European Journal of Scientific Research ISSN 1450-216X Vol.38 No.2 (2009), pp.296-305 © EuroJournals Publishing, Inc. 2009 http://www.eurojournals.com/ejsr.htm Development of Morphology in Hatchery-Reared Rutilus Frisii Kutum Larvae Mahdieh Jafari Department of Aquaculture, Faculty of Agriculture, University Putra Malaysia 43400 UPM Serdang, Selangor, MALAYSIA Mohd Salleh Kamarudin Department of Aquaculture, Faculty of Agriculture, University Putra Malaysia 43400 UPM Serdang, Selangor, MALAYSIA E-mail: msalleh@agri.upm.edu.my Tel: +603-89464171, Fax: +603-8946 4146 Che Roos Saad Department of Aquaculture, Faculty of Agriculture, University Putra Malaysia 43400 UPM Serdang, Selangor, MALAYSIA Aziz Arshad Department of Aquaculture, Faculty of Agriculture, University Putra Malaysia 43400 UPM Serdang, Selangor, MALAYSIA Shahrbanoo Oryan Department of Physiology, Faculty of Biology. Teacher Training University Karaj, Tehran, Iran. P.O. Box 31979- 37551 Mahmoud Bahmani Dadman International Sturgeon Research Institute Rasht, IRAN P.O. Box.14635-3464 Abstract Most fish are poorly developed at hatching. They undergo important functional and morphological changes during the early larval period. This study was conducted to monitor the morphological changes of Caspian kutum Rutilus frisii kutum larvae in early life stages. Fertilized kutum eggs were incubated at 16-19ºC in 8 l glass incubators for 9.5 days. At first feeding (3 days after hatch) larvae were fed two times per day with egg yolk for 5 days, and then Artemia nauplii and egg yolk until day 30. The juveniles were then transferred to an outdoor fish hapa nets and further reared until day 60. Larval development, growth, and some morphological changes were described from day 0 to end of experiment. The results showed that there were 3 main stages and 18 sub-stages of ontogenetic development in Caspian kutum from hatching to juvenile stage. The most important change in these stages was change from endogenous to exogenous feeding. During post hatch development, the various organs gradually differentiated and became identifiable. Development of Morphology in Hatchery-Reared Rutilus Frisii Kutum Larvae 297 Keywords: Early ontogenic development; Rutilus frisii kutum; Morphological changes; The Caspian Sea 1. Introduction Marine fish larvae undergo major morphological and cellular changes during the first months of life. The sensitivity of larvae to low food intake, especially following yolk absorption, can significantly influence their chances of survival (McFadzen et al., 1994). In relation to this, a study of morphological characteristics provides simple tools for detecting unfavorable culture conditions during larval development (Dettlaff et al., 1993). The greatest advantage of morphological indices is attributable to the short processing time, lost cost and ease of attainability (Martin et al., 1987; Theilacker, 1978). Morphological deformities occur in a wide range of aquaculture fish species including Dicentrarchus labrax (Barahona-Fernandes, 1982; Daoulas et al., 1991; Koumoundouros et al., 2001a), Pagrus major (Hattori et al., 2003; Kihara et al., 2001), Diplodus sargus (Sfakianakis et al., 2003), Dentex dentex (Koumoundouros et al., 2001b), Salmo gairdneri (Dabrowski, 1990), Chanos chanos (Gapasin et al., 1998), Solea senegalensis (Gavaia et al., 2002) and Oreochromis niloticus (Soliman et al., 1986). These deformities are often associated with the intensive hatchery production (Andrades et al., 1996; Barahona-Fernandes, 1982) and are a significant problem for aquaculture production (Andrades et al., 1996; Daoulas et al., 1991). Morphological characteristics of early life stages have been described for many fishes including Lepisosteus osseous, L. platostomus and L. oculatus (Abdoli, 1999).The Caspian kutum is a migratory anadromous fish which spawns on aquatic weeds, graveled and sandy substrates in rivers and lagoons between March to April (Azari Takami, 1990; Emadi, 1979). Caspian kutum is an important commercial fish species in the Caspian Sea in Iran. The sharp decline in its annual catch observed