Fisheries Science 60(4), 361-368 (1994)

Development of Sense Organs and Mouth and Feeding of Reared Marble Goby Oxyeleotris marmoratus Larvae*

Shigeharu Senoo,*1 Kok Jee Ang,*1 and Gunzo Kawamura*2 *1 Faculty of Fisheries and Marine Science , Universiti Pertanian Malaysia, 43400 UPM, Serdang, Selangor, Malaysia *2Faculty of Fisheries , Kagashima University, Shimoarata, Kagoshima 890, Japan (Received August 23, 1993)

To obtain the fundamental information required toooestablish an artificial seed production technique for the marble goby Oxyeleotris marmoratus, the development of sense organs and mouth and the feeding of the larvae were observed. The larvae exhibited a S-posture and horizontal swimming at 2 days after fertilization (d AF) and commenced feeding on phytoplankton (species unknown) at 3 d AF when the eyes, the otic vesicle with ciliated epithelium, the free neuromasts on the head and the trunk, and the ciliated olfactory epithelium were functional. The first taste buds appeared in the oral cavity at 6 d AF after the onset of feeding on Brachinous spp. The larvae commenced feeding on Cyclops sp. at 7 d AF, Moina sp. at 10 d AF, and Anemia salina nauplii at 15 d AF. This change of feeding might be due to the development of the mouth and mobility of larvae rather than the development of sense organs. At 20 d AF, the larvae developed free neuromasts on the caudal fin. Twin cones and rods appeared in the ratinae at 30 d AF when the larvae changed in phototaxis from positive to negative. The larvae became benthic in habit at 35 d AF.

Key words: Oxyeleotris marmoratus, sense organs, mouth, feeding, artificial seed production, larval fish

One principal requirement in fish culture is successful Science, Universiti Pertanian Malaysia, from June to August 1992. The mass-production of fry as seed stock. Heavy mortality larvae were obtained from naturally spawned eggs under artificial usually occurs early in the life history of fish if suitable food conditions.11) The eggs hatched during 35-60 h after fertilization (h AF), with a peak at 40-50 h AF at 28.5-30.5•Ž. The age of the fish was in hours is scarce, especially when the yolk sac is exhausted and the or days was based on the fertilized time. The next day of spawning was larvae must begin active feeding.1) The seed production defined as 1 day after fertilization (d AF). of the marble goby Oxyeleotris marmoratus has been at- Five thousand newly hatched larvae, 3.15-3.32 mm in total length (TL) tempted in Singapore,2) Thailand,3) and Malaysia, 4, 5) and at 40 h AF, were reared in a 1,000 1 tank following the method of Senoo et al." Various live organisms such as several species of phytoplankton a high mortality in the early stage prevented smooth rearing. (species unknown), Brachionus spp., Cyclops sp., and Monia sp. were Tan and Lam2) noted a high mortality of the larvae due to collected using a plankton net with 60ƒÊm mesh size from an earthen fish starvation. Senoo et al.') reported that the larvae had a pond in the university in which 0. marmoratus had been naturally sufficiently large mouth to feed on artificial powdered feed propagating. The collected organisms in the water were filtered using a and Brachinous spp., but some of the larvae did not and plankton net with 200ƒÊm mesh size and given to the larvae from 2 d AF. After 10dAF, Anemia salina nauplii were given in addition to the above eventually died. organisms. Feeds were given ad libitum at 0800 and 1700. The tank was Studies of the behavior and ecology of various stages of placed under natural light in the hatchery and the maximum illuminance fish development related to the causes of mortality may was 400 lx on the water surface during the daytime. During the rearing provide much needed information. The aquaculture of fish period, the water temperature, dissolved oxygen (DO), and pH in the tank ranged from 27.5--32.5•Ž, 6.3-7.8 mg/1, and 6.3-6.9, respectively. The depends on an adequate knowledge of the behavior of the feeding and swimming behavior of the fish in the tank were observed early life stages that cause the greatest technical difficulties several times a day. Some fish in the tank were transferred into a 1 1 glass in culture.6) The ontogenetic changes of behavior are closely beaker, and their reactions to water current and light from an electric related with the development of sense organs.1,7-10) It has torch were observed. Other specimens were anesthetized by MS 222 been suggested that the food preference of fish larvae is (m-aminobenzoic acid ethylester methanesulfonate) at 10-50 ppm to observe the morphological development, and body measurements were based on the differentiation of the teste buds in Paralichthys made with a dissection photomicroscope and a profile projector. The olivaceus, salmoides, and Tilapia nilotica.7,9) mouth size was represented by the upper jaw length since exact Information on the development of sense organs and mouth measurement of the gaped mouth was difficult. should facilitate a better understanding of the feeding Fish in the tank were sampled at random every day until 10d AF, and behavior of O. marmoratus larvae. thereafter at 5 day intervals until the end of the experiment (20th August, 1992). The sampled fish were preserved in 10% buffered formalin and processed for morphogenetic study of the sense organs. For the scanning electron microscopy (SEM), the preserved samples were post-fixed in 4% Materials and Methods glutaraldehyde and 0.1 M sodium cacodylate buffer with 1% osmium tetraoxide, dehydrated in an acetone series, critical dried, and spattered O. marmoratus larvae were reared at the Faculty of Fisheries and Marine with gold. For the photomicroscopy, the preserved larvae were embedded

