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Rapp. P.-v. Réun. Cons. int. Explor. Mer, 178: 495-500. 1981.

INITIAL SWIM BLADDER INFLATION IN THE LARVAE OF PHYSOCLISTOUS AND ITS IMPORTANCE FOR LARVAL CULTURE1

S. I. DOROSHEV, J. W. CORNACCHIA, AND K. HOGAN Aquaculture Program, University of California, Davis, California 95616, USA

Developmental abnormalities during the formation of a functional physoclistous swim bladder are identified as a major problem in larval culture of several important . The timing of the initial inflation of larval swim bladder is described for Morone saxalilis and Sarolherodon mossambica with respect to larval growth, specific gravity, and yolk sac and oil globule resorption. Swim bladder inflation and establishment of hydrostatic regulation appear at the onset of external feeding and coincide with completed (M. saxalilis) or partial (5. mossambica) yolk sac resorption. Rapid cytomorphosis in the swim bladder epithelium occurs during the process of inflation providing at least morphological evidence of its secretory activity. Two possible modes of the initial swim bladder inflation and gas transport in larvae of physoclistous fish are discussed.

INTRODUCTION study because it can be spawned easily throughout the year under laboratory conditions. High larval mortality in fish with pelagic spawning is a well-known phenomenon in larval culture and has been attributed mainly to an insufficient nutritional MATERIALS AND METHODS regime during the transition to external feeding. In many instances, however, this mortality has been Striped bass eggs were obtained by induced observed long before or far beyond the “point of no spawning (HCG) of adults caught in the Sacramento return” and was associated with a sinking or “descent” River in California, USA, and incubated in of pelagic larvae to the bottom of rearing tanks. We McDonald jars at 18°C. The larvae were reared in 18 propose that this is the result of a malfunction in the liter aquaria at 18.0 ± 0.5° C and fed with brine shrimp larval mechanism of hydrostatic regulation, since one nauplii beginning on the fourth day after hatching. of the major developmental anomalies that appears Tilapia were spawned without hormonal induction in concurrently is the abnormal initial inflation of the aquaria set at 25.5 ± 0.5°C. The eggs were extracted swim bladder usually observed in physoclistous fish from the female’s mouth prior to hatching, and the (Doroshev, 1970; Spectorova and Doroshev, 1976; larvae were held without feeding in 18 liter aquaria and Nash et al., 1977). (25.5 ± 0.1° C) until completion of swim bladder Although there are numerous studies concerning inflation. the adult swim bladder, little is known about the initial Tilapia larvae were sampled daily from hatching inflation of the larval physoclistous swim bladder. The through the sixth day of development; striped bass objective of this study was to investigate the timing larvae were sampled from hatching through the tenth and histological patterns of this event and discuss the day. Larvae were positioned longitudinally under a event’s importance with respect to larval culture. The dissecting microscope, examined for the presence of striped bass (Morone saxatilis) was selected because of gas in the swim bladder, and then photographed. The its importance in the California sport fishery and the yolk sac and oil globule areas as well as the body length present lack of an effective larval rearing technique for were measured from the negatives using an Image this fish. In contrast, tilapia (Sarotherodon Analyzing Computer (Quantimet 720). mossambica) is not a pelagic spawner, but was used for Development of the swim bladder was studied histologically during the period of inflation. Tilapia 1 Supported by a Grant from the Agency for International larvae were preserved in either Bouin’s or Karnovsky’s Development, U. S. State Department (A1D/DSAN-G0102). fixatives, and striped bass larvae were fixed in 2% 496 S. I. Doroshev, J. W. Cornacchia, and K. Hogan

buffered formaldehyde. Serial longitudinal sections of The process of swim bladder inflation begins about 6/um were stained with Delafield’s haematoxylin and the fourth day after hatching in tilapia, with 50% of the eosin. Plastic sections (0.5/um) were made from swim yolk sac remaining (Fig. 2). The larvae become bladders of the striped bass and stained with toluidine neutrally buoyant by the seventh day, which coincides blue. with the completion of yolk sac resorption. Initiation The specific gravity of striped bass larvae was esti­ of inflation cannot be observed easily in vivo due to mated daily. Ten anesthetized larvae were placed in a heavy pigmentation. Growth in length is linear saline solution and the number that sank was recorded (and expressed as a percentage). This was repeated for several groups over a range of salinities. By using least YOLK SAC (A) LENGTH OIL GLOBULE (mm) squares linear regression, the percentage that sank was (mm2) Inflation < %) 56 71 expressed as a linear function of specific gravity, and a 1.0 - 1«. specific gravity was calculated at which 50% of the 8 larvae were predicted to sink. The percentage of larvae . - with inflated swim bladders was determined in each .6 - sample. .4--

