Development of sense organs in the Japanese sardine Sardinops melanostictus

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1 FISHERIES SCIENCE 2001; 67: Original Article Development of sense organs in the Japanese sardine Sardinops melanostictus MASANOBU MATSUOKA* National Research Institute of Fisheries and Environment of Inland Sea, Ohno, Hiroshima , Japan ABSTRACT: The developmental processes of the olfactory organ, taste buds, lateral line system, and inner ear in the Japanese sardine Sardinops melanostictus were examined in reared and wild specimens. Both the ciliated and microvillous receptor cells in the olfactory organ were found shortly after hatching. Formation of the olfactory nostrils and lamellae began at about 20 mm standard length (SL). The calculated inflection points in the relationship between the number of olfactory lamellae and standard length were at 30.3 and 62.7 mm SL. Taste buds first appeared in a 16.1-day-old larva of 11.2 mm notochord length. Newly hatched larvae were equipped with 12 pairs of neuromasts on the head and trunk. The formation of the lateral line canal commenced at about 20 mm SL and the four canals had ossified by 32.5 mm SL. At hatching, the inner ear was an oval-shaped auditory vesicle with two otoliths. Three semicircular canals formed by the first-feeding stage, and larvae assumed an upright position. The pro-otic bulla was gas-filled and formation of the lagena pocket began at about 20 mm SL. The saccular sac was entirely formed by about 32 mm SL. From the present results, the basic structure of the sense organs was almost completed by 32 mm SL, and the adult condition might be attained at mm SL, as suggested by the second inflection point in the relationship between the number of olfactory lamellae and SL. KEY WORDS: development, inner ear, Japanese sardine, lateral line, olfactory organ, Sardinops melanostictus, sense organs, taste bud. INTRODUCTION Fish possess various kinds of sense organs and use them to detect many kinds of information in the ambient environment. Development of sense organs is very important for early stages of fish. In previous studies, Matsuoka 1 3 clarified the developmental processes of the osteological elements, lateral muscle, and the retina in eyes of the Japanese sardine Sardinops melanostictus. The present study describes the development of sense organs, except for eyes, including the olfactory organ, taste buds, lateral line system, and inner ear. The olfactory organ and taste buds operate as chemoreceptors and receive smell or taste substances saturated in water. The lateral line system and inner ear function as mechanoreceptors and receive water flow, body equilibrium, and auditory stimuli. The sense *Correspondence: Tel: Fax: yamame@nnf.affrc.go.jp Received 5 October Accepted 20 April organs are essential for fish survival, and their developmental processes may be closely related to larval survival through feeding, predator avoidance, vertical migration, and school formation. Most investigations of sense organ development have been conducted in salmonids, freshwater fishes, herring, and flatfish (see Blaxter 4 ). Recently, this knowledge has been increasing in the red sea bream Pagrus major, the Japanese parrot fish Oplegnathus fasciatus, the puffer Takifugu rubripes, 5,6 the flounder Paralichthys olivaceus, 7 the largemouth bass Micropterus salmoides, the Nile tilapia Oreochromis niloticus, 8 and the marble goby Oxyeleotris marmoratus, 9 and the red-spotted grouper Epinephelus akaara. 10 S. melanostictus larvae hatch at relatively undeveloped stage from pelagic eggs. Most sense organs appear thereafter as well as other organs, but no information exists about their development. Therefore, the present study was undertaken to clarify the development of the sense organs, except for eyes, in S. melanostictus from initial formation to completion, using a series of reared and wild specimens.

