Abstract

Larvae of the bluefin tuna Thunnus orientalis were reared from hatching to day 30, sampled every day, and examined under light and electron microscopy for morphological development of the sense organs. The larvae came from fertilized eggs collected from a coastal broodstock pen and transferred to the hatchery of the Amami Station of the Japan Sea Farming Association. Growing larvae were sequentially fed rotifers, brine shrimp, and fish larvae. In this paper, morphological development is given by larval size (total length TL) and age (hours or days from hatching). Newly hatched larvae were 3.0 mm long, hung suspended head down in the water column, and avoided glass pipets put in their way. Yolk-sac larvae had well developed mechanoreceptors a pair of large free neuromasts (45 m diameter, with cilia and cupulae 40-50 m thick and 250-310 m long) behind the eyes, seven pairs of small neuromasts on the head, and seven pairs on the trunk. Yolk-sac larvae also had an inner ear an ovate otic vesicle with two otoliths and an innervated ciliated epithelium; the inner ear formed three maculae after 18 h and three pockets after 2 d. The retina was innervated after 13 h, the retinal layers and the lens formed after 19 h, and the single cones and horizontal cells in the retina differentiated after 2 d. The olfactory pits also opened after 2 d and the olfactory epithelium had ciliated receptor cells, microvillous receptor cells, and ciliated nonsensory cells. Larvae 3.9 mm long (2 d) were rheotactic and could swim 10x their total length in a single burst. Larvae 4.0 mm long had well pigmented eyes when they started to feed at 3 d. Larvae of 4.5 mm (5 d) were slightly photopositive. Larvae 5.6 mm long (8 d) had a flexed notochord, small pelvic fins, and three partly ossified semicircular canals. The taste buds differentiated first in the epithelium of the upper pharynx of larvae 5.6 mm long, when canine-like teeth and pharyngeal teeth also appeared. Larvae of 16 mm (16 d) had complete fin rays and spines except in the pectorals, had nine pairs of neuromasts on the trunk and 11 pairs on the head, and showed optomotor reaction. The development of the sensory organs are accompanied by behavioral changes that have important implications to larval ecology at sea and to tuna hatchery operations.

Introduction
Ontogeny of the sense organs and behavior is important to feeding and predator avoidance of fish larvae at sea (Blaxter 1986). Such is true as well in fish farms. Successful farming depends on understanding the behavior of fish, especially during the early larval stages when technical difficulties result in high mortality in the hatchery. Changes in larval behavior are closely related to the development of the sense organs in marine fishes (Ishida 1987), in large-mouth bass Micropterus salmoides and Nile tilapia Tilapia nilotica (Kawamura and Washiyama 1989), and in reared marble goby Oxyeleotris marmoratus (Senoo et al. 1994). Tunas are among the most important species in world fisheries but also among the least known in terms of early life history. Most studies of the early life history of tunas have depended on ichthyoplankton net samples. Yabe et al. (1966) collected larvae of the bluefin tuna Thunnus orientalis at high density in western Pacific waters, indicating spawning in the area. Davis et al. (1990) found diel patterns in the vertical distribution of larvae of southern bluefin Thunnus maccoyii and other tunas in the east Indian Ocean. Margulies (1997) examined the visual system of larvae and juveniles of Euthynnus lineatus, Scomberomorus sierra, and Auxis spp. caught at sea and inferred their performance capabilities. The recent success in breeding and rearing bluefin tuna in Japan (Miyashita et al., 2001) has made available valuable experimental material for many studies on larval biology: the digestive system (Takii et al. 1996; Miyashita et al. 1998); oxygen consumption (Miyashita et al. 1999); rotifer-size selectivity and optimum rotifer density (Sawada et al., 2000); trauma caused by collision with walls of tanks or cages (Miyashita et al., 2000); morphological development (Miyashita et al., 2001); muscle development (Hattori et al., 2001); and retinomotor responses (Masuma et al., 2001).We studied the morphogenesis of the sense organs in bluefin tuna to improve the knowledge about the early life history of this fish.

Materials and Methods

Fertilized bluefin eggs were collected from the coastal broodstock pen and transferred to a 500 l rearing tank in the laboratory of the Amami Station. Larvae and juveniles were fed S-type rotifer Brachionus plicatilis and L-type rotifer B. rotundiformis from first feeding to 25 d, Artemia nauplii from 9 d to 30 d, larvae of striped knifejaw Oplegnathus fasciatus from 10 d to 30 d, and minced and chopped fish meat from 15 d to 30 d. Water temperature in the tank ranged from 27.3 to 29.0C. The juveniles were transferred to an outdoor fish cage after 30 d. Bluefin larvae were sampled every day for 33 d (the oldest specimens were from the outdoor

cage) and examined for morphological development of the sense organs. One or two specimens were anesthetized with MS 222 and illustrated with the aid of a camera lucida. Live yolk-sac larvae were anesthetized with MS 222 for observation of the cupulae of free neuromasts under a profile projector. Some specimens were preserved in Bouins solution, embedded in paraffin, cut into 6-8 m thick longitudinal and cross sections and stained with hematoxylin-eosin for histological examination under a photomicroscope. Other specimens were preserved in 4% glutaraldehyde or 4% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4), dehydrated in an ethanol series, freeze124 THE BIG FISH BANG

dried, and coated with platinum for examination of the olfactory epithelium, free neuromasts, and taste buds under a scanning electron microscope. As it was difficult to resolve the rod cells in the retina by photomicroscope, the nature of the visual cells was ascertained by making counts of the distinct ellipsoidal structures (the cone ellipsoids) in the visual cell layer, and of the cell nuclei in the outer nuclear layer in 3-5 adjacent cross-sections of the retina. To study the functional development of the eyes, free neuromasts, and olfactory epithelium, the behavior patterns of larvae of 3.7 mm to 24 mm in total length (1 d to 21 d old) were observed both in the rearing tank and in a 2 l glass beaker in the laboratory: phototactic response to a 5-watt flashlight (method earlier described by Kawamura et al. 1983), optomotor reaction to a moving pattern of alternate black and white stripes 1 cm wide (Kawamura and Hara 1980), rheotaxis (Kawamura et al. 1984).

