was raised at aquaculture species. Ontogenetic development a rate of 1°C/day and maintained at 26°C using of the larval swim bladder was histologically a thermostatically controlled titanium heater. examined. The larvae were reared in tanks with The tanks were aerated at a rate of 0.2-0.5 l/ and without treatments to remove oil film from min with air stones (KA-20, Tanaka Sanjiro, the water surface to understand the process Fukuoka, Japan) placed on the center and at the and timing of initial swim bladder inflation in four corners of the tank bottom. the larvae of these two grouper species. The red spotted grouper (RSG) larvae were Initial Swim Bladder Inflation in Grouper 351 cultured during July 2013 at the Tamano Larviculture and swim bladder inflation under Laboratory, National Research Institute of two different water surface conditions Fisheries and Environment of Inland Sea, FRA, The LG larvae were reared until 15 DAH Tamano, Okayama, Japan. Fertilized eggs of in two 3 kl square tanks with and without RSG were obtained from spontaneous spawning oil-film-removal treatments according to the of multiple broodstocks (115 male individuals, culture protocol in the Kamiura Laboratory 64 female individuals) in a 50 kl tank (2.5 m as described above. The oil film was removed deep). Floating and sinking eggs were sepa- by the overflow method used for rearing RSG rated by placing the eggs into a 500 l sea water larvae in the Tamano Laboratory. Eighty to 100 tank. The RSG larvae were reared in two 2 kl larvae were sampled from each tank every day square tanks with and without treatments to after 4 DAH, and the SL of the anesthetized remove oil film from the water surface. The oil larvae was measured to the nearest 0.1 mm film was removed from the water surface by using a profile projector. Then, the swim blad- overflowing the tank at a rate of 2 l/min using der was dissected to check for air bubbles a PVC pipe (120 cm length and 50 mm outer under a microscope to confirm inflation. The diameter) placed vertically on the tank drain. rate of swim bladder inflation (%) was calcu- In the other treatment, the oil film was allowed lated as follows: (number of larvae with an to accumulate on the water surface by not inflated swim bladder)/ (total number of larvae exchanging the tank water. About 30,000 float- examined)×100. ing fertilized eggs were stocked in each tank, Swim bladder inflation was monitored using and the larvae were fed with S-type rotifers at the RSG larvae that were reared for examining a density of 15 individuals/ml from 1 DAH until ontogenetic development of the swim bladder the end of the experiment (15 DAH). The roti- in the Tamano Laboratory as mentioned above. fers were enriched with SV-12 for 6-12 h before Forty fish were sampled from respective tanks feeding. The water temperature was main- with and without oil-film-removal treatments tained at 29°C until hatching and was lowered every day. The SL was measured, and the swim gradually and maintained at 27°C from 3 DAH bladder inflation rate was calculated. onwards. Aeration was conducted at a rate of The SL values and the incidence of swim blad- 0.2-0.5 l/min using an airstone (AS-1, Tanaka der inflation were compared using Student’s Sanjiro, Fukuoka, Japan) and air hoses (Uni t-test and chi-squared test, respectively, to Air Hose, 0.4 m long; Tanaka Sanjiro, Fukuoka, detect differences between the larvae in Japan). The airstone was placed on the center tanks with and without the oil-film-removal of the bottom of the rearing tank and air hoses treatments. A p-value < 0.05 was considered were set on the tank bottom under the edge of significant. each wall. Six LG larvae and three or four RSG larvae Results were collected every day from each rearing tank. The standard length (SL) of the larval Ontogenetic development of the swim bladder specimens was measured to the nearest 0.1 mm The posterodorsal wall of the digestive tract using a profile projector (V-12BSC, Nikon, of LG larvae protruded and the swim bladder Tokyo, Japan) under anesthesia (0.05-0.1 mg/l pouch (the swim bladder primordium) formed MS-222; Sigma, Tokyo, Japan). The larvae were completely by 3 DAH (2.8 mm SL, Fig. 1(a)). then fixed in Bouin’s solution for 1 day and then The swim bladder pouch was directly con- transferred into 70% ethanol. All specimens nected with the digestive tract, and the inner were embedded in paraffin and sectioned longi- cavity of the swim bladder pouch was very tudinally at 6 µm or in the serial direction, and narrow and solid. The swim bladder was dis- then stained with hematoxylin-eosin. tinctly observed at 4 DAH, but no pneumatic duct was detected (2.8 mm SL, Fig. 1(b)). The 352 T. Iwasaki, S. Mizuta, T. Kogane, J. Satoh, S. Dan and K. Hamasaki

