Fisheries Science 65(2), 224-229 (1999)

Histological Characteristics and Development of the Retina in the Japanese Sardine Sardinops melanostictus*

Masanobu Matsuoka•õ

Seikai National Fisheries Research Institute, Kokubu, Nagasaki 850-0951, Japan

(Received July 27, 1998)

Histological characteristics and the distribution pattern of the visual cells, single cones, twin cones, and rods in the retina of the eye of wild adult Japanese sardine Sardinops melanostictus were investigat ed. The developmental process of the visual cells was also examined in the reared and wild larvae. In the specialized part (area temporalis) of the adult retina, slender twin cones were regularly distributed in a square mosaic pattern with central single cones. In the unspecialized part, twin cones with stout ellip soids were regularly distributed and many rods were recognizable. A newly-hatched larva had develop ing lens and undifferentiated retinal cells. The retinal differentiation proceeded rapidly, with the pig ment epithelial cells and visual cells already formed in a 31.5 hour-old larva. In a 79.5 hour-old larva at the first-feeding stage, the visual cell layer consisted of only single cones. A so-called pure-cone retina was still recognized in a larva of 18.6 mm standard length (SL). In a 20.9 mm. SL larva, rod-like cells with thin ellipsoids and outer segments appeared and twin cones also observed. In larvae greater than about 20 mm SL, the rods and twin cones rapidly increased in number. The retina of a 35.6 mm SL juvenile basically did not differ from the adult one. Key words: Japanese sardine, Sardinops melanostictus, retina, development, single cone, twin cone, rod

The Japanese sardine Sardinops melanostictus is one of larval stages of the Clupeiformes such as the northern an the most important commercial fishes in Japan. Many in chovy E. mordax,15) the pilchard Sardina pilchardus,9) and vestigations have been conducted into the population dy the herring Clupea harengus,16,17) although Sardinops has namics of this species (see Kuroda11)), but biological not yet been examined. In contrast, the leptocephalus of knowledge, particularly regarding its developmental biolo the European eel Anguilla anguilla and the deep-sea larva gy, is insufficient.2) Recently, Matsuoka3,4) reported the de of the Macruridae have only rods as visual receptors.9) velopment of bony and lateral muscular systems of this The rods usually appear well after hatching and often as species, but more information of other organs is required late as metamorphosis. As it is relatively difficult to ob- to clarify larval survival strategy. serve the rods directly under light microscope, the number The eye is one of the most important sense organs for of cones is compared with that of the prominent nuclei of feeding and predator avoidance. The detailed structure of the outer nuclear layer.17) This indirect counting technique the eye of adult clupeoid fish has been investigated in the to examine the differentiation of rods has been used in Pacific sardine Sardinops caerulea, the northern anchovy many studies. 10-12,14-19)The twin cones are formed by the Engraulis mordax, the American shad Alosa sapidissima, combination of two single cones. They usually appear just and the deep-bodied anchovy Anchoa compressa by before or during metamorphosis.20) It is important to clari O'Connell,5) although there has been no information on fy the differentiation of rods and twin cones by species, rel that of S. melanostictus. The retina of the fish eye usually ative to their morphological and ecological development. includes three types of visual cells, single cones, twin The present study clarifies histological characteristics and cones, and rods. The cones are involved in color vision the distribution pattern of visual cells in adult S. and visual acuity, and the rods in dim light vision. The melanostictus, and the developmental processes of their twin cones are peculiar to teleosts,6) and Kawamura and visual cells are also investigated. Tamura7) suggested that they are functional in dim light en vironment. Materials and Methods In the early larval stages of many teleosts, including salmon Oncorhynchus spp., the haddock Melanogrammus Specimens aeglefinus, the sand goby Pomatoschistus minutus, the red An adult specimen, 190 mm in standard length (SL), seabream Pagrus major, the sole Solea solea, the flounder was caught at night in the waters off southern Kyushu by Paralichthys olivaceus, and the marble goby Oxyeleotris angling. Of the specimens examined, 27 larvae were labora marmoratus, a pure-cone retina without rods has been ob- tory-reared, using wild eggs collected with a plankton served.") A pure-cone retina has been also reported in the net.2) They were from 3.6 mm in notochord length (NL)

* Contribution from Seikai National Fisheries Research Institute, No. 570. •õ Present Address: National Research Institute of Fisheries and Environment of Inland Sea, Ohno, Hiroshima 739-0452, Japan. Retinal Histology and Development in Sardine 225