in 1970s and early 1980s due to declining natural population and other factors (Ghaninejad et al., 2007) had prompted the Iranian government to launch its restocking project in 1984. The kutum fry are now routinely produced in hatcheries. The stock conservation program for kutum which started in 1982 with the release of fingerlings of the spring form of this species into rivers that has significantly affected kutum stocks. At present the annual release of more than 150 million kutum fingerlings plays a vital role in the rehabilitation of kutum stocks (Caspian Environment. org., 2007). The information on the morphological development and changes of Caspian kutum, Rutilus frisii kutum larvae is still scarce. This study attempted to describe the morphological development of the kutum larvae. Some of morphological characteristics could be used as indicators of growth and nutritional condition in Caspian kutum larvae during early life stages and would be useful for the kutum hatchery operators. 2. Materials and Methods Fertilized eggs were collected from a pair of kutum broodstock and incubated at 16-19ºC in an 8l glass incubator with a 13h light:11h dark photoperiod regime. Upon hatching (9 days after fertilization), larvae were transferred into three 20 L aquaria at 50 larvae per liter. The kutum larvae began their first feeding on 3 days after hatching (DAH). The larvae were fed ad libitum twice a day with egg yolk for the first 5 days and followed with Artemia nauplii and egg yolk for another 25 days, After 30 days, the juveniles were transferred to an outdoor fish hapa nets (1 m X 1 m X1 m) and reared for another 30 days fed on pond natural food. Water temperature and dissolved oxygen were daily monitored while pH was estimated twice a week. The ranges of rearing water pH, dissolved oxygen and temperature during the study period were 7.9-8.5, 7.5-8.2 mg/l and 16.7-18.3ºC, respectively. During the study, fifteen healthy larvae or juveniles were sampled daily from hatching to 7 DAH, every 2 days to 15 DAH and then every 5 days until 60 DAH. The total length (TL) to nearest 0.1 mm of fish was measured using an ocular micrometer. All measurements were done using the Nikon Alphaphot YS microscope. Sequences of vertebral development were hand drawn. China myomere numbering was used as described by Siefert(1969). Ten larvae were also sampled and 298 Mahdieh Jafari, Mohd Salleh Kamarudin, Che Roos Saad, Aziz Arshad, Shahrbanoo Oryan and Mahmoud Bahmani preserved in 10% formalin, dehydrated in graded series of ethanol and embedded in paraffin, cut into 6-8 μm , air dried, and stained with hematoxylin-eosin (H&E) following Pearse (1985). 3. Results There were 3 main stages (Protolarval, Meoslarval and post larval) and 18 sub-stages of fish observed between hatching and the juvenile stage when the lateral line scales were completed (Fig. 1). Figure 1: Total Length (mm) at different stages of Post – hatch development of Rutilus frisii kutum larvae 35 25 20 15 10 Juvenile Squamation Pelvic - fin - formation Anal fin formation pelvic fin-bud Dorsal- fin - formation Two- chamber- gas bladder yolk absorption one- chamber- gas bladder Gas- bladder- emergence Melanoid- eye Gill filament Xantic- eye Gill- arch Rudimentary-pectoral- fin Hatching 0 Notochord- tip- lifting 5 dorsal- fin- differentiation Total body Lenght (mm) 30 Larval Stages During these stages, the fish underwent a change from endogenous to exogenous feeding. During post hatch development, the various organs gradually differentiated and became identifiable (Table 1). Generally these characters were seen in kutum larvae: Table 1: Primary diagnostic characteristics in the early development of Rutilus frisii kutum larvae Comparative character Stage A. Protrusion on the head Otic capsule to heart pulsation B. Caudal vein C. Snout shape and pigmentation of the dorsal surface of the head Hatching to gill filaments Gas bladder emergence to yolk absorption Differentiation of dorsal fin to juvenile D. Deep head pigment pattern One - chamber gas bladder to two chamber gas bladder Shape Development of Morphology