* Study of Artificial Seed Production of Marble Goby-‡U. 362 Senoo et al.

in paraffin, and 3ƒÊm thick horizontal, sagittal, and cross sections were made and stained by Heidenhain's azan method.

Results

Larval Development and Behavior The detailed morphological development of O. marmo ratns larvae has been described under artificial conditions.') Only the notable features of larval development, behavior, growth (Fig. 1), and mouth development (Figs. 2 and 3) obtained in this study are presented here. The larval development was slightly faster than in the previous observation5) probably due to the higher water temperature. The larvae 40 h AF (1 d AF) had neither open mouth (Fig. 2A) nor functional sense organs. These newly Fig. 1. Early growth of 0, marmoratus. hatched larvae lay on the tank bottom and did respond to Filled circle, mean; vertical line, standard deviation (n=10).

Fig. 2. Development in mouth morphology with growth of 0. marmoratus. Scale bar, 500 gm.

Fig. 3. Changes in upper jaw length of 0. marmoratus and in food organisms ingested . Filled circle, mean of upper jaw length (n= 10); horizontal line, feeding period. Sense Organs of Marble Goby Larvae 363 a glass pipette carefully inserted into the water, and were under the profile projector was 330 ƒÊm. The sizes of Cyclops easily caught. sp. ingested from the head by this specimen were measured At 55 h AF (2 d AF), the mouth was formed at an inferi and found to be 300-600 ƒÊm, total length, 200-400 ƒÊm, body or position (Fig. 2B). The larvae commenced vertical length and 100-200 ƒÊm body width, and not well digested. swimming, avoided the approaching pipette and were In the larvae which fed well on Cyclops sp., 2 teeth were positively rheotactic. At 65 h AF (2d AF) when the air formed on the upper jaw and 4 on the lower jaw (Fig. 2E). bladder was inflated, most of the larvae showed an At 10 d AF, a rudiment of the caudal fin appeared, and S-posture, horizontal swimming, and became positively the larvae congregated in the middle water column and phototactic. At 3 d AF, the S-posture, horizontal swimming, commenced feeding on small Moina sp. which measured and positive rheotaxis became active, and the larvae 200-300 ,ƒÊm in body length, and fed well on Brachionus spp. congregated in the surface water column. At this stage, and Cyclops sp. At 15 d AF, rudiments of the anal and mouth movements were observed (Fig. 2C) and the larvae second dorsal fins appeared, and the larvae commenced commenced feeding on phytoplankton of 30-60 ƒÊm in feeding on Artemia salina nauplii (Fig. 2F). Thereafter the dimeter. At thid point, the upper jaw length was 200-210 ƒÊm larvae preferred Anemia to the other organisms given. At (Fig. 3), and the yolk sac as absorbed almost the same as 20 d AF, rudiments of the pelvic and first dorsal fins the air bladder. While feeding on the phytoplankton, the appeared, the aggregation expanded from the middle to the mouth seemed large enough to feed on Brachionus. bottom water column, and the larvae were suspended At 5 d AF, the larvae showed an active S-strike and strong motionless but oriented themselves against the water flow phototaxis. The yolk sac was completely absorbed, the in the column, and were negatively phototactic. The teeth mouth developed at a superior position, and a pair of on the upper jaw were regularly arranged in a single row. canine-like teeth were found on the lower jaw (Fig. 2D). At 25 d AF, the aggregation dispersed and the fish moved At this stage, the larvae commented feeding on Brachinous solitarily regardless of the water flow in the bottom water spp. At 7 d AF, the gaped mouth size of a larva measured column. At 30 d AF, the head was roundish, the mouth