2 - RESULTS -i 1------1— i------1— h------1 y The larvae of the striped bass begin inflating their swim bladders at the completion of yolk sac resorption Sb INFLATION on the fifth day after hatching, which coincides with SPECIFIC GRAVITY (B) (X) the initiation of feeding (Fig. 1 A). Despite the fact that 1.008- ■ x 8 0 active feeding is established, growth in length ceases 1.007- - -60 between days five and eight. Resorption of the oil 1.006- - 1«. X- -40 globule is slow compared with that of the yolk sac, and / 1.005 only 55% of its original amount is resorbed by the 2o- --20 tenth day. The estimated specific gravity of the larvae -X-+- -t- -4- increases from hatching, reaching a maximum during H 1 T ~ t 8 9 10 AGE (DAYS) days five and six (Fig. IB). Figure I. (A) Resorption of the yolk sac (I) oil globule (2) and The primordial swim bladder in the striped bass is growth in length (3) of the larvae of the striped bass. Bars enclose observed easily in vivo beginning the third day after 95% confidence interval. (B) Estimated specific gravity (I) and hatching and is enlarged considerably the fifth day the percentage of striped bass with inflated swim bladders (2). (Fig. 3, No. 1). In those larvae that have not inflated, the swim bladder appears under the microscope as a YOLK SAC LENGTH flattened sac (Fig. 3, No. 2). The inflated condition is Imm^l (mm) characterized by a light-refractive bubble (Fig. 3, No. 7T Sb Inflation T 12 4) readily observable without magnification. As

shown in Figure 1, the percentage of larvae with 6 -- --11 inflated swim bladders increase from 14 to 20% on the fifth day, to 70% on the eighth day, and to 80% on the 5-- --1 0 tenth day. Prior to inflation, the ventral wall of the swim bladder is lined with a prominent glandular epithelium, while the dorsal aspect is composed of 3-- --8 cuboidal cells (Fig. 3, No. 2 and 3). At the initiation of inflation, the glandular tissue becomes vacuolated and 2 - --7 transforms into a cuboidal epithelium. The dorsal lining apparently flattens until the majority of the --6 bladder wall is composed of a squamous epithelium (Fig. 3, No. 5). In larvae that fail to inflate, the development of the swim bladder is arrested at a stage resembling that prior to inflation, and with time, the AGE (DAYS) glandular tissue degenerates (Fig. 3, No. 6). A Figure 2. Resorption of the yolk sac ( 1 ) and growth in length (2) of pneumatic duct is present during inflation. the larvae of tilapia. Bars enclose 95% confidence interval. Swim Bladder Inflation in Larvae 497

%

ri#i •**: % *

Figure 3. Larval swim bladder of the striped bass: (1) Day 5, posthatch, (30X); (2) Day 5 (300X); (3) Day 6, 0.5/nm section, toluidine blue ( IOOOX); (4) Day 5, dark field (30X); (5) Day 6 (3000X); (6) Day 23 (350X). Symbols: sb — swim bladder; og — oil globule; i — intestine; n — notochord; 1 — lumen; ge — glandular epithelium; v — vacuole; cp — capillary; pd — pneumatic duct. through the sixth day. contiguous with the basal portion of the columnar Major cytoarchitectural modifications occur during cells. By the fifth day, when cytomorphosis is inflation in the swim bladder epithelium of tilapia. completed, the columnar cells transform into a Prior to inflation on the fourth day, the bladder wall is squamous epithelium as the lumen swells with gas composed of one row of large columnar cells that line a (Fig. 4, No. 2, 3, and 4). An eosinophilic material lines narrow lumen (Fig. 4, No. 1). A highly developed the lumen of the swim bladder during initial inflation capillary bed is opposed ventrally to the swim bladder, and in swim bladders that did not inflate successfully. while numerous capillaries surround and are Tilapia with uninflated swim bladders are rare by 498 S. I. Doroshev, J. W. Cornacchia, and K. Hogan