2 Sense organ development in sardine FISHERIES SCIENCE 1037 The present study was mainly conducted in the Seikai National Fisheries Research Institute. MATERIALS AND METHODS Light microscopic observation Twenty-four larvae examined ( mm notochord length (NL)) were laboratory reared, using wild eggs collected with a plankton net. 11 These larvae were reared at C and fed on small type rotifers after the first-feeding stage. Four wild larvae examined (8.95 mm NL mm standard length (SL)) were collected with a plankton net and 19 specimens examined (14.5 mm NL 35.6 mm SL) with a scoop net using a fish lamp. Most specimens were fixed in Bouin s solution and some were fixed in Zenker s solution, and embedded in paraffin. Serial transverse sections were cut at 4 6 mm thick with a microtome, and stained with Alcian blue hematoxylin eosin. Sections were observed under a light microscope. Scanning electron microscopic observation Twelve laboratory-reared larvae examined ( mm NL) and three wild specimens examined ( mm SL) were fixed in a mixed solution of Karnovsky s paraformaldehyde and glutaraldehyde. Specimens were dehydrated, dried by the critical point drying technique, and ion-coated. They were observed under a scanning electron microscope. Stereomicroscopic observation The numbers of olfactory lamellae of 65 specimens were counted. Thirty-five specimens examined ( mm SL) were caught with a seine net and 30 specimens examined (12.7 mm NL 33.5 mm SL) were caught with a scoop net using a fish lamp. The olfactory epithelia of both right and left sides were removed, then the olfactory lamellae were exposed, and counted. Development of the nostril, especially the internostril epidermis, was also observed under a stereomicroscope. RESULTS Olfactory organ In 4 h-old larvae after hatching, the olfactory placode was located on the anterior front of the snout (Fig. 1a). Developing ciliated receptor cells and microvillous receptor cells were observed in the olfactory placode of a 3.65-mm NL larva (Fig. 1b). In a 3.9-mm NL larva of the same age, the ciliated receptor cells and microvillous receptor cells were clearly distinguishable, and the developing kinocilia of ciliated non-sensory cells were also found (Fig. 1c). With the mouth formation, the olfactory placode was transferred upward and located on the dorsal side of the snout in an 82-h-old larva of 5.6 mm NL at the first-feeding stage (Fig. 1d). Morphological characteristics of the olfactory organ in larvae smaller than 18 mm SL were basically similar to these in the first-feeding stage larvae, with numerous kinocilia of ciliated nonsensory cells (Fig. 1e). Development of the nostril, especially the internostril epidermis, was differentiated into three stages by the extent of formation of the epidermis dividing the anterior and posterior nostrils: not formed (I), developing (II), and completed (III). The internostril epidermis was not formed in larvae up to mm SL (stage I, Fig. 2). It developed as two projections at each middle side of the nostril (Fig. 1f). The smallest specimen with a developing internostril epidermis was 18.9 mm SL and the largest was 24.2 mm SL (stage II). Two projections of the internostril epidermis coalesced in the specimen larger than 21.8 mm SL (stage III, Fig. 2). The olfactory lamellae were formed as folds of the olfactory placode. A lamella first appeared in the posterior lateral region of the placode in an 18.9-mm SL larva. The number of lamellae increased along the posterior margin of the placode (32.5 mm SL; Fig. 1g). Numerous cilia of receptor cells and kinocilia of non-sensory cells existed on the lateral surfaces of the lamellae (Fig. 1h). Figure 3 shows the relationship between the numbers of the olfactory lamellae and standard length on log/log coordinates. The allometric equations for the relationship calculated by least squares regression (Log y = a + b Log x, where y = lamella number, x = standard length, a and b = constants) were (1) Log y = Log x (18.9 mm x 33.5 mm, open circles and triangles), (2) Log y = Log x (24.5 mm x 29.0 mm, solid circles and triangles), (3) Log y = Log x (31.0 mm x 60.5 mm, solid circles and triangles), and (4) Log y = Log x (67 mm x 188 mm, solid circles and triangles). The calculated inflection points from (2) to (3) and from (3) to (4) were 30.3 mm and 62.7 mm, respectively. The maximum number of lamellae was 28 in an 182-mm SL specimen, fully grown adult.