in Hatchery-Reared Rutilus Frisii Kutum Larvae 299 Gas bladder emergence to yolk absorption E. Pigment around the rudimentary pectoral fins One - chamber gas bladder to differentiation of dorsal fin Notochord tip lifting to anal fin formation F. Pigment on caudal fin G. Pigmentation of the preanal and anal fin folds One - chamber gas bladder to differentiation of dorsal fin One - chamber gas bladder to two chamber gas bladder Pelvic fin bud to squamation H. Anal fin shape Anal fin formation to juvenile 3.1. Protolarval stage Stage 1: Hatching stage Hatching occurred at 216 hours (9 DAF) and total length mean was 6.0 ±0.26 mm. The body was transparent and the heart was at the top of the anterior edge of the yolk sac. The yolk sac was a lengthened tear drop shape with a bright- blue color on the posterior end. The posterior aorta, the main vein, the caudal vein, and myomere blood vessels were differentiated. The blood color was apricotyellow. The eyes were transparent. Below the eye, there was a triangular black spot(Fig.2). Figure 2: Photo micrograph of eye Rutilus frisii kutum embryo in hatching stage (H&E. Bouin’s and formalin fixation ×400). The pigment layer (PL) of the eye extends to the lens and Gill filaments (GF) are forming. The branchiostegal membrane (BM) covers the first pair of gills, L, lens; PlL, plexiform layer, the retina (Re) of the eye is well-developed. The otic capsule was relatively small. The myomere numbering was 8+18+13=39. The larvae remained on their side at the bottom of the container most of the time, but occasionally they darted to the surface ( Fig. 3.1). Stage two: Rudimentary - pectoral- fin stage Total length was 6.3±0.18 mm at 1 DAH. The rudimentary pectoral fin was crescent shaped and located below myomeres two or three. The heart moved slightly ventrally and was located anterior to 300 Mahdieh Jafari, Mohd Salleh Kamarudin, Che Roos Saad, Aziz Arshad, Shahrbanoo Oryan and Mahmoud Bahmani the middle of the yolk sac. The cuvierian duct appeared at the top of the yolk sac. The caudal vein was large and wide. The black spot below the eye became oval. The heartbeat was 90 items per minute. The larva usually rested at the aquarium bottom but occasionally swam (Fig. 3.2). The myomere numbering was 8+19+13=40. Stage three: Gill - arch stage Total length was 6.5 ±0.19 mm at 1.5 DAH. The body color was butter yellow. The head extended straight out from the body. The indentation of the mouth appeared. The pectoral fin enlarged. The blood vessels were clearly evident. Ventral to the otic capsule and posterior to the eye, four-gill arches appeared. The caudal vein expanded and was apricot-yellow. The operculum extended. The diameter of the eye was about 0.40 ±0.015 mm and the myomere numbering was 8+19+14= 41 (Fig. 3.3). Stage four: Xanthic – eye stage Total length was 6.84 ±0.16 mm at 2 DAH. The yellow pigmentation of the eye appeared. The caudal vein was thick and the color was loquat-yellow. The mouth was slightly open. The indention of the mouth appeared. The rudiments of the gill filaments appeared. The myomere numbering was 8+19+14=40 (Fig. 3.4). Stage five: Gill filament stage Total length was 7.2 ±0.12 mm at 2.5 DAH. The gill filaments were complete and the operculum appeared. The blue color of the posterior end of the yolk sac disappeared. The head and body straightened. Melanophores on the front edge of eyes increased. The caudal vein and the cuvierian duct narrowed (Fig. 3.5). Stage six: Melanoid – eye stage Total length was 7.42 ± 0.20 mm at 3 DAH. Black pigment appeared in the eye at the top front and later extended all the way around (Fig. 3.6). The black spot below the eye disappeared. The operculum was clearly evident. The rudimentary cleithrum appeared. The yolk sac became narrow and elongated. Two or three stellate melanophores appeared on the anterior portion of the yolk sac. The mouth moved forward and was located below the front margin of the eye. The caudal vein became thinner. The opening mouth in this stage was a main morphological change during fish larvae, therefore the first main stage ended in this stage. 