Fig. 1. Serial (a, b, and g–i) and longitudinal (c–f) cross-sections of swim bladder in longtooth grouper larvae; (a), non-inflated swim bladder 3 days after hatching (DAH), 2.8 mm standard length (SL); (b), non-inflated swim bladder 4 DAH, 2.9 mm SL; (c–f), non-inflated swim bladder 6 DAH, 3.6 mm SL; (g), non-inflated swim bladder 11 DAH, 5.0 mm SL; (h), inflated swim bladder 16 DAH, 5.0 mm SL; (i), non-inflated swim bladder 16 DAH, 4.8 mm SL. Scale bars indicate 50 µm. Abbreviations: dt, digestive tract; gg, gas gland; nch, notochord; pd, pneumatic duct; sb, swim bladder; sbe, swim bladder epithelium; sbp; swim bladder pouch.

pneumatic duct connected the digestive tract swim bladder were connected with the pneu- to the swim bladder by 6 DAH, but no air was matic duct at 5 DAH (3.0 mm SL, Fig. 2(a-d)). detected in the swim bladder (3.6 mm SL, Figure 2(e) shows a non-inflated swim bladder Fig. 1(c-f)). Figure 1(g) shows the non-inflated at 8 DAH (3.3 mm SL), in which air was present swim bladder of a larva on 11 DAH (5.0 mm in the pneumatic duct at the back of the swim SL), in which the pneumatic duct was separate bladder. The inflated swim bladder lumen of from the swim bladder, and an air space was larvae > 2.7 mm SL was widely expanded, the present in the anterior terminal of the pneu- gas gland was situated on the ventral side of matic duct. Figure 1(h) shows the inflated swim the epithelium, and the pneumatic duct had bladder of a larva at 16 DAH (5.0 mm SL), in degenerated (9 DAH; 4.3 mm SL, Fig. 2(f)). which the swim bladder lumen was a wide In contrast, the swim bladder lumen of larvae open space, epithelial cells lined the bottom > 4.0 mm SL with a non-inflated swim bladder of the swim bladder, and the pneumatic duct was filled with hyperplastic epithelial cells, and had degenerated. In contrast, in a larva with a the pneumatic duct had disappeared (11 DAH; non-inflated swim bladder at 16 DAH (4.8 mm 5.6 mm SL, Fig. 2(g)). SL), the swim bladder epithelial cells had prolif- erated to fill the lumen, and the pneumatic duct Larval growth and swim bladder inflation under was gone (Fig. 1(i)). two different water surface conditions In larvae of RSG, the digestive tract and the The changes in mean SL and the swim Initial Swim Bladder Inflation in Grouper 353

Fig. 2. Longitudinal cross-sections of red spotted grouper larvae swim bladder (a–d), non-inflated swim bladder 5 days after hatching (DAH), 3.0 mm standard length (SL); (e), non-inflated swim bladder 8 DAH, 3.3 mm SL; (f), inflated swim bladder 9 DAH, 4.3 mm SL; (g), non-inflated swim bladder 11 DAH, 5.6 mm SL. Scale bars indicate 50 µm. Abbreviations: dt, digestive tract; gg, gas gland; nch, notochord; pd, pneumatic duct; sb, swim bladder; sbe, swim bladder epithelium.

