Fish Sci (2010) 76:457–461 DOI 10.1007/s12562-010-0237-3
ORIGINAL ARTICLE
Biology
Feeding behavior under dark conditions in larvae of sutchi catfish Pangasianodon hypophthalmus Yukinori Mukai • Audrey Daning Tuzan Leong Seng Lim • Syahirah Yahaya
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Received: 11 December 2009 / Accepted: 10 March 2010 / Published online: 8 April 2010 Ó The Japanese Society of Fisheries Science 2010
Abstract Sutchi catfish Pangasianodon hypophthalmus hatch with morphologically immature sensory organs; however, sensory organs develop rapidly with larval growth. Two-day-old larvae commenced ingesting Artemia nauplii. The larvae displayed many taste buds on the barbels, the head surface, and in the buccal cavity. Other sense organs were also well developed at this stage. Feeding experiments revealed that 2-day-old larvae ingested Artemia under both light and dark conditions, moreover, the larvae could ingest frozen dead Artemia. The ingestion rates for 4- and 7-day-old larvae were significantly higher under dark conditions than under light conditions. The rates using frozen dead Artemia were mostly higher than the rates using live Artemia. Therefore, feeding behavior under dark conditions is most likely not mediated by visual or mechanical senses, but rather by chemosensory senses, such as taste buds. Larval fish are vulnerable to predators; thus, if they can search for and eat food at night, they can avoid diurnal predators. The behavior observed here appears to represent their survival strategy. Moreover, these results suggest a new possibility that sutchi catfish larvae can be reared under dark or dim light conditions in order to improve survival and growth rates as in the case of African catfish Clarias gariepinus. Keywords Feeding behavior Larvae Pangasianodon hypophthalmus Sensory organs
Y. Mukai (&) A. D. Tuzan L. S. Lim S. Yahaya Borneo Marine Research Institute, Universiti Malaysia Sabah, 88999 Kota Kinabalu, Sabah, Malaysia e-mail:
[email protected]
Introduction The sutchi catfish Pangasianodon hypophthalmus (synonym Pangasius hypophthalmus) originates from the area extending from the Mekong River basin of Vietnam to the Chao Phraya River of Thailand [1, 2]. The sutchi catfish is a popular fish for aquaculture; it is cultured widely in Asia in countries such as Vietnam, Malaysia, Indonesia, Laos, Cambodia, and China [1–5]. The production of sutchi catfish in aquaculture is considerable. For instance, Vietnam is the largest production country of sutchi catfish in Southeast Asia [6]. There, in 2007, 1,200,000 tons of catfish was produced of which 95–97% was sutchi catfish [7]. A total of 387,000 tons of fillets of sutchi catfish was exported to over 80 countries [7]. Therefore, sutchi catfish is an important species for aquaculture. Artificial seed production is succeeding in fulfilling the requirements from fish farms; however, larval rearing still has the problem of high mortality due to cannibalism at the larval stage [8]. Moreover, there are not enough studies about the ecological and biological characteristics of this species, for instance the early life history is still not clear [9, 10]. According to some studies [9, 10], eggs are spawned at the roots of Gimeila asiatica trees. After the larvae hatch, they drift downstream with the water current and eventually enter their rearing and feeding habitats on the floodplains. However, early larval behavior in the floodplains is still unclear. Information on the morphogenesis of sensory organs will contribute to our understanding of the early life history of fish larvae in the wild [11]. Feeding behavior is essential for fish larvae to survive in their habitats [12]; therefore, understanding the feeding behavior helps us to understand their early life history, as well as to improve the efficiency of larval-rearing techniques in fish hatcheries.
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There are many studies of feeding behavior in catfish at the larval and juvenile stages [13–15]. However, there are no detailed studies about the feeding behavior in the larval stage of sutchi catfish, and also there is no study concerning their feeding behavior under light and dark conditions that is useful to understand fish senses for prey detection. Therefore, this study examined the feeding behavior of the early larval stage of sutchi catfish under light and dark conditions to elucidate their feeding habits and the morphological development of sensory organs as related to the feeding behavior.
Materials and methods Larvae for experiments Fertilized eggs from sutchi catfish were obtained from brood fish reared in a hatchery at the Borneo Marine Research Institute, Universiti Malaysia Sabah. The eggs hatched at 24 h after artificial fertilization. The larvae were reared in a 1 m3 FRP tank at a temperature of 28–30°C for histology and feeding experiments. The larvae were fed with rotifers Brachinonus sp., Artemia nauplii, and artificial compound feed (Gemma micro 150, Skretting) from 2 days old. The mean total lengths of the larvae were determined from the measurement of ten larvae at 0, 1, 2, 5, and 10 days old.