Figure 4. Swim bladder inflations in the larvae of tilapia: ( 1) (2) (3) Day 4, posthatch, (200X); (4) Day 5 (70X); (5) Day 6(200X); (6) Day 20 (200X). Symbols: ce — columnar epithelium; arrows indicate eosinophilic material, see Fig. 3 for the explanation of other symbols. Swim Bladder Inflation in Larvae 499 the sixth day after hatching (<1%). When examined The histological description of the larval histologically, they resemble the stage prior to physoclistous swim bladder provides the anatomical inflation (Fig. 4, No. 5); the columnar cells are less basis for at least two modes of inflation. A temporary elongate, slightly amorphic, and the nuclei appear pneumatic duct exists during inflation in many faintly basophilic. The epithelium of uninflated swim physoclistous larvae and may act as a passageway for bladders becomes highly necrotic by the twentieth day gas transfer from the digestive tract to the swim posthatch (Fig. 4, No. 6). There is no evidence of a bladder (Johnston, 1953; Duwe, 1955; Schwarz, 1971). pneumatic duct in the larvae during the stages Ledebur and Wunder (1938) prevented inflation in described. Gasterosteus aculeatus by denying larvae access to the surface: they concluded that larvae accomplished inflation by gulping atmospheric gas. The pneumatic duct in physoclistous larvae eventually occludes and DISCUSSION degenerates; however, little is known about the timing The timing of swim bladder inflation demonstrates of this event. The region of occlusion is located in the the significance of this event during the transition from mucosa of the intestine in largemouth black bass yolk resorption to external feeding. Inflation began (Johnston, 1953) and haddock (Schwarz, 1971). Little when yolk reserves were exhausted and specific gravity is known about the functional significance of this had increased. The development of a functional larval structure in physoclistous larvae. The larvae of two swim bladder provides the capability for hydrostatic cichlids, Sarotherodon mossambica and Hemichromis regulation and the ability to overcome increasing bimaculata, lack a pneumatic duct (McEwen, 1940; specific gravity. The achievement of neutral buoyancy Doroshev and Cornacchia, 1979). greatly reduces the energetic cost of swimming and Other evidence suggests a second possible mode: improves predatory efficiency (Hunter, 1972). This is that physoclistous larvae may secrete gas into the swim particularly important for pelagic larvae, such as the bladder during initial inflation. The pronounced striped bass, in facilitating the initiation of feeding. glandular epithelium observed in tilapia, striped bass, The period of development following yolk sac largemouth black bass (Johnston, 1953), and the resorption is characterized by a growth plateau for haddock (Schwarz, 1971) is a transitory larval many marine pelagic larvae as well as for the striped structure and appears secretory about the time of swim bass (Farris, 1959). During swim bladder inflation and bladder inflation. The cytomorphological changes of the transition to exogenous nutrients, yolk sac reserves the epithelium during inflation resemble are not available for further growth; and apparently, morphological and histochemical changes that occur only maintenance energy requirements are met in the hypersecreting gas gland of the physoclistous (Warren and Davis, 1967). Even with an adequate adult (Copeland, 1969). In addition, the cichlids nutritional regime, one would predict that a delay or Hemichromis bimaculata and tilapia inflated their complete failure of inflation would further depress swim bladders although they were denied access to growth, ultimately producing inviable larvae. For atmospheric gas, and inflation appeared to be example, in the demersal spawner (Gasterosteus depressed in tilapia under hypoxic conditions aculeatus) growth was depressed considerably in (McEwen, 1940; Doroshev and Cornacchia, 1979). artificially prevented from inflating their The above evidence suggests that the glandular tissue swim bladders (Ledebur and Wunder, 1938). The oil is secretory and that inflation may be accomplished by globule provides a major source of energy reserve after gas secretion in some physoclistous larvae. completion of yolk sac resorption, and the low specific Clearly, it would be valuable to understand the gravity of the oil may aid in larval flotation (Rogers, mechanism of larval swim bladder inflation and the 1978). effects of culture methods on this process. In many The timing of initial swim bladder inflation in cases, pelagic larvae reared under artificial conditions species with demersal eggs is somewhat different from have demonstrated highly abnormal inflation. For that in pelagic spawners. The larvae of tilapia and example, nearly an entire population of largemouth black bass began inflation when a maeoticus either failed to inflate or developed considerable portion of the yolk sac remained excessive inflation (Spectorova and Doroshev, 1976). (Johnston, 1953), however, in both these species and in Nash et al. (1977) reported excessive inflation in the the striped bass, a pelagic spawner, neutral buoyancy larvae of the grey mullet (Mug/7 cephalus). The appears to be attained near the end of yolk sac percentage of uninflated swim bladders in the larvae of resorption. It is interesting that abnormal inflation striped bass may be as high as 90%, but variations of generally has not been reported in the larvae of 20-70% are encountered normally (Doroshev, 1970). demersal spawning fish. This anomaly in the development of pelagic larvae 500 S. I. Doroshev, J. W. Cornacchia, and K. Hogan may be common in artificial rearing systems and might Valenciennes. Aquaculture 8(4): 365-370. explain the second peak of larval mortality described Hunter, J. R. 1972. Swimming and feeding behavior of larval for M ugil curema (Houde et al., 1976), Dicentrarchus anchovy Engraulis mordax. Fish. Bull., U. S., 70(3): 821-838. labrax (Girin, 1975), and Mugil cephalus (Nash and Johnston, P. M. 1953. The embryonic development of the swim Kuo, 1975). Further work is in process to elucidate bladder of the large mouth black bass Micropterus salmoides both the mechanism of larval inflation and the salmoides (Lacepede). J. Morphol., 93: 45-67. Ledebur, J. T. Von, and Wunder, W. 1938. Beitrage zur Physiologie environmental conditions responsible for inducing der Schwimmblase der Fische. IV. Beobachtungen an Stichlingen swim bladder abnormalities during larval culture. die ihre Schwimmblase nicht mit Gas füllen Konnten. Z. vergl. Physiol. Bd., 25(5): 149-155. McEwen, R. S. 1940. The early development of the swim bladder REFERENCES and certain adjacent parts in Hemichromis bimaculata. J. Morphd., 67(1): 1-57. Copeland, D. E. 1969. Fine structural study of gas secretion in the Nash, C. E., and Kuo, C-M. 1975. Hypothesis for problems physoclistous swim bladder of Funclulus heleroclilus and Gadus impeding the mass propagation of the grey mullet and other callarias and in the euphysoclistous swim bladder of Opsanus tau. finfish. Aquaculture 5(2): 119-134. Z. Zellforsch., 93: 305-331. Nash, C. E., Kuo, C-M„ Madden, W. D.,and Paulsen, C. L. 1977. Doroshev, S. I. 1970. Biological features of the eggs, larvae and Swim bladder inflation and survival of Mugil cephalus to 50 days. young of the striped bass, Roccus saxalilis Walbaum. J. Aquaculture 12(1): 89-94. Ichthyol., 10(2): 235-248. Rogers, B. A. 1978. Temperature and the rate of early development Doroshev, S. 1., and Cornacchia, J. W. 1979. Initial swim bladder of striped bass, Moronesaxatilis (Walbaum). Ph.D. Dissertation, inflation in the larvae of Tilapia mossambica (Peters) and University of Rhode Island, 193 pp. Morone saxalilis (Walbaum). Aquaculture 16: 57-66. Schwarz, A. 1971. Swim bladder development and function in the Duwe, A. E. 1955. The development of the gas bladder in the green haddock, Melanogrammus aeglefinus L. Biol. Bull., 141: sunfish, Lepomis cyanellus. Copeia 2: 92-95. 176-188. Farris, D. A. 1959. A change in the growth rate offour larval marine Spectorova, L. V., and Doroshev, S. I. 1976. Experiments on the fishes. Limnol. Oceanogr. 4: 29-36. artificial rearing of the black sea (Scophthalmus maeoticus Girin, M. 1975. Point des technique d’elevage larvaire du bar. maeoticus). Aquaculture 9(3): 275-286. Contrib. No. 426, Dept. Scient, du Centre Oceanol. de Bret. Warren, C. E., and Davis, G. E. 1967. Laboratory studies on the CNEXO: 133-141. feeding, bioenergentics, and growth of fish. In The biological Houde, E. D., Berkeley, S. A., Klinovsky, J. J., and Schekter, R. C. basis for freshwater fish production. Ed. by S. D. Gerking. 1976. Culture of larvae of the white mullet, Mugil curema Blackwell Scientific Publications, Oxford, pp. 175-214.