3.2. Mesolarval Stage Stage seven: Gas bladder – emergence stage Total length was 8.0 ±0.17 mm in 6 DAH. The gas bladder began to appear. The yolk sac continued to elongate. The gut was narrow and continued to extend, the mouth moved forward, and the gill filaments grew longer. The otic capsule enlarged to almost the diameter of the eye. The yolk sac remained only as a narrow strip. The pectoral fins extended; near the insertion of the fin there was one stellate melanophore. From the dorsal surface of the gas bladder, there were two lines of pigment between the gut and myomeres. The pigment lines extended to caudal end of the vertebral column. The body was light-yellow. The myomer numbering was 9+19+14=42 (Fig. 3. 7). Stage eight: one - chamber - gas - bladder stage Total length was 8.3 ± 0.26 mm in 10 DAH. The inside of the gut appeared wavy, gut folds appeared, and the exogenous feeding began. The yolk sac was mostly disappeared as this was the transitional period of both endogenous and exogenous feeding. The mouth was terminal. The body was lemonyellow, covered by many melanophores; on the body side, there were four lines of pigment and another one along the gut. Many stellate melanophores were visible in the dorsal view of the head. The stellate melanophore on the dorsal fin enlarged. Many melanophores were grouped together inside the deep posterior portion of the head. From the dorsal view these melanophores appeared vase-shaped (Fig. 3.8). There were a few melanophores below the caudal end of the notochord. At this time the fish could swim normally. The myomer numbering was10+19+14=42. Development of Morphology in Hatchery-Reared Rutilus Frisii Kutum Larvae 301 3.3. Post larval stage Stage nine: Yolk absorption stage Total length was 9.8 ±0.25 mm at 20 DAH (days after hatch) (Fig. 3.9). The yolk sac was completely exhausted. The gut folds were more developed. In the nature, the fish began feed on plankton. The anterior margin of the head was flattened. The pigment on the body side and dorsal head surface increased, but the deep pigment inside did not change (Fig. 4). The stellate melanophores on the pectoral fin numbered two or three. There were two or three stellate melanophores on the lower caudal fin fold. Figure 4: Pigmentation form on the head and surface body of Rutilus frisii kutum larvae Scal bar: 1mm Stage ten: Dorsal–fin–differentiation stage Total length was 13 ±0.20 mm at 25 DAH. The dorsal margin of the anterior part of the dorsal fin fold had a sigmoid shape (Fig. 3.10). There were stellate melanophores and some small melanophores on the lower part of the caudal fin; with the naked eye these together appeared as a gray spot. Myomere numbering was 11+18+14= 43. Stage eleven: Notochord-tip-lifting stage Total length was 16 ±0.25 mm at 30 DAH (Fig. 3.11). The anterior portion of the dorsal had a pronounced triangular from, which was the rudimentary dorsal fin. The end of the vertebral column curved upward. The caudal fin fold began to differentiate; the edge was crenulated. The anal fin fold began to differentiate. The caudal vein was still visible and was loquat-yellow. The pigmentation on the surface of the head between the eyes enlarged. Pigmentation also appeared on the maxillary. The anterior margin of the dorsal fin fold moved backward. The operculum enlarged and covered the entire gill chamber. Myomere numbering was 12+17+15=44. Stage twelve: Two – chamber – gas bladder stage Total length was 20±0.26 mm at 35 DAH. The anterior gas bladder appeared and formed a ball shape (Fig. 3.12).The posterior gas bladder moved backward, lengthened, and tapered towards the rear. On the rudimentary dorsal fin and anal fin, there were six seven initial rays and several stellate melanophores. The caudal fin continued to differentiate; 16 rays were apparent. The vertebras were clearly visible. Myomere numbering was 13+16+15=44. Stage thirteen: Pelvic-fin-bud stage Total length was 22.2 ±0.26 mm at 40 DAH. The pelvic fin bud appeared in the mid part of the preanal fin fold (Fig. 3.13).The rudimentary dorsal fin grew larger; the numbers of rays and melanophores increased. The total fin fold shrank. The numbers of rays and melanophores on the anal fin fold increased. The caudal fin was forked; between the rays there were melanophores. The mandible and maxillary were developed. The mouth was terminal. The anterior gas bladder enlarged and became oval. Below the end of the urostyle, there was a large and obvious stellate melanophore. Myomere numbering was 13+17+15=44. 