Fig. 4. Photomicrographs of 0. marmoratus retinase. A, harizontal section showing the densely pigmented retinae at 3d AF, scale bar, 200ƒÊm; B, sagittal section showing a regular arrange ment of single cones at 3 d AF, scale bar, 100ƒÊm; C, sagittal section showing the presence of twin cones (arrow) at 30d AF, scale bar, 100ƒÊm; D, sagittal section of the cone layer showing square arrangement of twin and single cones at 35d AF, scale bar, 100ƒÊm. 364 Senoo et al. became adult like (Fig. 2G) and all fins were formed. At the same retinae at 30 d AF, the ratio of the number of 35 d AF, the fish repeated an active intermittent swimming cone ellipsoids to that of photoreceptor nuclei exceeded 1, near the bottom and were resting on the bottom. They were inplving the appearance of the rods at this stage. capable of feeding without exhibiting the S-posture on 2. Lateral line system: The change of the arrangement the tank bottom. In the fish at 40d AF, yellow-ocher of free neuromasts is shown in Fig. 5. Free neuromasts were pigmentation with scales appeared on the body, and fish not observed in the newly hatched larvae at 40 h AF. In were resting motionless on the bottom all the time except the larvae at 55 h AF, well-developed free neuromasts with for when feeding and being disturbed. cupula were found in the epidermis around the olfactory During the experiment, the feeding was diurnally active pits and the eyes and on the trunk. Thereafter, the free and the larvae did not feed on the live organisms at night. neuromasts increased in number on the head and the trunk. Some of the free neuromasts were found along the caudal Ontogenetic Development of Sense Organs fin rays at 20 d AF and many at 25 d AF (Fig. 6A). The 1. Eyes: In the newly hatched larvae at 40 h AF, the free neuromasts on the trunk were submerged in fish at 40 d eyes were not pigmented and the cornea was not developed. AF, implying the first formation of the lateral line canal at The retinal cells were arranged radially and were not yet differentiated into different retinal elements. At 55 h AF, the eyes were slightly pigmented and the stratification of the retinal elements began; photoreceptor nuclei (cone nuclei) were formed, and the inner plexiform layer was differentiated to separate the ganglion layer from the inner nuclei layer. In the larvae at 3 d AF, the eyes were deeply pigmented, horizontal cells and cone ellipsoids were formed, and cone nuclei were long and thick in the temporal retina showing a regional differentiation of the retina (Fig. 4A). At this time, the nerve fibers projected the optic tectum. The photoreceptors were all single cones with no rods or twin cones (Fig. 4B). Twin cones were first found in the retinae at 30d AF (Fig. 4C) and formed a square mosaic without an additional single cone at 35 d AF (Fig. 4D). In

Fig. 6. Free neuromasts on the caudal fin of 0 . marmoratus. A, free neuromasts are radially arranged along the caudal fin rays Fig. 5. Change in arrangement of free neuromasts with growth of 0. in a larva at 25d AF, scale bar, 400ƒÊm; B, scanning electronmicro marmoratus. graph showing a free neuromast in a larva at 40 d AF, scale bar, 6 ƒÊm. Sense Organs of Marble Goby Larvae 365

Fig. 7. Scanning electronmicrograph showing free neuromasts on the lower jaw of O. marmoratus at 40 d AF. Scale bar, 330ƒÊm.