bladder inflation rate in LG larvae are shown at 7-15 DAH). in Fig. 3 (a, c). The mean SL of larvae in the Changes in the mean SL and the swim blad- tank with the oil-film-removal treatment was der inflation rate of RSG larvae are shown in 5.3 mm at the end of rearing (15 DAH), sig- Fig. 3 (b, d). The mean SL of larvae at the end nificantly larger than that of larvae (5.1 mm) of rearing (15 DAH) was 6.6 mm and 6.1 mm in in the tank without the treatment (t-test, p < the tanks with and without the oil-film-removal 0.01). Larvae with an inflated swim bladder treatments, respectively. The larvae tended to were first observed at 6 DAH in the tank with be larger in the tank with the oil-film-removal the oil-film-removal treatment, and the smallest treatment than in the tank without, and the inflated larva was 3.6 mm SL. The swim blad- differences in larval size between tanks were der inflation rate increased during 6-9 DAH significant at 7, 8, and 12-15 DAH (t-test, p < to reach 60%-80% in the treatment tank. The 0.01). Larvae with inflated swim bladders were swim bladder inflation rate in the tank without first observed at 5 DAH in both tanks, and the the treatment was about 30% during 9-15 DAH, smallest larva with an inflated swim bladder and remained at this rate throughout the exper- was 2.7 mm SL. The swim bladder inflation iment. The incident of swim bladder inflation of rate in the treatment tank increased during 5-8 larvae in the tank with the oil-film-removal treat- DAH and was 90%-100%. In contrast, the swim ment was significantly higher than that of larvae bladder inflation rate in the tank without the in the tank without the treatment after 6 DAH treatment was about 30% during 5-15 DAH. (chi-squared test, p < 0.05 at 6 DAH and p < 0.01 The incident of swim bladder inflation of larvae 354 T. Iwasaki, S. Mizuta, T. Kogane, J. Satoh, S. Dan and K. Hamasaki

6 ** 8 (a) (b) ** ** ** 5 * 6 **

** 4 4 **

3 2 Standard length (mm) Standard length 2 0 456789101112131415 ** ** ** ** ** 100 (c) Days after hatching 100 (d) ** ** ** ** ** ** 80 80 ** ** ** ** 60 ** 60

(%) ** ** 40 40

20 ** 20 *

Swim bladder Swiminflation bladder rate 0 0 4 5 6 7 8 9 10 11 12 13 14 15 3 4 5 6 7 8 9 101112131415 Days after hatching

Fig. 3. Changes in mean standard length (bar indicates standard deviation) of longtooth grouper (a) and red spotted grouper (b) larvae and swim bladder inflation rates of longtooth grouper larvae (c) and red spotted grouper (d). Black circles with solid line represent data from larvae in the tank with the oil-film-removal treatment; white circles with a broken line represent data from larvae in the tank without the oil-film-removal treatment. Asterisks indicate significant difference between larvae in tanks with and without treatments (＊p < 0.05, ＊＊p < 0.01).

in the tank with the oil-film-removal treatment swim bladder is connected to the digestive tract was significantly higher than that of larvae in by the pneumatic duct, and air is transported the tank without the treatment after 6DAH (chi- to the swim bladder via the pneumatic duct squared test, p < 0.01). (Woolley and Qin 2010). In the present study, development of the swim bladder was exam- Discussion ined histologically in two grouper species. In both species, the swim bladder was connected Fish species with a swim bladder can be to the digestive tract by a pneumatic duct divided into physostomous and physoclistous (Fig. 1(c-f) and Fig. 2(a-d)), and the pneumatic species (Itazawa 1991; Woolley and Qin 2010). duct disappeared soon after the initial inflation Physostomous species have a swim bladder and of the swim bladder. The pneumatic duct of a pneumatic duct that connects the swim blad- non-inflated larvae separated from the swim der to the digestive tract throughout life (Itazawa bladder (Fig. 1(g) and Fig. 2(e)) and eventually 1991; Woolley and Qin 2010). Physoclistous disappeared as the fish grew. In both species, species have a swim bladder that is connected the swim bladder lumen filled with hyperplas- to the digestive tract by a temporary pneu- tic epithelial cells (Fig. 1(i) and Fig. 2(g)) in matic duct only during the early life stage, and non-inflated larvae. There have been no pre- is isolated from the digestive tract thereafter vious studies on the ontogenetic development (Itazawa 1991; Woolley and Qin 2010). It is of the swim bladder in grouper, but our his- believed that physoclistous fish species inflate tological results are similar to those reported their swim bladders by gulping air from the for other physoclistous fish species such as water surface during a finite period when the greater amberjack, Seriola dumerili (Imai et al. Initial Swim Bladder Inflation in Grouper 355