3 Fig. 1 Scanning electron micrographs showing development of the olfactory organ in the larval and early juvenile Japanese sardine. (a) Head of a 4-h-old larva of 3.9 mm in notochord length (NL) showing the olfactory placode. An arrow shows anterior direction. (b) Developing ciliated olfactory epithelium of a 4-h-old larva of 3.65 mm NL. (c) Ciliated olfactory epithelium of a 4-h-old larva of 3.9 mm NL. (d) Head of an 82-h-old larva of 5.6 mm NL showing the olfactory placode. (e) Ciliated olfactory epithelium of a 10.2-day-old larva of mm NL showing numerous kinocilia. (f) Nostril of a larva of 21.7 mm in standard length (SL). (g) Olfactory lamellae of a 32.5-mm SL specimen. An arrow shows anterior direction. (h) High-power view of an olfactory lamella in (g). AN, anterior nostril; CR, ciliated receptor cell; E, eye; IE, inner ear; KC, kinocilia; M, mouth; MR, microvillous receptor cell; OL, olfactory lamella; OP, olfactory placode; PN, posterior nostril; RE, rudimentary eye. Scale bars indicate 100 mm (a,d,f,g), 10 mm (e,h), and 1 mm (b,c).

4 Sense organ development in sardine FISHERIES SCIENCE 1039 Matured taste buds with sensory hairs on their apical region were first recognized in a 16.1-dayold larva of 11.2 mm NL (Fig. 4b). Taste bud distribution extended to the dorsal and ventral epitherium of the oral cavity in larger specimens. Lateral line system Fig. 2 Relationship between developmental stages of the nostril and standard length in the Japanese sardine. Stage I, the internostril epidermis is not formed. Stage II, developing. Stage III, completed. Fig. 3 Logarithmic plots showing relationship between the number of olfactory lamellae and standard length in the Japanese sardine. Solid circle and triangle: specimens caught with a seine net in 1988 and Open circle and triangle: specimens caught with a scoop net in Triangles joined by a vertical line indicate different lamella numbers from the two sides of the nostrils in the same specimen. Taste buds Several taste bud rudiments covered by the epithelium (Fig. 4a) were first observed on the basal part of the lower branchial arches, the posterior part of the upper and lower branchial arches, and the pharynx in a 16.1-day-old larva of 10.6 mm NL. In 3.7-h-old larvae of 3.6 and 3.65 mm NL, two pairs of free neuromasts were observed on the head and 10 pairs on both sides of the trunk (Fig. 4c,e). The number of free neuromasts on the head increased to four pairs in a 31.5-h-old larva of 4.75 mm NL, seven pairs in a 53-h-old larva of 4.85 mm NL, and 10 pairs in a 77.5-h-old larva of 4.8 mm NL at the first-feeding stage. The number of free neuromasts on the trunk was 11 or 12 pairs on both sides in a 31.5-h-old larva and 12 pairs in a 53-h-old larva (Fig. 4d, 58.5-h-old larva). A 77.5-h-old larva also had 12 pairs on both sides of the trunk. A 12.2-day-old larva of 9.65 mm NL had 14 pairs of free neuromasts on the head and 26 or 30 pairs on both sides of the trunk. Most of these neuromasts on the trunk were located around the myoseptum between adjacent myomeres, and became flatter than those at earlier stages (Fig. 4f). A 21.7-mm SL larva had distinct free neuromasts surrounded by grooves, although neuromasts in a 22.0-mm SL larva were still of rudimentary appearance. It was difficult to detect apparent free neuromasts on the trunk in the 31.8 mm SL specimen, as if they had disappeared. The head lateral line canal began to form in the region of the lateral recess in a 20.9-mm SL larva, and free neuromasts started to sink into the canal (Fig. 4g). Formation of the infraorbital and mandibular canals preceded that of the supraorbital and preopercular canals. These four canals were completed in a 32.5-mm SL specimen (Fig. 4h). Inner ear In a 5.7-h-old larva of 3.75 mm NL, the inner ear was an oval-shaped auditory vesicle with two otoliths, the lapili and sagitta. The sensory epithelium was situated ventrally, consisting of the rudiments of the utricular and saccular maculae rudiments. No apparent ciliation of the epithelium was observed (Fig. 5a). The upper and lateral protrusions of the semicircular canals were observed in a 32-h-old larva of 5.5 mm NL (Fig. 5b). The auditory vesicle was

5 1040 FISHERIES SCIENCE M Matsuoka Fig. 4 Transverse histological sections of the taste buds and scanning electron micrographs showing development of the lateral line system in the Japanese sardine. (a) Taste bud rudiment of a 16.1-day-old larva of 11.2 mm notochord length (NL). (b) Taste bud with sensory hairs of a 16.1-day-old larva of 11.2 mm NL. (c) Free neuromasts (arrows) of a 4-h-old larva of 3.85 mm NL. (d) Newly formed free neuromasts (arrows) of a 58.5-h-old larva of 5.4 mm NL. (e) Magnified free neuromast on the trunk of a 4-h-old larva of 3.65 mm NL. (f) Free neuromasts (arrows) of a 28.2-day-old larva of 13.8 mm NL. (g) Neuromast (arrow) sinking into the infraorbital canal of a 21.7-mm standard length (SL) larva. (h) Pores of the preopercular canal (arrows) of a 32.5-mm SL specimen. BA, branchial arch; FN, free neuromast; SH, sensory hairs; TB, taste bud. Scale bars indicate 100 mm (c,d,f,h), 10 mm (a,b,g), and 1 mm (e).