(newly-hatched) to 9.65 mm NL (12 days old) in 1989 and quarters of the retina showed almost the same histological from 3.75 mm NL to 11.2 mm NL (16 days old) in 1991. features, called the unspecialized part here. Twin cones These larvae were reared at 17.0-17.5•Ž and fed on small with stout ellipsoids were regularly distributed and type rotifers after the first-feeding stage. In addition, 20 numerous very thin rods were recognizable among them, wild specimens, from 6.1 mm NL to 35.6 mm SL collected although single cones were not observed (Fig. 1A, B). The with a plankton net and a scoop net using a fish lamp, were outer nuclear layer was thick, indicating the existence of examined. numerous rod nuclei (Fig. 1B). The ventro-temporal quarter of the retina included the Histology specialized area, area temporalis, which contained densely The adult specimen was fixed in Bouin's solution. The re cones. Slender twin cones were regularly distributed in a tina was dissected out, cut into four quarters, dorso-nasal, square mosaic pattern with central single cones. Few rods ventro-nasal, dorso-temporal, and ventro-temporal, and were detected in this part (Fig. IC, D). The outer nuclear embedded in paraffin. Transverse and tangential sections layer of the area temporalis was thinner than that of the un- 4 Mm thick were cut with a microtome and stained with Al specialized retina (Fig. 1D). cian blue-Hematoxylin-Eosin. Larvae and juveniles were mainly fixed in Bouin's solu Retinal Development tion and partly in Zenker's solution. Transverse sections In a 3.5 hour-old larva of 3.6 mm NL, the lens was com- 4-6 ƒÊm thick were cut and stained with Alcian blue- posed of two layers, the inner fibrous layer and the outer Hematoxylin-Eosin. Sections were observed under a light single cell layer. The retinal cells were not yet differentiat microscope. ed (Fig. 2A). In a 31.5 hour-old larva of 4.75 mm NL, the differentiation of the retinal cells had rapidly proceeded, Results and the pigment epithelium and visual cell layer were formed (Fig. 2B). In a 53 hour-old larva of 4.85 mm NL, Retinal Structure of Adult the oculomotor muscles were formed and pale pigmenta The dorso-nasal, ventro-nasal, and dorso-temporal tion appeared (Fig. 2C).

Fig. 1. Histological sections of the retina in the adult Japanese sardine Sardinops melanostictus. A: tangential section of unspecialized part showing regularly distributed twin cones with stout ellipsoids and very thin rods. B: transverse sec tion of unspecialized part showing thick outer nuclear layer with many rod nuclei. C: tangential section of specialized part (area temporalls) show ing slender twin cones regularly distributed in a square mosaic pattern with central single cones. D: transverse section of specialized part showing a relatively thin outer nuclear layer. ONL, outer nuclear layer; PEL, pigment epithelial layer; R, rod; SC, single cone; TC, twin cone. Scale bars indicate 50 ƒÊm. 226 Matsuoka

In a 79.5 hour-old larva of 5.15 mm NL at the first-feed- stage. ing stage, the pigment epithelial layer was fully pigmented Figure 3A shows the visual cells of the unspecialized (Fig. 2D). The visual cell layer consisted of only single area of the retina in an 18.6 mm SL larva. Many darkly cones and the ratio of cellular nuclei of the outer nuclear stained small nuclei were observed in the basal part of the layer to cone ellipsoids was 1:1 (Fig. 2E). Figure 2F indi outer nuclear layer. The ratio of cellular nuclei to cone el cates a pure-cone retina of a 12 day-old larva of 8.9 mm lipsoids was about 2:1, although the rod ellipsoid or outer NL. It appeared similar to the retina of the first-feeding segment was not recognizable. The tangential section