2011), Japanese sea bass, Lateolabrax japonicus inflation rates and lower growth rates in tanks (Makino et al. 1995), and striped trumpeter, without the oil-film-removal treatment than in Latris lineata (Goodsell et al. 1996). tanks with the treatment (Fig. 3a, b). Similar To demonstrate that certain fish species ini- trends have been reported for larvae of several tially inflate their swim bladder by gulping air other fish species, including greater amberjack from the water surface, fish larvae have been (Hashimoto et al. 2012), yellow perch, Perca experimentally reared in tanks in which the flavescens (Czesny et al. 2005), and zebrafish, water surface was separated from atmospheric Danio rerio (Goolish and Okutake 1999). The air by a sealant, such as liquid paraffin, oil low growth rates of larvae that are unable to film, or a mesh screen on the water surface inflate their swim bladders are attributed to (Doroshev and Cornacchia 1979; Kitajima et al. the greater energy demand for feeding and 1981; Chatain and Ounais-Guschemann 1990; maintaining body position against negative Kawabe and Kimura 2008; Imai et al. 2011; buoyancy (Czesny et al. 2005; Hashimoto et al. Tsuji et al. 2016). Initial swim bladder inflation 2012). In addition, larvae of several finfishes in many Perciformes is generally prevented in such as Pacific bluefin tuna, Thunnus orientalis tanks with a sealant because the sealant pre- (Miyashita 2006; Tanaka et al. 2009) and greater vents air gulping by the larvae (Doroshev and amberjack (Teruya et al. 2009), which are nega- Cornacchia 1979; Kitajima et al. 1981; Chatain tively buoyant, tend to sink to the tank bottom, and Ounais-Guschemann 1990; Kawabe and causing mass mortality due to bottoming death. Kimura 2008; Imai et al. 2011; Tsuji et al. 2016). The body densities of LG larvae and juveniles In contrast, tilapia, Tilapia mossambica, can with a non-inflated swim bladder increase with inflate their swim bladder in a tank containing growth, whereas the larvae and juveniles with a sealant, suggesting that tilapia inflate their an inflated swim bladder maintain body density swim bladder through gas exchange in the at nearly neutral buoyancy (Hirata et al. 2009). water (Doroshev and Cornacchia 1979). In the Therefore, promoting larval swim bladder infla- present study, the swim bladder inflation rates tion is an effective way to maintain body density were significantly higher in the tanks with at near neutral buoyancy and prevent mortality the oil-film-removal treatment than in those associated with bottoming death, consequently without. Our results confirmed that these two improving larval survival during seed produc- grouper species are physoclistous and that tion of both grouper species. Furthermore, sev- they inflate their swim bladder by gulping air eral fish species (including LG and sevenband from the water surface. In the present study, grouper) that fail to inflate their swim bladder about 30% of larvae in the tanks without the have been reported to develop morphological oil-film-removal treatment had an inflated swim malformations, particularly lordosis (Chatain bladder at the end of rearing experiment (15 1994; Kitajima et al. 1994; Tsuji et al. 2016; Uji DAH) in both grouper species. A similar phe- 2014). Consequently, inflation of the swim blad- nomenon was reported for blacktip grouper, der is important for normal larval growth and E. fasciatus and the authors suggested that the development, and better survival during mass larvae may have gulped the aeration air or were seed production of grouper. able to gulp air from the water surface despite Our larval rearing results demonstrated the oil film. More research is required to deter- that an oil film on the water surface should be mine how some larvae of LG and RSG inflate removed to promote initial swim bladder infla- their swim bladders in tanks without the treat- tion of LG and RSG larvae. However, oil film on ment to remove oil film from the water surface. the water surface is often stimulated by adding Buoyancy control is an important role of a commercially available lipid oil to larviculture the swim bladder in finfish (Woolley and Qin tanks to prevent water surface tension-related 2010). In the present study, the larvae of both death, one of the biggest causes of mortality grouper species showed lower swim bladder during early stage grouper seed production 356 T. Iwasaki, S. Mizuta, T. Kogane, J. Satoh, S. Dan and K. Hamasaki