6 Sense organ development in sardine FISHERIES SCIENCE 1041 Fig. 5 Transverse histological sections showing development of the inner ear in the Japanese sardine. (a) Auditory vesicle with the otolith and sensory macula of a 5.7-h-old larva of 3.75 mm notochord length (NL). (b) Auditory vesicle of a 32-h-old larva of 5.5 mm NL showing the initial formation of the semicircular canals. (c) Auditory vesicle of a 49.5-h-old larva of 4.65 mm NL. (d) Inner ear of a 79.5-h-old larva of 5.4 mm NL. (e) Inner ear of a 16.1-day-old larva of 10.6 mm NL. (f) Inner ear of a 20.9-mm standard length (SL) larva showing the formation of the lagena. (g) Complete saccule in a 35.6-mm SL juvenile and the recessus utriculi (arrow). AC, anterior vertical canal; B, brain; C, crista; CA, cartilage; HC, horizontal canal; L, lagena; LA, lapillus; M, macula; N, notochord; PC, posterior vertical canal; S, saccule; SA, sagitta; U, utricle; VC, vertical canal. Scale bars indicate 100 mm.

7 1042 FISHERIES SCIENCE M Matsuoka apparently expanded and three semicircular canals (anterior vertical, horizontal, and posterior vertical) with rudimentary cristae formed in a h-old larva of 4.65 mm NL (Fig. 5c). The auditory vesicle (inner ear) was fully expanded in a 79.5-h-old larva of 5.4 mm NL at the first-feeding stage (Fig. 5d). The utricular and saccular maculae possessed apical sensory hairs, and the cristae of the semicircular canals were accompanied by cupula. The structure of the inner ear in larvae smaller than 18 mm SL was characterized by the ventral wall of the saccular region being slightly concave and cartilage already beginning to cover the inner ear (Fig. 5e). In an 18.6-mm SL larva, the saccular pocket was clearly concave. The sensory macula of the lagena was first observed, although the lagena pocket was not yet formed. The pro-otic bulla formed and was connected with the gas bladder through the precoelomic capillary, although it was not yet gas filled. The lagena pocket formed in a 20.9-mm SL larva (Fig. 5f), and the pro-otic bulla was entirely gas filled and connected with the utricular macula and lateral recess. The saccular pocket developed into the saccular sac and it was connected to the utricle through the recessus utriculi (Fig. 5g). The diameters of the recessus utriculi were 66, 26, and 15 mm in 29.5, 32.15, and 35.6 mm SL specimens, respectively. In the latter two larger specimens, the inner ear structure was thought to be completed in the adult condition. DISCUSSION In the olfactory organ of Sardinops melanostictus, both the ciliated and microvillous receptor cells were found in 4-h-old larvae. Two types of receptor cells were observed in the early stages such as 1 2- day-old larvae of the Dover sole Solea solea, 12 newly hatched Oplegnathus fasciatus larvae, 5 3-day-old Micropterus salmoides larvae, and 1.5-day-old Oreochromis niloticus larvae. 8 So, the olfactory system in S. melanostictus seems to function shortly after hatching, as found for the northern anchovy Engraulis mordax. 13 Kawamura 14 suggested that the olfactory pits expand and then the olfactory sense becomes functional at an earlier stage than retinal pigmentation and mouth opening in fish. In larval S. melanostictus, two types of receptor cells, ciliated and microvillous, and ciliated nonsensory cells were distinguishable, but rod cells were absent. Rod cells have been reported in the larvae of Pagrus major, O. fasciatus, 5 and Paralichthys olivaceus, 7 while they were not observed in M. salmoides, O. niloticus, 8 and the rainbow trout Oncorhynchus mykiss. 15 Eller et al. 16 suggested that rod cells may be generated by artifactual alteration of the cell surface of ciliated cells due to fixation. Yamamoto and Ueda 17 reported that a small number of ciliated non-sensory cells (type 1 ciliated cells) are present in an approximately 12 cmlong specimen of S. melanostictus, while the present study revealed that numerous kinocilia of these cells are present in the larval stage of the same species. As kinocilia are thought to produce water current through the olfactory organ during the less mobile stages, 8 they may decrease in number as swimming activity increases and ventilation system by the accessory nasal sacs is developed. 18 The olfactory lamellae in fish increase in number during ontogeny to attain species-specific count. 