Results
Larval development and behavior. The typical pattern of early larval development of bluefin tuna is shown in Figure 1. The newly hatched yolk-sac larvae averaged 3.0 mm in total length TL. The pectoral fin buds appeared after one day.When the larvae were 3.9 mm long (2 d), the pectoral fins were formed, the yolk and oil globule were nearly resorbed, and the mouth and anus opened. Larvae avoided a moving glass pipet well; the single-burst avoidance movement increased to as much as 4 cm (ca. 10x TL) after 2 d. Larvae were also capable of horizontal movement, staying upright, and swimming against the current (positive rheotaxis). Larvae at 4.0 mm (3 d) had well pigmented eyes, a bent and swollen rather than straight gut, and small amounts of yolk and oil globule still remaining; these larvae started to feed on rotifers. Larvae 4.5 mm long (5 d) were positively phototactic and some had swimbladders inflated for the first time. The rudiments of the hypural plate and the anal fin appeared in larvae 5.8 mm (9 d), at the same time as four teeth on the upper jaw and three on the lower jaw. The notochord was slightly flexed in 5.8 mm larvae and fully flexed in 8.2 mm (12 d) larvae. All larvae 8.2 mm long had inflated swimbladders and forked caudal fins. Cannibalism was first observed among the flexion larvae. Larvae 16 mm (16 d) had the full complement of rays and spines in all fins except the pectorals, and showed optomotor reaction for the first time. The pectoral fin in most larvae larger than 24 mm (21 d) attained the full complement of 33 soft rays, and this marked the start of the juvenile stage. Scale formation began with the lateral line scales behind the operculum in 18 mm larvae (18-20 d), proceeded over the trunk, but was still not completed by 33 d in juveniles 29.8-45.5 mm. Table 1 shows the chronology of morphological, sensory, and behavioral development of bluefin larvae. Details are given in the following sections. Lateral line system. Bluefin larvae hatched with unpigmented eyes, closed olfactory pits, no taste buds, but well developed free neuromasts. Yolk-sac larvae hung suspended head down in the water column but were capable of avoiding a glass pipet gently placed in their way in the rearing tank. A pair of extremely large free neuromasts (45 m diameter, with cilia and cupulae 40-50 m thick and 250-310 m long) was present behind the eyes, seven pairs of smaller neuromasts (cupulae 20 m thick and 30-40 m long) on the trunk, one pair on the snout, and five pairs around the eyes (Figure 2). Bluefin larvae did not seem hindered by their large cupulae during burst swimming. Electron microscopy of the free neuromasts showed that the neighboring hair cells were arranged in opposite directions and a single neuromast had four polarities, cranial-caudal and dorsalventral (Figure 3). The neuromasts with four polarities in bluefin can detect four-directional water displacements. The free neuromasts with four polarities are also found around the eyes in Japanese parrotfish Oplegnathus fasciatus (Ishida and Kawamura 1985). The four polarities might be more advanced in detecting moving objects than two polarities, cranial-caudal or dorsal-ventral, which can detect only bidirectional water displacements. The free neuromasts increased to 9 pairs on the trunk and 11 pairs on the head in larvae 16.0 mm long (16 d). In juveniles 30.4 mm (23 d), the epidermis folded to form the lateral recess in which the free neuromasts were lodged, but some free neuromasts remained exposed in the open

grooves on the head. Juveniles 29.8-45.5 mm long and 33 d old no longer had free neuromasts in the epidermis, but almost complete lateral line canals. nner ear. At hatching, the inner ear of bluefin larvae was an ovate vesicle with two otoliths and an innervated ciliated epithelium (Figure 4). Three maculae were formed 18 h from hatching, and the semicircular canals and crus commune after 2 d. The canals were partly ossified in 5.6 mm larvae (8 d) and completely ossified in 8.2 mm larvae (12 d) when the notochord was fully flexed. The third otolith was not seen under a dissection microscope in larvae during the preflexion and flexion stages. Eye. The visual system of bluefin larvae also developed rapidly (Figure 5). In larvae 3.4 mm long (13 h from hatching), the eye lens was formed, the outer nuclear layer of the retina had a single layer of cells and a thin inner plexiform layer was already recognizable. After 25 h, the 3.7 mm larvae had short but identifiable cone ellipsoids in the retina and a single layer of horizontal cells. The eyes were well pigmented in 4 mm larvae (3 d) and the visual system was morphologically complete, although the retina had only single cones. In larvae 4.5 mm (5 d), the retina thickened in the temporal area into an area lateralis, indicative of acute vision in the nasal direction. The horizontal cells formed three layers in the temporal and ventro-temporal regions of the retina in 10.5 mm larvae (16 d) and along the entire retina with an area lateralis in the ventro-temporal area in 30.4 mm larvae (23 d).