Fig. 2. Histological sections of the eye and retina in the early larval Japanese sardine Sardinops melanostictus (all transverse sections) . A: a 3.5-hour-old larva of 3.6 nun in notochord length (NL) showing the developing lens and undifferentiated retinal cells (arrows). B: a 31.5-

hour-old larva of 4.75 mm NL showing differentiated pigment epithelium and visual cell layer. C: a 53-hour-old larva of 4 .85 mm NL showing pale pigmentation. D: a 79.5-hour-old larva of 5.15 mm NL at the first-feeding stage showing a thick pigment epithelial layer. E: enlargement of D showing a pure-cone retina. F: a 12-day-old larva of 8.9 mm NL showing a pure-cone retina. L, lens; N, nucleus of visual cell; OM , oculomotor muscle; PE, pigment epithelium; PEL, pigment epithelial layer; SC, single cone; V, visual cell layer. Scale bars indicate 50 ƒÊm (A, B, C, D) and 10 ƒÊ m (E, F). Retinal Histology and Development in Sardine 227

Fig. 3. Histological sections of the retina of the Japanese sardine Sardinops melanostictus in the larvae around the time of metamorphosis. A: transverse section of an 18.6 mm larva in standard length (SL) showing a pure-cone retina. Arrows indicate darkly stained small nuclei. B: tangential section of an 18.6 mm SL larva showing single cones. C: transverse section of a 20.9 mm SL larva showing appearance of rods. D: tan

gential section of a 20.9 mm SL larva. Twin cones and thin rods are distinguishable. E: transverse section of a 26.8 mm SL larva. F: tangential sec tion of the retina of a 26.8 mm SL larva. Note numerous rods. R, rod; SC, single cone; TC, twin cone. Scale bars indicate 10ƒÊm.

showed single cone ellipsoids (Fig. 3B). from a larval size of about 20 mm SL. Figures 3E and F In a 20.9 mm SL larva, visual cells with thin ellipsoids show a well-differentiated duplex retina including twin and outer segments were observed, which appeared to be cones and numerous small rods in a 26.8 mm SL specimen. initial rods (Fig. 3C). The tangential section of the nasal The retina of a 35.6 mm SL juvenile contained more region of the retina showed pairing of single cones to form numerous rods, resembling the adult retina. twin cones and slender rods (Fig. 3D). In the temporal region of the retina, twin cones were not formed, while Discussion some slender rods were observed. The number of rods and twin cones rapidly increased O'Connell5) investigated the retinal structure of six 228 Matsuoka pelagic marine teleosts including Sardinops caerulea. S. formed in C. harengus larvae over about 25 mm and the melanostictus examined in the present study possessed the rods develop thereafter,16) although Sandy and Blaxter17) area temporalis in the ventro-temporal part of the retina found young rods in the retina of a 22 mm C. harengus lar like S. caerulea. The histological features and distribution va. In the zebrafish Brachydanio rerio, possible rod nuclei pattern of single cones, twin cones, and rods in the un- were seen in larvae eight days after fertilization and rods specialized part and the specialized part (area temporalis) were first identified in 12-day-old larvae.22) From these find of the retina in S. melanostictus were quite similar to those ings, two-tiered nuclei observed in the larval retina might in S. caerulea, although the latter has a few single cones in not necessarily indicate the appearance of functional rods. the unspecialized part. In the area temporalis, the very However, O'Connell151 thought that the rods started to ap- high density of cones gives high acuity, so that the main pear in E. mordax larvae of 10 mm SL, coinciding with the visual axis is in the upper-fore direction in both S. start of vertical migration, 21) with larvae moving into and caeruleasl and S. melanostictus. becoming more active in dimmer light. It is likely that func A pure-cone retina is observed in most teleost larvae in tional rods in E. mordax may appear somewhat later, so cluding S. melanostictus except for eel leptocephalus and early vertical migration may occur irrespective of rod deep-sea Macruridae larvae.) Recently, Omura et al.21) differentiation. Although the indirect method has been demonstrated that both seven-day-old cultivated larvae used in many studies, 10-12,14-19)the direct observations are and an 11.0 mm wild caught leptocephalus (ca. 2 weeks clearly needed to identify rod differentiation. old) of the Japanese eel Anguilla japonica had cone-like The twin cones of S. melanostictus appeared in larvae of photoreceptor cells. They suggested that cone-like pho about 20 mm SL, simultaneously with the rods. According toreceptors develop first with rod-like cells appearing later to Sandy and Blaxter,17) in Solea solea, rod-like cells ap in the retina of A. japonica, because the 11.0 mm wild peared and twinning of cones began when migration of the specimen had longer rod-like photoreceptors in the left eye and metamorphosis started. The formation of twin peripheral retina. It is possible that the leptocephalus of cones and rods almost coincides also in other species, such A. anguilla and the deep-sea larvae of the Macruridae also as Pagrus major10) and Oxyeleotris marmoratus.14) In con possess cone-like photoreceptors in earlier stages, such as trast, the twin cones of C. harengus were found just before other teleosts. metamorphosis (28-30 mm),16) and cone twinning took O'Connell15) reported for Engraulis mordax that the out- place suddenly after rod differentiation.17) Moreover, the er nuclear layer remains two-tiered until larvae of 10 or 12 twin cones were formed a little later than the rods in some mm SL, with the layer soon increasing to three and more other species, such as Paralichthys olivaceus11) and Tilapia tiers of nuclei, undoubtedly indicating rod recruitment. In nilotica. 13) S. melanostictus, the second layer of darkly stained small Matsuoka3) osteologically defined the ontogenetic inter nuclei was recognized in larvae over 15 mm NL. However, vals in S. melanostictus. According to this definition, the these nuclei had neither ellipsoids nor outer segments, larval period consists of three phases, the larva I phase which were first observed in larvae over 20 mm SL. Blaxter (from the first-feeding stage to 10 mm NL), the larva ‡U and Jones16) demonstrated by an electron microscopic phase (from 10 mm NL to 20 mm SL), and the larva ‡V study that rod-like structures are not present at least for phase (from 20 mm SL to 34 mm SL, corresponding to larvae up to a length of about 20 mm in the retina of metamorphosis) (Fig. 4). Matsuoka4) also clarified the later Clupea harengus. Darkly stained small new nuclei were al muscle development of the species. At the first-feeding