19 Yamamoto and Ueda 17 reported that 12 cm-long S. melanostictus specimen had 24 lamellae. In the present study, the number of lamellae reached 28 in an 182-mm SL specimen. Considering equation (4) had a slope of 0.213, it is possible that larger specimens have more lamellae. However, equations (1) and (2) also indicate that the numbers of olfactory lamellae in specimens collected with a seine net were more than those with a scoop net within the range of equivalent body length (Fig. 3), implying that body length may have greatly shrunk by capture with a seine net. No taste buds were observed in first-feeding stage larvae of S. melanostictus and the complete taste buds were first developed in a 16.1-day-old larva of 11.2 mm NL. In P. major, 15-day-old larvae first possessed the taste buds, 20 and in P. olivaceus, 12-day-old larvae. 7 These indicate that gustation is not concerned with the first feeding in these marine fish larvae, as suggested by Ishida and Kawamura. 5 Clupeoid fishes have a developed lateral line system on the head, but lack it on the trunk. The development and distribution of the system of free neuromasts in clupeoids is not well known. 21 Newly hatched larvae of the pilchard Sardina pilchardus possessed seven or eight free neuromasts on both sides of the trunk, 22 while those of E. mordax had a row of neuromasts on the trunk and three or four pairs on the head, with the numbers increasing with size. 13 Newly hatched larvae of the herring Clupea harengus had 10 pairs of free neuromasts on the trunk and six to eight pairs on the head. 21 The present study showed that S. melanostictus larvae shortly after hatching had 10 pairs of free neuromasts on the trunk and two pairs on the head. Notably, the number of free neuromasts on the head increased to 10 pairs by the

8 Sense organ development in sardine FISHERIES SCIENCE 1043 first-feeding stage, with 12 pairs on the trunk. It is considered that newly hatched larvae of S. pilchardus, E. mordax, and S. melanostictus from pelagic eggs may be at a less advanced condition of neuromast development than C. harengus larvae hatched from demersal eggs. Kuroda 23 reported that the number of free neuromasts on the trunk in S. melanostictus decreased after larvae reached about 7 mm TL (total length) and almost disappeared in the mm TL specimen. In the present study, scanning electron microscope (SEM) observations revealed that a 13.8-mm NL larva had apparent free neuromasts, although they were to be flatter than those at earlier stages. By stereomicroscopic observation, they should not be found. Considering that a mm SL larva had rudimentary free neuromasts and no apparent neuromasts were observed in a mm SL larva, further observations are required to answer the question whether neuromasts disappear or change in form. The head lateral line in S. melanostictus developed at the lateral recess as in other clupeoid fishes. 13,24,25 It appeared from 20.9 mm SL in S. melanostictus, a little larger than mm in C. harengus, mm in E. mordax, 13 and 17 mm in the Atlantic menhaden Brevoortia tyrannus. 25 The canal formation in S. melanostictus was completed by 32.5 mm SL, at a smaller size than in C. harengus 24 (50 60 mm). The teleost inner ear consists of two functionally discrete regions, the utricle and three semicircular canals as the vestibular region and the saccule and lagena as the auditory region. 26 Little is known about the development of the inner ear in fish, especially in the histology and/or cytology. 4,26 In newly hatched larvae of P. olivaceus, 7 M. salmoides, 8 and O. marmoratus, 9 the inner ear is an oval-shaped auditory vesicle with two otoliths, as found in S. melanostictus. In S. melanostictus, three semicircular canals with cristae had formed in a 79.5-h-old larva. Thereafter, larvae first assumed an upright position at this time and began to swim forward. The formation of the three semicircular canals with cristae seems necessary for body e quilibrium. The pro-otic bulla was gas filled and connected with the utricle at 20.9 mm SL. As the bulla-swimbladder system probably functions in detecting and analyzing small vibrational pressures and Fig. 6 Relationship between sense organ development and ontogenetic intervals in the Japanese sardine.