Fig. 8. Scanning electronmicrograph showing distribution of the taste buds on the tongue of O. marmoratus at 40 d AF. Scale bar, 250ƒÊm. this stage. In these specimens, the free neuromasts were found still in the epidermis of the head (Figs. 5 and 7) and the caudal fin (Fig. 6B). Fig. 9. Scanning electronmicrographs showing the nares of O. marmoratus. 3. Inner ear: The inner ear was an oval-shaped otic A, olfactory pits are elongated at 35 d AF, scale bar, 300ƒÊm; B, vesicle containing two otoliths but no apparent ciliation of the anterior (AN) and posterior (PN) nares are formed at 40d AF, the epithelium was found in the newly hatched larvae at free neuromasts around the eye and nares are pointed by arrowheads, 40 h AF. Ciliation was found in the specimens at 55 h AF. scale bar, 200 ƒÊm. The otic vesicle formed a pocket of the anterior semicircular canal at 5 d AF. The anterior and posterior semicircular in the epithelium of the oral branchial cavity, gill arches, canals were formed at 6 d AF, and lagena was found at 12 d and phyaryngeal region at 9 d AF. In the juveniles at 40 d AF. The inner ear was partially ossified at 6 d AF and the AF, many teste buds were observed on the tongue but none ossification was complete at 30d AF. on the lips (Fig. 8). 4. Taste buds: The first taste bud was observed in the 5. Olfactory organ: Olfactory pits were opened in the epithelium of the oral cavity of the larvae at 6 d AF, then larvae at 55 h AF. Observation of the olfactory epithelium 366 Senoo et al. with the SEM was possible only 65 h AF when the olfactory larvae is diagrammatically shown in Table 1, pits expanded horizontally. The olfactory epithelium was Many studies have shown that vision is the prinicipal well-ciliated at 3 d AF, but the types of cell were not sense used for feeding by fish larvae.7,9,10,12-17) Fish larvae discriminated. In most of the larvae at 30-35 d AF, the feed actively during the hours of daylight, seeking out food olfactory pits elongated in shape (Fig. 9A), and were organisms by sight, but cease active feeding at night.l0.18) completely separated into the anterior and posterior nares This is also true in 0. marmoratus larvae. While the mouth at 35-40d AF (Fig. 9B). was opened at 55 h AF and the first S-posture and horizontal swimming were observed at 65 h AF, the larvae commenced Discussion feeding on phytoplankton at 3 d AF when the eyes were pigmented, and no feeding was observed at night. At this The correlation between bahavioral changes and onto- stage, the yolk sac had been absorbed to almost the same genetic development of sense organs on 0. marmoratts size as the air bladder, and the S-posture and horizontal