(Yamaoka et al. 2000; Tsuchihashi et al. 2003). National Research Institute of Aquaculture, Thus, grouper seed production has the dilemma and the Tamano Laboratory, National Research as to whether to include an oil film or not. The Institute of Fisheries and Environment of timing of initial swim bladder inflation varies Inland Sea for their kind assistance with labo- among fish species and is species-specific ratory work. We also thank R. Miyagi, Tokyo (Woolley and Qin 2010). Therefore, the timing University of Marine Science and Technology of initial swim bladder inflation must be deter- for assistance with the rearing trial and the red mined for each fish species. The present his- spotted grouper larval sample collection. tological results suggested that the pneumatic duct was connected with the digestive tract References and swim bladder during 6-11 and 5-8 DAH in LG and RSG, respectively. These periods Battaglene, S. C. and R. B. Talbot (1993) Effects of salinity match the main periods of initial swim blad- and aeration on survival of and initial swim bladder inflation in larval Australian bass. Prog. Fish-Cult., 55, - der inflation in the cultured larvae (LG, 6 10 35-39. DAH; RSG, 5-8 DAH). Therefore, the oil Chatain, B. and N. Ounais-Guschemann (1990) Improved film on the rearing water surface should be rate of initial swim bladder inflation in intensively removed only during these times to promote reared Sparus auratus. Aquaculture, 84, 345-353. Chatain, B. (1994) Abnormal swimbladder development initial swim bladder inflation, but should be and lordosis in sea bass (Dicentrarchus labrax) and allowed to reform at other times to prevent sea bream (Sparus auratus). Aquaculture, 119, water surface tension-related larval death. 371-379. However, the period of swim bladder inflation Czesny, S. J., B. D. S. Graeb and J. M. Dettmers (2005) Ecological consequences of swimbladder noninflation could vary depending on water temperature, for larval yellow perch. Trans. Am. Fish. Soc., 134, as described for several fish species, includ- 1011-1020. ing greater amberjack (Iwasaki et al., 2011b), Doroshev, S. I. and J. W. Cornacchia (1979) Initial swim red sea bream, Pagrus major (Iseda et al., bladder inflation in the larvae of Tilapia mossambica (Peters) and Morone saxatilis (Walbaum). 1979), and striped trumpeter (Trotter et al., Aquaculture, 16, 57-66. 2004). Therefore, further research is required Fielder, D. S., W. J. Bardsley, G. L. Allan and P. M. to investigate the relationship between water Pankhurst (2002) Effect of photoperiod on growth temperature and the timing of initial swim and survival of snapper Pagrus auratus larvae. Aquaculture, 211, 135-150. bladder inflation in grouper. In the tanks with Fukuhara, O. (1989) A review of the culture of grouper in the oil-film-removal treatment in this study, Japan. Bull. Nansei Reg. Fish. Res. Lab., 22, 47-57. almost all RSG larvae inflated their swim blad- Goodsell, A., D. Wikeley and L. Searle (1996) Histological der but some LG larvae did not. The success of investigation of swim-bladder morphology and inflation in cultured larval striped trumpeter (Latris lin- initial swim bladder inflation by larvae can be eata) (Teleostei, Latridae). Mar. Freshwater Res., 47, affected by other environmental factors, such 251-254. as water temperature, salinity, photoperiod, Goolish, E. M. and K. Okutake (1999) Lack of gas bladder light intensity, aeration rate, water turbulence, inflation by the larvae of zebrafish in the absence of an air-water interface. J. Fish Biol., 55, 1054-1063. and tank color (Battaglene and Talbot, 1993; Hashimoto, H., A. Imai, T. Iwasaki, K. Hamasaki, K. Martin-Robichaud and Peterson, 1998; Fielder Teruya, K. Hamada and K. Mushiake (2012) Feeding et al., 2002; Trotter et al., 2003a, b; Teruya et and growth of larval greater amberjack Seriola al., 2009). Thus, further research is required to dumerili with non-inflated, normal inflated and over-inflated swim bladders. Aquacult. Sci., 60, 99-106 determine the optimal environmental conditions (in Japanese with English abstract). for successful swim bladder inflation in grouper Hirata, Y., K. Hamasaki, K. Teruya and K. Mushiake larvae. (2009) Ontogenetic changes of body density of larvae and juveniles in seven-band grouper Epinephelus septemfasciatus Epinephelus bruneus Acknowledgements and kelp grouper . Nippon Suisan Gakkaishi, 75, 652-660 (in Japanese with English abstract). We thank the staff of the Kamiura Laboratory, Imai, A., T. Iwasaki, H. Hashimoto, Y. Hirata, K. Hamasaki, Initial Swim Bladder Inflation in Grouper 357