9 1044 FISHERIES SCIENCE M Matsuoka displacements, 25 larvae beyond this stage can use their auditory and pressure sense with the system. The lagena, the last organ to be formed in the inner ear, first appeared at 20.9 mm SL and the complete saccular sac was formed by about 32 mm SL. These two organs and the bulla-swimbladder system are concerned with the auditory sense, so auditory ability may increase at later larval and juvenile stages. Matsuoka 1 defined the ontogenetic intervals in S. melanostictus from osteological observations. The larva period consists of three phases: larva I (from the first-feeding stage to 10 mm NL), larva II (from 10 mm NL to 20 mm SL), and larva III (from 20 mm to 34 mm SL), corresponding to metamorphosis. The development of lateral muscle 2 and retina 3 seems to proceed along with these osteologically defined intervals. Development of the olfactory organ, taste buds, lateral line, and inner ear in S. melanostictus also should be applied to the above ontogenetic intervals. Recently hatched larvae contain olfactory receptor cells, so they seem capable of perceiving smell. They are also equipped with several free neuromasts on the head and trunk, and respond to external stimuli such as water vibration. They usually float with their head down, except for occasional swimming bursts, in the free embryo phase of the embryo period. By the first-feeding stage, some of the skeleton, 1 red fibers of the lateral muscle, 2 and the pure-cone retina of the eye 3 have developed, and the larva I phase of the larva period has started. The inner ear is fully expanded and three semicircular canals with cristae are formed. This development enables larvae to maintain an upright position, swim forward, and start feeding. Prey perception in early feeding larvae may be dependent on vision, the olfactory sense, and water flow recognition by free neuromasts. Taste buds appear at mm NL and they enable larvae to search and select prey organisms by gustation. This stage corresponds with the beginning of the larva II phase, defined by the formation of fin rays and pterygiophores. At about 20 mm SL, the beginning of the larva III phase, many changes in sense organ development occur as well as in the skeleton, 1 lateral muscle, 2 and retina. 3 Formation of the olfactory nostrils and lamallae begins, free neuromasts on the trunk become obscure, and ossification of the head lateral line canals begins. The saccular and lagena pockets are formed, and the pro-otic bulla is gas filled. These are metamorphic changes in sense organ development and are almost completed by about 32 mm SL. It is the end of the larva III phase of the larva period and the start of the juvenile phase of the juvenile period. Quantitative changes continue until the adult condition is reached at mm SL, as suggested by the second inflection point in the relationships between the number of olfactory lamellae and SL. Considering that the osteological structure is also completed at mm SL, 1 the development of the skeleton, lateral muscle, retina, and sense organs seems to occur in parallel (Fig. 6). This size indicates the end of the juvenile phase and the start of the young phase of the juvenile period. ACKNOWLEDGMENTS I express my sincere thanks to Mr Takumi Mitani of the National Research Institute of Fisheries Science for help in collecting sardine eggs. This work was supported in part by a Grant-in-Aid (Bio Cosmos Program) from the Ministry of Agriculture, Forestry, and Fisheries. REFERENCES 1. Matsuoka M. Osteological development in the Japanese sardine, Sardinops melanostictus. Ichthyol. Res. 1997; 44: Matsuoka M. Development of the lateral muscle in the Japanese sardine Sardinops melanostictus. 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