Fig. 4. Diagrammatic developmental pattern including retina and ontogenetic intervals in the Japanese sardine Sardinops melanostictus, altered from Matsuoka.3) Retinal Histology and Development in Sardine 229 stage, when the pure-cone retina is well differentiated, a Sardinops melanostictus. Ichthyol. Res., 44, 275-295 (1997). part of the head skeleton and pectoral fin supports are also 4) M. Matsuoka: Development of the lateral muscle in the Japanese formed,3) and superficial fibers of the lateral muscle have sardine Sardinops melanostictus. Fisheries Sci ., 64, 83-88 (1998). aerobic metabolism as red fibers.) These morphological 5) C. P. O'Connell: The structure of the eye of Sardinops caerulea, En changes are linked to first-feeding success. The differentia graulis mordax, and four other pelagic marine teleosts. J. Morph., 113, 287-329 (1963). tion of rods and twin cones, which is the most drastic 6) H. Somiya and H. Niwa: Vision, in "Fish Physiology" (ed. by Y. change of the retina in the larval period, begins in larvae Itazawa and I. Hanyu), Koseisha-Koseikaku, Tokyo, 1991, pp. of about 20 mm SL at the start of metamorphosis. The 403-441 (in Japanese). number of rods and twin cones rapidly increases during 7) G. Kawamura and T. Tamura: Morphological studies on the retina metamorphosis. Ossification of various essential skeletal of two teleosts Scomber tapeinocephalus and Halichoeres poecilop elements, such as the neurocranium and vertebral column, terus. Nippon Suisan Gakkaishi, 39, 715-726 (1973). 8) M. A. Ali: The ocular structure, retinomotor and photobehavioural also occurs at the start of metamorphosis.3) The clear responses of juvenile Pacific salmon. Can. J. Zool., 37, 965-996 stratification of red fibers of the lateral muscle, the differen (1959). tiation of tonic-like fibers, and the appearance of small 9) J. H. S. Blaxter and M. Staines: Pure-cone retinae and retinomotor white fibers among the large fibers also occurs in larvae responses in larval teleosts. J. Mar. Biol. Ass. U.K., 50, 449-460 greater than about 20 mm SL.) The histological features (1970). of retina of a 35.6 mm SL juvenile did not differ from the 10) G. Kawamura, R. Tsuda, H. Kumai, and S. Ohashi: The visual cell morphology of Pagrus major and its adaptive changes with shift adult ones, except for the adult retina having considerably from pelagic to benthic habitats. Nippon Suisan Gakkaishi, 50, more rods. The greater part of the osteological develop 1975-1980 (1984). ment has been completed in juveniles of 34 mm SL3) and 11) G. Kawamura and K. Ishida: Changes in sense organ morphology the lateral muscle structure of a 37.0 mm SL juvenile is and behaviour with growth in the flounder Paralichthys olivaceus. similar to large young specimens.4) Thus, the development Nippon Suisan Gakkaishi, 51, 155-165 (1985). of bones, lateral muscle, and retina seems to occur in 12) G. Kawamura, H. Mori, and A. Kuwahara: Comparison of sense organ development in wild and reared flounder Paralichthys parallel to one another. olivaceus larvae. Nippon Suisan Gakkaishi, 55, 2079-2083 (1989). A pure-cone retina may be adequate for first-feeding, be 13) G. Kawamura and N. Washiyama: Ontogenetic changes in behavior cause light is required for feeding by many species, at least and sense organ morphogenesis in largemouth bass and Tilapia in the early larval stages.24) Larvae possessing a pure-cone nilotica. Trans. Amer. Fish. Soc., 118, 203-213 (1989). retina might be unable to feed in dim or dark conditions 14) S. Senoo, K. J. Ang, and G. Kawamura: Development of sense or and more susceptible to attack by predators. Larvae would gans and mouth and feeding of reared marble goby Oxyeleotris mar be able to take food organisms in dim light after rod moratus larvae. Fisheries Sci., 60, 361-368 (1994). 