302 Mahdieh Jafari, Mohd Salleh Kamarudin, Che Roos Saad, Aziz Arshad, Shahrbanoo Oryan and Mahmoud Bahmani Stage fourteen: Dorsal-fin-formation stage At the beginning of this stage (Fig. 3.14), total length was 24.12 ±0.22 mm at 45 DAH. The dorsal fin was separated from the dorsal fin fold (ray numbering= ii, 7). The anal fin extended and the number of rays increased. The pelvic fin bud enlarged. The dorsal fin fold and anal fin fold shrank to the caudal peduncle. The ribs and vertebral processes began to appear. The myomeres further developed from a single chevron shape to chevrons both above and below the lateral line. The operculum became thicker. Myomere numbering was 13+18+15=46. Stage fifteen: Anal-fin-formation stage Total length was 25.7 ±0.37 mm at 50 DAH (Fig. 3.15). The anal was formed (ray numbering=iii, 8). The anal fin fold continued to shrink. The caudal fin was thoroughly developed. The pelvic fin lengthened and the pre anal fin fold shrank. Stage sixteen: Pelvic-fin-formation stage Total length was 28 ±0.71 mm at 55 DAH. The pelvic fin formed. The preanal fin fold remained only in a narrow strip. The pectoral fin continued to develop; the whole body was pigmented (Fig. 3.16). Stage seventeen: Squamation stage Total length was 30 ±0.79 mm at 60 DAH (Fig. 3.17). The morphology was similar to that of an adult fish; very little remained of the pre anal fin fold. Lateral line scales and several rows of the back. The growth of scales depended on environmental conditions. Stage eighteen: Juvenile stage Total length was 33 ±1.48 mm (Fig 3.18). This stage lasted from 62 to 70 DAH. Squamation was completed. The lateral line scales numbered 55-57. Other than the larger eyes, the fish resembled an adult fish. Figure 3: The stages of post- hatch development of Rutilus frisii kutum. (1)Hatching,(2) Rudimentary – pectoral- fin,(3) Gill –arch,(4) Xanthic – eye,(5) Gill filament,(6) Melanoid – eye,(7) Gas bladder – emergence,(8) one – chamber – gas- bladder,(9) Dorsal – fin – differentiation,(10) Dorsal – fin – differentiation,(11) Notochord- tip- lifting,(12) Two – chamber – gas –bladder,(13) Pelvic- fin – bud,(14) Dorsal – fin- formation,(15) Anal fin formation,(16) Pelvic – fin- formation,(17) Squamation,(18) Juvenile Development of Morphology in Hatchery-Reared Rutilus Frisii Kutum Larvae 303 4. Discussion Early life history of fishes is a complex phenomenon of growth and differentiations. The results of this study should contribute to a better understanding of the biology of kutum larvae. The new information can be used to explain some aspects of the early life history at hatchery conditions and help develop better larval rearing techniques in the hatchery. Results showed the anterior margin of the dorsal myomeres increased gradually, and central myomere counts reduced correspondingly. The increase in total number of myomeres during development resulted from an increase in post anal myomeres. During the developmental stages from the melanoid eye stage to two – chamber gas bladder, there were large inter connected patterns of stellate melanophores close to the otic capsule and gill arch in the inner part of the head, and anterior and dorsal to the pectoral fin base, viewed dorsally through the transparent top of the head, these pigments made patterns that were diagnostic to species. The pigment pattern on the deep head of kutum resembled heart shape. This character in the kutum is special and generally different to the other carp. The presence, absents, or abundance of stellate melanophores on the loose of the rudimentary pectoral fin of larvae and juvenile also could be used as a characteristic to identify the species. At