Table 1. Correlation between behavioral changes and ontogenetic development of the sense organs on 0. marmoratus larvae Sense Organs of Marble Goby Larvae 367 swimming were active. These morphological and behavioral the benthic habit rested motionless most of the time on the characteristics can be used to indicate the timing of the first bottom. Therefore, in this fish, the free neuromasts on the intensive feeding with phytoplankton to ensure the survival caudal fin could be functioning as a mechanoreceptor. of the larvae. Among the zoo plankton given, 0. marmoratus Based on the timing of appearance and submergence, the larvae fed on Brachionus spp. first then on Cyclops sp., free neuromasts of 0. marmoratus seemed to be grouped Moina sp., and Artemia salina nauplii in order. While the into three; free neuromasts on the head, free neuromasts visibility of plankton is known to influence their vulner on the trunk, and free neuromasts on the caudal fin. The ability to predators, 19,20) the difference in pigmentation of differences in the timing and position on body might imply these plankton can be excluded as a possible reason for differences in the function and role in their behavior betwen food selection, since the most invisible Cyclops sp. was eaten the three groups as suggested by the schooling of red sea earlier than the slightly pigmented Moina sp. and these two bream Pagrus major and Lepomis macrochirus.25) were eaten earlier than the deeply pigmented Anemia. In young marine fish, the first taste buds appear sev Gustation can also be excluded as a reason because the eral days after the onset of feeding wheras freshwater larvae fed on Brachionus along with Cyclops and Moina fish larvae develop taste buds before the first feeding.1) The after developing taste buds. Possible factors governing food timing of the appearance of the taste buds of 0. marmoratus selection might be the size and moving manner of the larvae is similar to that of marine fish; the teste buds plankton and the development of the larval jaws rather than appeared in the oral cavity after the onset of feeding on the development of the sense organs. Brachionus would be phytoplankton and Brachionus spp. This implies that the easy to capture for it is the smallest, and moves showly and gustatory sense plays only a small role in feeding.1) Although smoothly. Cyclops and Moina are larger than Brachionus the role of olfaction in larval behavior is not examined here, and move swiftly and in an irregular zigzag. The larvae fed olfaction might be related to aggregation rather than feed on these two fast-moving plankton when they were capable ing. Since the olfactory organ developed at a very early of stong S-strike and jaw movement. The developed stage. canine-like teeth facilitate gripping the plankton in the As the larvae grew, taste buds developed in the epithelium mouth, and larvae with a large developed mouth preferred of the oral branchial cavity, gill arches, and pharyngeal Artemia, probably because Artemia was large and moved region. This developmental change did not appear related less quickly when transferred from salt water to freshwater to changes in the food organisms ingested by the larvae in the rearing ta. since the later change seemed to be related to the jaw de Cycloops was found to be an unfavorable food for the velopment and mobility of the larvae rather than the sense larvae at 7-9d AF, since this plankton in the gut remained organ. undigested near the anus. Therefore, during this period, the larvae should be fed with Brachionus spp. to improve Acknowledgments The authors wish to thank Mrs. M. Kartini, Mr. A. their growth and survival. For this purpose, the sea water Rosli, and the staff who supported the experiment in the Faculty of Brachionus or some proper artificial diet should be used Fisheries and Marine Science, University Pertanian Malaysia. We are also most grateful to the Japan International Cooperation Agency for their since the mass-production and supply of freshwater financial support of this study. Brachionus is not reliable at present. In general, once the eyes are deeply pigmented, the visual References system is functional at least for light perception, and fish larvae are phototactic.7,9,10,14,16) When the eyes of 0. 1) T. Iwai: Sensory anatomy and feeding offish larvae, in "Fish Behavior marmoratus larvae became deeply pigmented, the imma and its Use in the Capture and Culture of Fishes" (ed. by J. E. tured visual system was morphologicaly completed and the Bardach, J. J. Magnuson, R. C. May, and J. M. Rinhart), larvae showed positive phototaxis. The appearance of twin International Center for Living Aquatic Resources Management, cones and rods increases the sensitivity of the retina,9,10) Manila, 1980, pp. 124-145. 2) O. K. K. Tan and T. J. Lam: Induced breeding and early development and regular square mosaic of single and twin cones is of the marble goby (Oxyeleotris marmorata, Blk.). Aquaculture, 2, advantageous for acute vision.21) 0. marmoratus larvae 411-423 (1973). with the twin cones and rods exhibited a benthic habit. This 3) P. Tavarutmaneegul and C. K. Lin: Breeding and rearing of sand retinal change in morphology and sensitivity might be goby (Oxyeleotris marmoratus, Blk.) fry. Aquaculture, 69, 299-305 preprogrammed for their wild benthic habitat16,22) and (1988). would prolong feeding time in dim light.23) 4) S. H. Cheah, S. Y. Lam, and K. J. 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