SUISANZOSHOKU 49(4), 451-460 (2001) Skeletal Anomalies in Cultured Flounder, Paralichthys olivaceus, with Shortened Upper Jaw Yoshifumi SAWADA*1, Manabu HATTORI*2, Ryouta SUZUKI*2, Hirofumi MIYATAKE*3, Michio KURATA*4, Tokihiko OKADA*1, and Hidemi KUMAI*5 (Accepted October 15, 2001) Abstract: Skeletal anomalies in cultured flounder, Paralichthys olivaceus, were described for speci - mens with shortened upper jaw (104.2-129.0 mm SL). Each eight specimens with shortened upper jaw and without it were analyzed with cartilages and bones double-stained. Shape and relative size change was observed in neurocranium (prevomer, preethmoid, mesethmoid, lateral ethmoid, and nasal), in branchiocranium (premaxilla, maxilla, palatine, metapterygoid, ectopterygoid, endoptery - goid, quadrate, preopercle, and subopercle). No deficiency of osteological elements in the head region was observed in specimens with shortened upper jaw. Other bones in neurocranium, bran -chiocranium, vertebrae, and appendicular skeleton showed no anomaly related to the shortened upper jaw. Key words: flounder; Paralichthys olivaceus; skeletal anomaly; upper jaw The flounder, Paralichthys olivaceus, is cul -tured in large quantities in Japan and Korea1,2). Since mass rearing beyond the stage of meta - morphosis was firstly succeeded3), biological information on the early life stage of P. olivaceus has been accumulated and rearing techniques have been largely improved4). Many research has been focused on describing deformed bones and finding out the cause of such defor - mities5-9). However, as in other species, bone deformities still decrease the efficiency of aqua -culture production. To elucidate the cause of skeletal anomalies and to prevent them, it is first necessary to identify and sort their symp -toms. In this study, skeletal anomalies in the head region of P. olivaceus specimens with shortened upper jaw were described. Materials and Methods *1 Fisheries Laboratory of Kinki University Ohshima Experiment Station, Ohshima 1790-4, Kushimoto, Wakayama 649-3633, Japan. *2 Graduate School of Kinki University, Nakamachi 3327-204, Nara 631-8505, Japan. *3 Kinki University Fish Nursery Center Shirahama Station, Sakata 1-5, Shirahama, Wakayama 649-2200, Japan. *4 Kinki University Fish Nursery Center Ohshima Station, Ohshima 1790-4, Kushimoto, Wakayama 649-3633, Japan. *5 Fisheries Laboratory of Kinki University Shirahama Experiment Station, Shirahama 3153, Wakayama 649-2211, Japan.
452 Y. Sawada, M. Hattori, R. Suzuki, H. Miyatake, M. Kurata, T. Okada, and Hidemi Kumai Fig. 1. Growth and rearing condition of a flounder, Paralichthys olivaceus. Closed circles: with shortened upper jaw. Table 1, Body part lengths and number of vertebrae of Paralichthys olivaceus specimens (112 days posthatch)
Skeletal Anomaly of P. olivaceus 453 Fig. 2. Photographs of skull skeleton a flounder, Paralichthys olivaceus, with and without shortened upper jaw. Specimens were cleared and double-stained the cartilages and bones with alcian blue and alizarin red S. A, ocular side of the speci - men without shortened upper jaw; B, blind side of the same specimen as in A; C, ocular side of the specimen with short - ened upper jaw; D, blind side of the same specimen as in C. Bar in each photos indicates 10 mm. SL was measured and branchiocranium. of skeletal al.12), Hosoya system for bones of neurocranium Osteological mainly followed and Kawamura8), terminology Helfman et and Hosoya13). Results Incidence rate of shortened upper jaw The result of the sorting of fish on 50-d posthatch gave the incidence rate of all kinds of deformities at 18.6-19.1%in the four 30 m3 inter - vening tanks. Among them, the incidence rate of shortened upper jaw was 1.9-3.9%.Examples of specimens with and without shortened upper jaw were shown in Figure 2. Relative UJL offish with shortened upper jaw Relative UJL were less than 10% SL (4.8-9.8% SL) in specimens with shortened upper jaw (Fig.3). In contrast, specimens without short - ened upper jaw had relative upper jaw length of 10.4-11.1%SL. Fig. 3. Upper jaw length (%) in relation to standard length of Paralichthys olivaceus specimens with and without shortened upper jaw. Open and closed circles indicate UJL of specimens without and with shortened upper jaw, respectively.