15) C. P. O'Connell: Development of organ systems in the northern differentiation. Rods seem to be involved in movement per anchovy, Engraulis mordax, and other teleosts. Amer. Zool., 21, ception and are perhaps important in predator 429-446 (1981). avoidance.24) The differentiation of rods and twin cones in S. 16) J. H. S. Blaxter and M. P. Jones: The development of the retina melanostictus larvae seems to rapidly increase their ability and retinomotor responses in the herring. J. Mar. Biol. Ass. U.K., to survive through feeding and predator avoidance, with 47, 677-697 (1967). the aid of the development of bony elements, lateral mus 17) J. M. Sandy and J. H. S. Blaxter: A study of retinal development in cle, and others. larval herring and sole. J. Mar. Biol. Ass. U.K., 60, 59-71 (1980). 18) G. Kawamura, Y. Mukai, and H. Ohta: Change in visual threshold with development of rods in ayu Plecoglossus altivelis. Nippon Sui Acknowledgments I express my sincere thanks to Professor Dr. Gunzo san Gakkaishi, 50, 2113 (1984). Kawamura of Kagoshima University for critical reading of the 19) G. Kawamura and M. Munekiyo: Development of the sense organs manuscript. I am grateful to Mr. Takumi Mitani of Nansei National Fish of ribbonfish Trichiurus lepturus larvae and juveniles. Nippon Sui eries Research Institute for help in collecting sardine eggs. This work was san Gakkaishi, 55, 2075-2078 (1989). supported in part by a Grant-in-Aid (Bio Cosmos Program) from the 20) K. Ishida and G. Kawamura: Development of sense organs. Aquabi Ministry of Agriculture, Forestry, and Fisheries. ology, 36, 8-14 (1985) (in Japanese). 21) Y. Omura, K. Uematsu, H. Tachiki, K. Furukawa, and H. Satoh: References Cone cells appear also in the retina of eel larvae. Fisheries Sci., 63, 1052-1053 (1997). 1) K. Kuroda: Studies on the recruitment process focusing on the early 22) T. Branchek and R. Bremiller: The development of photoreceptors life history of the Japanese sardine, Sardinops melanostictus in the zebrafish, Brachydanio rerio. I. Structure. J. Comp. Neurol., 224, 107-115 (1984). (Schlegel). Bull. Natl. Res. Inst. Fish. Sci., 3, 25-278 (1991) (in Japanese). 23) J. R. Hunter and C. Sanchez: Diel changes in swim bladder infla 2) M. Matsuoka and T. Mitani: Hatching and rearing experiments on tion of the larvae of the northern anchovy, Engraulis mordax. Fish. eggs of Japanese sardine, Sardinops melanostictus, collected from Bull., U.S., 74, 847-855 (1976). adjoining area of the Nagasaki Harbor (Preliminary study). Bull. 24) J. H. S. Blaxter: Development of sense organs and behaviour of Seikai Reg. Fish. Res. Lab., 67, 15-22 (1989) (in Japanese). teleost larvae with special reference to feeding and predator 3) M. Matsuoka: Osteological development in the Japanese sardine, avoidance. Trans. Amer. Fish. Soc., 115, 98-114 (1986).