the gas – bladder – emergence- stage kutum had one melanophore on the pectoral fin. From the one – chamber- gas bladder stage to the dorsal – fin differentiation stage, the number of stellate malanophores on the rudimentary pectoral fin increased to two or three. From the one- chamber- gas bladder stage to the dorsal – fin– differentiation stage, the pigment on the caudal fin between species had different characteristic. In some of them caudal could be seen by the naked eye and proved to be a rapid and efficient way to identify the species. There was a small grouping of melanophores on the anterior ventral portion of the caudal fin of the kutum. With the naked eyes, this grouping appeared as a large black point. The caudal vein was located along the base of the postanal fin fold. It was the most diagnostic morphological characteristic of the larval circulatory system over the period from the rudimentary – pectoral- fin stage to the gas- bladder- emergence stage. The caudal vein of kutum was the obvious and straight with the end looked like thin (narrow). At the gas- bladder- emergence stage, the larva could orient itself and swim normally. It was possible to observe the pigment distribution on the dorsal surface of the head as well as the snout shape to identify the species. After the pelvic- fin bud- stage, the head pigmentation did not change, expect there was some development of pigment on the dorsal surface of the head and between the eyes of the kutum. By the juvenile stage, the scales and fins were similar in shape to the adult fish. During and after the one – chamber – gas - bladder stage, some stellate melanophores appeared on the preanal and anal fin folds of kutum. This was an important and stable characteristic to identify the species. The stellate melanophores were large and had many branches. 304 Mahdieh Jafari, Mohd Salleh Kamarudin, Che Roos Saad, Aziz Arshad, Shahrbanoo Oryan and Mahmoud Bahmani The abundance of melanophores increased gradually in the anterior direction on the preanal fin fold; melanophores were distributed over the posterior half of the preanal fin fold. The stellate melanophores were small and darkly pigmented. In contrast, the melanophores of the anal fin folds of kutum appeared at one - chamber - gas - bladder stage and gradually formed a dark wavy line. At hatching stage, the kutum digestive tract was a straight tube of undifferentiated cells. The mouth was closed and esophagus unconnected with the intestine. During the endogenous feeding period, a very fast development of digestive tract was observed. Studies of larval development of bony fishes show that yolk sac material absorption is accompanied by an intense development of digestive system(Buddington et al., 1985). In this species, the mouth opened on the third day post hatching and the fish started active feeding but yolk sac complete absorption was observed 17 days later, 20 day of life. The Caspian kutum larvae were able to pinocytose exogenous food before the full yolk sac resorption. From the beginning of mixed feeding (3- 4 DAH), the larval intestinal valve created the intestinal valve plays an important role at early larval stage, preventing enzyme escape from the intestine. Acknowledgement The authors wish to thank of The Head and staffs of The Fish Disease Section of Dr. Dadman International Sturgeon Research Institute. Mr. Mohammad Hossein Tolouei of Shahid Dr. Beheshti Fish Hatchery and Mr. Moghaddasi of Shahid Rajaii fish Culture Center. Refferences [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] Abdoli, A. 1999. The Inland Water Fishes of Iran, Natural and Wild Life Museum of Iran, (in Persian), Tehran, Iran. 198–200. Andrades, J.A., J. Becerra, and P. Fernandez-Llebrez. 1996. Skeletal deformities in larval, juvenile and adult stages of cultured sea bream (Sparus aurata L.). Aquaculture. 141:1-11. Azari Takami, G. 1990. The nutritional value of Artemia in feeding Acipenseridae. In In: The Collection of Papers on Proper Exploitation of Caspian Fish Reserves, National Conferences in Iranian Shilat Company, Iran- Tehran. 