Y. Sawada, 454 Fig. 4A. Photographs of M. Hattori, double-stained R. Suzuki, H. Miyatake, neurocranium and M. Kurata, osteological T. Okada, and Hidemi elements in Kumai branchiocranium of a flounder, Paralichthys olivaceus. Left and right rows in each photo show bones of specimens without and with shortened upper jaw, respectively. Bars with arrows at both ends indicate the length used in the quantitative analysis (See Fig. 5). Bar without arrow indicates 10 mm in each photo. a, neurocranium (view from left overhead); b, premaxilla ocular side: c, premaxilla blind side; d, maxilla ocular side; e, maxilla blind side; f, palatine ocular side. The separation of parasphenoid and frontal in the neurocranium in photo a is the artifact which arose in the process of dissection. Fig. 4B. Photographs of double-stained branchiocranium of a flounder, Paralichthys olivaceus. For the explanation of figure, see legend of Fig. 4A. g, palatine blind side; h, metapterygoid ocular side; i, metapterygoid blind side; j, ectopterygoid ocular side; k, ectopterygoid blind side; 1, endopterygoid ocular side; m, endopterygoid blind side; n, quadrate ocular side; o, quadrate blind side. The cracks in metapterygoids in photo i, endopterygoid in photo m, and in quadrate in photo n are the artifacts which arose in the process Fig. 4C. Photographs see legend - ercle of double-stained of Fig. 4A. p, preopercle in photo r is the artifact which branchiocranium ocular arose side; of dissection. of a flounder, q, preopercle in the process blind of dissection. Paralichthys side; olivaceus. r, subopercle For the explanation ocular side. The crack of figure, in subop
Skeletal Anomaly of P. olivaceus 455
456 Y. Sawada, M. Hattori, R. Suzuki, H. Miyatake, M. Kurata, T. Okada, and Hidemi Kumai Fig. 5. Relative size (%) of neurocranium and osteological elements in branchiocranium in relation to standard length of a flounder, Paralichthys olivaceus, specimens with and without shortened upper jaw. Letters O and B indicate ocular and blind side, respectively. Specimens with shortened upper jaw, open circles; specimens without shortened upper jaw, closed circles. Changes of shape and size in bones of neurocrani - um and branchiocranium in deformed fish The prevomer, preethmoid, mesethmoid, lat -eral ethmoid, and nasal showed the shape change in the neurocranium of deformed speci - mens (Figs. 4A, 4B, and 4C). These bones con -stitute the rostral region of the neurocranium. Other bones of neurocranium showed no remarkable anomaly in the deformed speci - mens. In branchiocranium, shape change were observed in premaxilla (distortion and shrink -age), maxilla (distortion, shrinkage, and split), metapterygoid (distortion) (Fig. 4A, 4B and 4C), ectopterygoid (distortion), endopterygoid (distortion), palatine (distortion), and preoper-
Skeletal Anomaly of P. olivaceus 457 Fig. 5. -Continued cle (distortion). The premaxilla and maxilla on the blind side were remarkably more deformed than those on the ocular side. In addition, deformities in nasal and lachrymal were also observed. In contrast, no remarkable difference was observed between specimens with and without shortened upper jaw in dentary, angular, hyomandibular, quadrate, symplectic, interoper - cle, subopercle, hypohyal, ceratohyal, and epi - hyal. No deficiency of bone elements in neuro -cranium and branchiocranium was observed in deformed specimens. Quantitative analysis showed the difference of relative bone size in more bones of neurocra -nium and branchiocranium than in bones which showed the shape change (Fig.5). The follow -ing bones remarkably differed in relative size between fishes with and without shortened upper jaw; neurocranium, bones of upper jaw (maxilla and premaxilla), palatine arch (ectopterygoids, endopterygoids, and metaptery - goids), some bones of the hyoid arch (quadrate), opercular apparatus (preopercle and subopercle of ocular side). No difference in relative size was observed for dentary, angular, hyomandibula, symplectic, opercle, interopercle, subopercle of blind side, and bones of hyoid complex (hyp o- hyal, ceratohyal, and epihyal). Anomaly in other bones and number of vertebral columns Anomaly of the second epural (deficiency and fusion with the first epural) was observed both specimens with and without shortened upper jaw (Table 1). All the specimens, with and with -out deformity, had 11 abdominal and 26 caudal vertebrae both of which showed no anomaly.