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handling stress. Following the recovery period, 30 Artemia were placed into each beaker, and the larvae were left in the beakers for 1 h to feed on the Artemia. The larvae were subsequently anesthetized with Transmore (100 ppm, Nika Trading), and the remaining Artemia were counted in order to determine the number of Artemia eaten by each larva. In the experiments under light conditions (n = 70), the beakers were placed under 600 lux (Light meter; no. 401036, Extech Instruments), and under dark conditions (n = 70), beakers were covered with black plastic and placed in a darkened room (0.00 lux). The experiments on light and dark conditions were conducted on 2- to 7- and 10-day-old larvae, with ten replicates performed for each experiment. The number of Artemia eaten by a larva was expressed as the ingestion rate of Artemia per hour. Other trials were conducted under both light and dark conditions, using frozen dead Artemia that had been stored in a freezer (10 replicates, light, n = 70; dark, n = 70). The procedure for the experiments using frozen dead Artemia was the same as that used for the feeding experiments using live Artemia. Water temperature during feeding experiments was 28.0–29.0°C. An overall difference among the four groups (under light or dark with live or frozen dead Artemia) for each day was determined by Kruskal–Wallis test (SPSS v. 16). If the Kruskal–Wallis test was significant, differences among individual groups were estimated using Steel-Dwass multiple comparison test (KyPlot 5.0).
Experiments of sensory organs
Results
Larvae were sampled every day until 10 days after hatching to study sensory organ development. Larval specimens were anesthetized with 3-aminobenzoic acid ethyl ester (MS222, 200 ppm) and preserved in Bouin’s solution (n = 100). The specimens were embedded in paraffin, cut into 6 lm thick sections and stained with hematoxylineosin for histological examination (n = 45). Other specimens from the same group of larvae were anesthetized with MS222 (200 ppm) and preserved in Karnovsky’s fixative (n = 50). The specimens were dehydrated in an ethanol series, freeze-dried, and coated with platinum for examination of the larval body surface under a scanning electron microscope (SEM, JSM 5610; JEOL, Tokyo).
Changes in total length of the larvae
Feeding behavior experiments Feeding experiments were conducted under light and dark conditions by using Artemia nauplii as live feed and frozen Artemia as dead feed. Prior to the experiments, the larvae were starved for 6 h, and each larva was transferred to a 500 ml beaker containing 300 ml fresh water. The larvae were left for 20 min in order to allow recovery from the
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The larvae showed steady growth, and the total lengths (mean ± SD, mm) of newly hatched larvae were 3.7 ± 0.1, 5.7 ± 0.2 at 1 day, 6.8 ± 0.3 at 2 days, 8.4 ± 0.3 at 5 days and 11.9 ± 0.6 at 10 days (each age, n = 10). Sensory organs Newly hatched larvae had immature eyes with no pigment. The eyes of 1-day-old yolk-sac larvae were pigmented, and the retina was separated into several layers. In 4-day-old larvae, all layers of the retina were recognizable except for the rod cells; thus the eyes were morphologically complete (Fig. 1a). The nasal pits of sutchi catfish were open in newly hatched larvae, and the pits expanded with the growth of fish. In 2-day-old larvae, many cilia were observed in the sensory epithelium (Fig. 1b). According to Yamamoto [16], ciliated nonsensory cells and ciliated receptor cells were recognized in the sensory epithelium of 2-day-old larvae.
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There were no taste buds in the newly hatched larvae. In 2-day-old larvae, numerous taste buds were observed on the barbels (Fig. 1c), around the mouth, and in the buccal cavity. In 3-day-old larvae, taste buds were observed on the gills. One free neuromast (sensory receptors that respond to mechanical stimuli) was observed on the head in the newly hatched larvae. Several free neuromasts were observed on both the head and trunks of 1-day-old yolk feeding larvae. In 2-day-old larvae, many free neuromasts with welldeveloped features were observed on the head and trunk (Fig. 1d).
Ingestion rates (median +quartile deviation)
Fig. 1 Light micrograph (a) and scanning electron micrographs (b–d) of sensory organs from sutchi catfish larvae. a Eye of a 4-day-old larva, b sensory epithelium of the olfactory organ in a 2-dayold larva (CR ciliated receptor cell, CN ciliated nonsensory cell), c taste buds on the barbel of a 2-day-old larva, d free neuromast on the head of a 2day-old larva. Scales: a 50 lm, b 5 lm, c 20 lm, d 2 lm
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Feeding behavior Ingestion rates of Artemia and frozen dead Artemia are shown in Fig. 2. The results of Kruskal–Wallis test among four groups (under light or dark conditions with live Artemia, and light or dark conditions with frozen dead Artemia) are shown in Table 1. There were significant differences among the four groups except for the 2-day-old groups. The larvae commenced feeding on Artemia and frozen dead Artemia at 2 days old under both light and dark conditions in feeding experiments (Fig. 2). The number of Artemia eaten by a larva increased with fish growth. SteelDwass multi comparison test was performed except for the 2-day-old groups. Interestingly, the ingestion rates under dark conditions with live Artemia were significantly higher than those under light conditions at 4 and 7 days old. The results of the experiments using frozen dead Artemia showed higher tendency than the results using live
Live Artemia Live Artemia Frozen dead Artemia Frozen dead Artemia
Fig. 2 Growth-related changes in the ingestion rates (median ? quartile deviation) of larvae under light and dark conditions with live and frozen dead Artemia. The ingestion rate is the number of Artemia eaten by a larva per hour. Steel-Dwass multiple comparison test was conducted, P \ 0.05. Identical lowercase letters indicate that there is no significant difference Table 1 The results of Kruskal–Wallis test among four groups, under light or dark conditions with live or frozen dead Artemia Age (days)
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Artemia. For example, at 3, 4, 5, 6, and 7 days old, the ingestion rates under light or dark conditions with frozen dead Artemia were significantly higher than the rates under light or dark conditions with live Artemia. Moreover, at 7 days old, the results of the experiments with frozen dead
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Artemia revealed that the ingestion rate under dark conditions was significantly higher than the rate under the light conditions (Fig. 2).