509-523. Barahona-Fernandes, M.H. 1982. Body deformation in hatchery reared European sea bass Dicentrarchus labrax (L). Types, prevalence and effect on fish survival. Fish Biology J. 21:239249. Buddington, R.K., and J.P. Christofferson. 1985. Digestive and feeding characteristics of the conondrostreans. In North American Sturgeons.Biology and Aquaculture Potential. F. P.Binkowski and S. I., Dordrecht, Netherlands. Caspian Environment. org. 2007. Caspian Sea, general background. http://www.caspianenvironment.org/caspian.htm. Dabrowski, K. 1990. Absorption of ascorbic acid and ascorbic sulfate and ascorbate metabolism in comon carp (Cyprinus carpio L.). Comparative Physiology J B 160:549–561. Dabrowski K. 1990. Absorption of ascorbic acid and ascorbic sulfate and ascorbate metabolism in comon carp (Cyprinus carpio L.). Comparative Physiology J B 160:549–561. Daoulas, C., E.N. Economou, and I. Bantavas. 1991. Osteological abnormalities in laboratory reared sea bass (Dicentrarchus labrax) fingerlings. Aquaculture. 97:169-180. Dettlaff, T.A., A.S. Ginsburg, and O.I. Schmalhausen. 1993. Development of prelarvae. In: Sturgeon Fishes, Developmental Biology and Aquaculture. 155-221 Springer-Verlag Ed., Berlin, Germany. Emadi, H. 1979. The state of the fishing and reproduction of the Kutum, Rutilus frisii kutum, in The Caspian Sea of Iran. Journal of Ichthyology. 19:151-154. Development of Morphology in Hatchery-Reared Rutilus Frisii Kutum Larvae [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] 305 Gapasin, R.S.J., R. Bombeo, P. Lavens, P. Sorgeloos, and H. Nelis. 1998. Enrichment of live food with essential fatty acids and vitamin C effects on milkfish (Chanos chanos) larval performance. Aquaculture. 162:269-286. Gavaia, P.J., M.T. Dinis, and M.L. Cancela. 2002. Osteological development and abnormalities of the vertebral column and caudal skeleton in larval and juvenile stages of hatchery-reared Senegal sole (Solea sengalensis). Aquaculture. 211:305-323. Ghaninejad, D., and S. Abdulmaleki. 2007. Annual stocks assessment of bony fish in the Caspian Sea. Iranian Fisheries research Institute (IFRO), Tehran, Iran. 65pp. Hattori, M., Y. Sawada, Y. Takagi, R. Suzuki, T. Okada, and H.J. Kumai. 2003. Vertebral deformities in cultured red sea bream, Pagarus major, Temminck and Schlegel. Aquaculture research. 34:1129-1137. Kihara, M., S. Ogata, N. Kawano, I. Kubota, and R. Yamaguchi. 2001. Lordosis induction in juvenile red sea bream, Pagrus major, by high swimming activity. Aquaculture. 212:149-158. Koumoundouros, G., P. Divanach, and M. Kentouri. 2001a. The effects of rearing conditions on development of saddleback syndrome and caudal fin deformities in Dentex dentex (L.). Aquaculture. 200:285-304. Koumoundouros, G., E. Maingot, P. Divanach, and M. Kentouri. 2001b. Kyphosis in reared sea bass (Dicentrarchus labrax L.): ontogeny and effects on mortality. Aquaculture. 209:49-58. Martin, F.D., and D.A. Wright. 1987. Nutritional state analysis and its use in predicting striped bass recruitment: laboratory cahbration. In American Fisheries Society Symposium Vol. 2. 1109-114. McFadzen, I.R., D.M. Lowe, and S.H. Coombs. 1994. Histological changes in starved turbot larvae (Schophtalmus maximus) quantified by digital image analysis. Journal of Fish Biology. 44:255-262. Pearse, A.G.E. 1985. Histochemistry,Theoretical and Applied, Analytic Technology. Churchill Livingstone, New York 624 pp. Sfakianakis, D.G., G. Koumoundouros, L. Anezaki, P. Divanach, and M. Kentouri. 2003. Development of a saddleback-like syndrome in reared white sea bream (Diplodus sargus). Aquaculture. 217:673-676. Siefert, R.E. 1969. Biology of the white crappie in lewise and clarck lake.Technical paper. U.S. Bureau of Sport Fisheries and Wild life, Washington. Soliman, A.K., K. Jauncey, and R.J. Roberts. 1986. The effect of varying forms of dietary ascorbic acid on the nutrition of juvenile tilapias (Oreochromis niloticus). Aquaculture. 52:110. Theilacker, G.H. 1978. Effect of starvation on the histological and morphological characteristics of jack mackerel, Trachurus symmetricus, larvae. Fisheries Bulletin. 76:403-414.