458 Y. Sawada, M. Hattori, R. Suzuki, H. Miyatake, M. Kurata, T. Okada, and Hidemi Kumai Discussion The shortened upper jaw sometimes occurs at a considerably high rate in the seedling pro - duction of P. olivaceus. In this study, the inci -dence rate was the second highest (1.9-3.9%) to the vertebral deformity (3.9-7.0%) as well as the deformity of gill cover (0.3-5.4%). The external appearance of fish with shortened upper jaw is distinctly strange (Figs. 2 and 3), and such fish have no commercial value. Therefore, the pre - vention of shortened upper jaw is the urgent problem to be attained as well as above two deformities. The only anomaly of P. olivaceus in the exter - nal morphology was the shortened upper jaw, but anomalies were found in many bones in neurocranium and branchiocranium in addition to the bones in upper jaw (Figs. 4 and 5). Some of these bones exhibited the shape change of shrinkage, distortion, and split, in comparisons with bones of specimens without shortened upper jaw. However, some bones showed no remarkable such shape change, and in these bones only the relative size was different from bones of not deformed specimens. This sug -gests that such a quantitative analysis used in this study is necessary for the examination of skeletal anomalies of fish in a more precise character analysis. Another characteristics of skeletal anomaly in specimens with shortened upper jaw was that no deficiency or excess for - mation of bones was recognized in the head region. It is concluded that the shortened upper jaw of P. olivaceus was the bone anomaly restricted to the head region, from the fact that no mor - phological difference was observed in the verte - brae and appendicular skeleton between speci - mens with and without shortened upper jaw. The anomaly of the second epural in P. olivaceus reportedly occurs at a high rate in the artificial seedlings by Hosoya and Kawamura8). In this study, this deformation was observed in speci - mens of both with and without shortened upper jaw. Therefore, anomaly of the second epural would not be caused by the same cause as the The cause of bone anomalies in this study is unclear. Deformity of the upper jaw has been reported in many species, such as red sea bream, Pagrus major14), the jack mackerel, Trachurus japonicus15), the pike eel, Muraemesox cinereus16), and etc. In most of these cases, the deformity in upper jaw is so-called pugheadness and its cause has not yet been clear. However, the shortened upper jaw in P. olivaceus would not be the pugheadness because anomalous bones were different from pugheadness, although its external appearance resembles each other. Recently, we examined the pughead - ness of red sea bream, P. major, osteologically and histologically17). The results showed that the deformity of ethmoid and parasphenoid was the main bone anomaly of pugheadness in P. mayor. characteristics from wild ones as a result of selective breeding for generations. In such fish, various morphological and physiological pheno -types have changed from those of wild ones as in the red sea bream20,21). In addition, cultured fish experience different environmental condi -tions, food, rearing temperature, etc. from wild ones. This also causes the morphological and physiological changes in cultured fish22). Cultured P. olivaceus in Japan, whose seedling production started more than 35 years ago23), is the fish which comes under above situation as well as the red sea bream, P. major. Moreover, wild fish sometimes have geographical variations in their meristic characters columns as in the number of vertebral and fin-ray counts of P. olivaceus24,25). In such a case, it becomes a problem which
Skeletal Anomaly of P. olivaceus 459 geographical population should be used as the standard. The above-mentioned situation requires establishment of the standard P. olivaceus tribe both for wild and cultured in the study of teratology. Among target species in aquaculture, PP olivaceus is one of the species with much information available on its skeletal system and osteological development8,9,18,26-29). Such information will be useful to teratology of P. olivaceus. Teratology also necessitates the information of histology, physiology, genetics, and nutrition. In this study, the quantitative bone shape analysis, although it was preliminary in it, was useful to detect the bone anomalies. The quantitative analysis of bone shape should be more used in the study of fish teratology, and the more advanced arith - metic method in bone shape analysis should be developed, as introduced by Bookstein30). Acknowledgments We would like to thank the staff of the Fisheries Laboratory of Kinki University for their invaluable support and technical assistance during the experiment. References 3) Harada, T., S. Umeda, O. Murata, K. Kumai, and K. Mizuno (1966): On the growth and rearing methods of the fry of Hirame (Paralichthys olivaceus) obtained by artificial fertilization. Bull. Fish. Lab. Kinki Univ., 1, 289-303. (in Japanese). 