Discussion The histological study revealed that the features of newly hatched sutchi catfish larvae were morphologically immature; however, the sensory organs developed rapidly with fish growth. Larvae commenced feeding on Artemia and an artificial compound feed at 2 days old. At this point, the larvae already had many fully developed taste buds, and other sense organs were also well developed (Fig. 1). Feeding experiments demonstrated that when the larvae commenced feeding, they were able to catch Artemia under both light and dark conditions (Fig. 2). Therefore, the larvae were able to detect Artemia using not only their eyes but also other sensory organs. Feeding experiments using frozen dead Artemia showed higher ingestion rates under both light and dark conditions than using live Artemia (Fig. 2). Sutchi catfish larvae had free neuromasts, and free neuromasts are sensory receptors that respond to mechanical stimuli [12, 17, 18]. Since frozen dead Artemia produce no vibrations, the feeding on frozen dead Artemia under dark conditions would have been dependent not on free neuromasts but on the chemosensory sense in the present study. Frozen dead Artemia have no movement; therefore the ingestion rate might be higher than with live Artemia if the larvae are using chemosensory sense. Sutchi catfish larvae had many taste buds that were distributed around the mouth, on the barbels and on the head surface. These taste buds were also found in the buccal cavity and gill archs. Similar observations have been made in the African catfish Clarias gariepinus [19]. When these fish commence feeding, they possess numerous taste buds on the barbels and gill arch and in the buccal cavity. Kiyohara and Caprio [20] and Kiyohara and Tsukahara [21] examined the fish taste bud functions and argued that the taste buds of the sea catfish Plotosus lineatus and the goatfishes Parupeneus trifasciatus and Parupeneus pleurotaenia are closely related to feeding behavior. In the case of African catfish, Hecht and Appelbaum [13] conducted behavioral experiments that showed that the feeding rate of the larvae in which barbels had been removed was lower than in the control group and in the eye-cauterized group. The larvae of African catfish therefore use taste buds on the barbels. Other studies demonstrate that larvae and juvenile African catfish can be reared under continuous dark conditions [14, 15, 22–25]. Valentincˇicˇ et al. [26] demonstrated that olfactory detection of a conditioned amino acid increased the searching
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time for test stimuli in intact channel catfish Ictalurus punctatus as compared to the searching time in anosmic catfish guided by gustation alone. While distinguishing the roles of olfaction from that of gustation is complicated, the taste buds on the skin surface in the present study of sutchi catfish must have played an important role to detect Artemia. Moreover, previous studies on African catfish larvae and juveniles showed higher survival and growth rates reared under the dark or dim light conditions than those reared under the light conditions owing to lower level of cannibalism under dark conditions [15, 22]. Sutchi catfish larvae also had numerous taste buds on the head surface. Further studies are necessary to understand the behavior, however, the results of the present study suggest the possibility that sutchi catfish larvae also may show higher survival and growth rate under dark or dim light conditions. In the results of the present study, sutchi catfish larvae under dark conditions showed similar feeding rates or higher feeding rates than those under light conditions. The reason for higher larval feeding rates under dark conditions is difficult to understand. For instance, at the preliminary observation during larval rearing, larval swimming at night was more active than during daytime. Although further experiments are needed, the reason for this trend may be that higher activity under dark conditions increases feeding rates. Although there is no detailed report regarding larval habits of sutchi catfish, it can be asserted that larvae eat foods at night in their habitats. Previous studies [4, 10, 27] mentioned that after sutchi catfish larvae hatch from eggs attached to the roots of the Gimenila asiatica trees, the larvae drift downstream with the water current and eventually enter their rearing and feeding habitats on the floodplains. In the present study, we determined that the larvae feed on zooplankton and other foods both during the day and at night in the floodplains. Larval fish are vulnerable to predators; if they can search for and eat foods at night, they can avoid diurnal predators. This behavior represents a strategy to survive in their habitat. Moreover, this finding may contribute to the improvement of seed production techniques. Acknowledgments We would like to express our sincere thanks to Assoc. Prof. Dr. Abdul Hamid Ahmad, Director of the Institute for Tropical Biology and Conservation of Universiti Malaysia Sabah for his cooperation in using SEM. This study was supported by a Fundamental Research Grant (FRG 0002-ST-1/2006) from the Ministry of Education of Malaysia and the e-Science Fund (05-01-10-SF0054) of the Ministry of Science, Technology and Innovation of Malaysia.
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