Process and Timing of Initial Swim Bladder Inflation Inlongtooth
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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).