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K. Tsumoto, T. Nigou, T. Kasuya, T. Katou, T. Uji, S. (2014) Control of malformations. Nippon Suisan Kawamura, K. Okada, S. Uji and Y. Sawada (2014) Gakkaishi, 80, 996 (in Japanese). Effect of temperature on survival, growth and malfor- Woolley, L. D. and J. G. Qin (2010) Swimbladder inflation mation of cultured larvae and juveniles of the seven- and its implication to the culture of marine finfish band grouper Epinephelus septemfasciatus. Fish. Sci., larvae. Rev. Aquacult., 2, 181-190. 80, 69-81. Yamaoka, K., T. Nanbu, M. Miyagawa, T. Isshiki and A. Tsuji, M., S. Uji, Y. Tsuchihashi, K. Okada, T. Kawamura, Kusaka (2000) Water surface tension-related deaths T. Katoh, T. Kasuya, T. Nigo, T. Hamabe, A. in prelarval red-spotted grouper. Aquaculture, 189, Miyamoto, S. Nakamura, A. Nakano and Y. Sawada 165-176. (2016) Promotion of initial swim bladder inflation Yoseda, Y., K. Teruya, T. Sugaya and S. Sekiya (2006) and effect of initial inflation failure on skeletal malfor- Effects of delayed initial feeding on larval feeding, mation in cultured seven-band grouper Hyporthodus early survival, and growth of red spotted grouper septemfasciatus (Thunberg). Aquacult. Res., 47, Epinephelus akaara larvae. Nippon Suisan Gakkaishi, 1954-1971. 72, 702-709 (in Japanese with English abstract).

クエおよびキジハタの鰾の開腔過程と時期

岩崎隆志・水田翔・小金隆之・佐藤純・團重樹・浜崎活幸

クエおよびキジハタの鰾の開腔に関する組織学的観察および飼育試験を行った。鰾の組織観察ではクエでは 6～ 10日齢（体長3.6 ～ 5.0 mm）に，キジハタでは 5～ 8日齢（体長3.0 ～ 3.3 mm）に鰾と消化管が気管で繋がっている状態が確認された。飼育試験では両種共に水面の油膜除去区の方が油膜非除去区よりも鰾の開腔率が高く，油膜除去区では，クエでは 6～ 9日齢（体長3.2 ～ 3.9 mm）に，キジハタでは 5～ 8日齢（体長2.6 ～ 3.6 mm）にかけて，鰾の開腔率が急激に増加した。以上のことから，クエおよびキジハタはそれぞれ 6～ 10日齢および 5～ 8日齢頃に水面からの空気呑み込みによって鰾に空気を導入し，開腔するものと推察され，鰾の開腔を促進するためには，この期間に飼育水面の油膜を除去することが効果的であると考えられた。