5) Oda, T. and Y. Kayano (1988): Effect of several commercial diets on growth, occurrence of color anomaly, and vertebral malformation in hatchery-reared flounder Paralichthys olivaceus. Bull. Fish. Exp. Stn. Okayama Pref, 3, 41-46. 6) 7) Deji, J., T. Takeuchi, T. Seikai, T. Watanabe, and K. Hosoya (1997): Hypervitaminosis A during vertebral morphogenesis in larval Japanese flounder. Fish. Sci., 63(3). 466-473. 8) Hosoya, K. and K. Kawamura (1998): Skeletal formation and abnormalities in the caudal complex of the Japanese flounder, Paralichthys olivaceus (Temminck & Shlegel). Bull. Natl. Res. Inst. Fish. Sci.,12, 97-110. 9) Suzuki, T., I. Oohara, and T. Kurokawa (1998): Hoxd-4 expression during pharyngeal arch development in flounder (Paralichthys olivaceus) embryo and effects of retinoic acid on expression. Zool. Sci.,15, 57-67. 10) National Institutes of Health (1997): NIH image analysis software. NIH, Bethesda, Maryland, U. S. A.,104 p. 11) Kawamura, K. and K. Hosoya (1991): A modified double staining technique for making a transparent fish -skeletal specimen. Bull. Natl. Res. Inst. Aquacult., 20, 11-18 (in Japanese with English abstract). 12) 13) Hosoya, K. (1991): Osteological development, and development of feeding and swimming abilities in fish, A course to master the basin theory in the practice of fish farming technology in 1991, the series of development in larval and juvenile periods, 3, 1-18 (in Japanese). 14) Noda, M., T. Katano, and Y. Honma (1985): A pugheaded specimen of genuine porgy, Pagrus major (Temminck et Schlegel), caught from Ryotsu Bay of Sado Island in the Sea of Japan. J. Niigata Pref. Biol. Edu. Soc., 20, 41-43. 15) Yamakawa, F. and T. Yasuda (1967): A deformed specimen of jack mackerel, Trachurus japonicus (Temminck et Schlegel) from Tsuruga Bay in the coast of the Japan Sea. Collecting and breeding, 29(11), 405. 16) Hotta, H. and Y. Honma (1958): A case of a pug headed apodal fish, Muraemesox cinereus (Forskal). Collecting and breeding, 20(4),120-122. 17) Sawada, Y., M. Hattori, N. Hattori, T. Okada, S. Miyashita, O. Murata, and H. Kumai (2000): Pugheadness in red sea bream, Pagrus major. Nippon Suisan Gakkaishi, 66(6),1072-1073 (in Japanese). 18) Hosoya, K. and K. Kawamura (1997): Osteological evaluation in artificial seedlings of Paralichthys olivaceus (Temminck and Schlegel). U S.-Japan Cooperative Program in Natural Resources (UJNR) Technical
460 Y. Sawada, M. Hattori, R. Suzuki, H. Miyatake, M. Kurata, T. Okada, and Hidemi Kumai Report, 24, 107-114. 19) Matsuoka, M. (1987): Development of the skeletal tissues and skeletal muscles in the red sea bream. Bull. Seikai Reg. Fish. Res. Lab., 65,1-114. 20) Taniguchi, N., S. Matsumoto, A. Komatsu, and M. Yamanaka (1995) : Differences observed in quantita -tive and qualitative traits of five red sea bream strains propagated under the same rearing conditions. Nippon Suisan Gakkaishi, 61(5), 717-726 (in Japanese with English abstract). 21) Kato, K., O. Murata, T. Nakaarai, T. Nasu, S. Miyashita, and H. Kumai (1998) : Genetic variability, external morphology, and feeding of selected and non-selected strains of red sea bream. Suisanzoshoku, 46(2), 203-212. 22) Kinoshita, I., T. Seikai, M. Tanaka, and K. Kuwamura (2000): Geographic variations in dorsal and anal ray counts of juvenile Japanese flounder, Paralichthys oli - vaceus, in the Japan Sea. Env. Biol. Fishes, 57, 305-313. Tokyo, pp.1163-1165 (in Japanese). 26) Amaoka, K. (1969) : Studies on the sinistral flounders found in the waters around Japan. Taxonomy, anato - my and phylogeny. J. Shimonoseki Univ. Fish., 18(2), 65-340. 27) Hosoya, K. and K. Kawamura (1993) : Skeletal structure of the branchial arches in Paralichthys olivaceus (Temminck et Schlegel). Bull. Natl. Res. Inst. Aquaculture, 22,1-10. 28) Hosoya, K. and K. Kawamura (1998): Skeletal formation and abnormalities in the caudal complex of the Japanese flounder, Paralichthys olivaceus (Temminck & Schelegel). Bull. Natl. Res. Inst. Fish. Sci., 12, 97-110. 29) Balart, E. (1985) : Development of median and paired fin skeleton of Paralichthys olivaceus (Pleuronectiformes: Paralichthyidae). Japan. J. Ichthyol., 31(4), 398-410. 30) F. L. Bookstein (1991): Morphometric tools for land - mark data geometry and biology. Cambridge University Press, New York, 435 p.