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This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information
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Determination of appropriate feeding schedules from diel feeding rhythms infinfish larviculture
Tomonari Kotani ⁎, Hiroshi FushimiDepartment of Marine Biotechnology, Faculty of Life Science and Biotechnology, Fukuyama University, 452-10 Innoshima-Ohama, Onomichi, Hiroshima 722-2101, Japan
a b s t r a c ta r t i c l e i n f o
Article history:Received 30 September 2009Received in revised form 15 October 2010Accepted 27 October 2010Available online 3 November 2010
In finfish larviculture, feeding regimes and schedules vary with hatchery and species. They have no biologicalor technological foundation and are dependent of operator expediency and previous experience. Inadequatefeeding regimes and/or inappropriate food intake, especially during early larval stages, may result in a declinein health and/or quality and high mortalities. Fish have species-specific diel feeding rhythms; therefore,feeding schedules in larviculture cannot be determined uniformly among target species. To improve survivaland quality, it is important to establish feeding schedules corresponding to the diel feeding rhythms of larvalfish species. In fish, the feeding and circadian rhythms are linked; most species have diurnal feeding peaks,especially at dusk and dawn, such as flat fishes, groupers, sparids, devil stinger Inimicus japonicas and ayuPlecoglossus altivelis. These fish do not ingest food at night and the larvae actively feed at dawn and dusk.Differences in this trend have not been reported during the co-feeding period of rotifer and Artemia nauplii.Although ocellate puffer larvae also have diurnal peaks of food intake, they commenced ingestion before daybreak.The delay in first feeding causes serious problems in finfish larviculture and in many cases, it is performed toprevent this delay. Aftermouth opening, larvae do not have awell-developed functional jaw and, thus, rotifers orArtemia nauplii remain in the rearing water. Theymetabolize the enriched nutriments, resulting in deteriorationin the nutritional quality of the residual live food. Therefore, the timing of first feeding is important.In conclusion, the feeding schedule of a particular fish species should be determined on active diel ingestion.Delays in active ingestion result in un-ingested live food remaining in the rearing water and consequentnutritional deterioration. Therefore, feeding schedules in finfish larvae and juveniles should be matched to thepeak of food intake.
Feeding can have a significant impact on the success of finfishlarviculture. With technological developments in the mass culture oflive food, feeding regimes have been changing (Fushimi, 2001). SinceIto (1960) introduced euryhaline rotifers Brachionus on larviculture,they have been an important live feed for finfish larvae. Nevertheless,Artemia nauplii have still played an important role as a subsequentlive feed of rotifers. Shirota (1970) demonstrated that finfish larvaeselect food based on mouth size and, thus, the rearing period of thelarvae fed rotifers and Artemia nauplii is growth-dependent.
Optimum feeding regimes have been established for some finfishspecies (Oozeki et al., 1992; Fernández-Diaz et al., 1994; van derMeeren, 1991; Giri et al., 2002; Yilmaz et al., 2006), including thoseextensively cultured in Japan such as Japanese flounder Paralichthysolivaceus, red sea bream Pagrus major and yellowtail Seriolaquinqueradiata. However, it has never been verified whether thecurrently used diel feeding schedules are appropriate.
Previous studies have reported that larvae do not display constantfood ingestion under natural or laboratory conditions but have dielfeeding rhythms (Kitajima et al., 1976; Okauchi et al., 1980; Boujardand Leatherland, 1992; Yamamoto, 1996; Shoji et al., 1999; Dou et al.,2000; Yamamoto et al., 2003, 2005). In larviculture, this means thatfeeding on larvae which havemissed their period of active ingestion isinefficient due to deterioration in the nutritional quality of un-ingested live food or indirect starvation. Therefore, feeding schedulesshould be determined from the diel feeding rhythm and changes inlarval growth. Diel feeding schedules in finfish larviculture are usuallydetermined independently for each hatchery, even for the same fishspecies. They are not based on any biological or technologicalfoundation and feeding decision are usually made by hatcheryoperators, considering working hours, etc. There is a lack of conclusiveindicators for feeding schedules in the scientific literature dealingwith larval production and rearing.
This paper reviews studies on feeding rhythms in finfish larvaeunder laboratory condition and suggesting optimum species-specificfeeding schedules for some common species cultured in Japaneseaquaculture industries.
2. Feeding rhythm
In their review, Boujard and Leatherland (1992) reported that fishdisplay a diel feeding rhythm. The rhythms are under endogenouscontrol and light-entrainable, though many sampled larvae from openseawater (reviewed in Spieler, 1992). Generally, feeding rhythm isstrongly related to the circadian rhythm and a number of studies havereported the diel feeding rhythm of larvae (Yamashita et al., 1985;Tomiyama et al., 1985; Shoji et al., 1999). Feeding rhythms vary amongspecies (Yamashita et al., 1985; Tomiyama et al., 1985; Shoji et al., 1999)and, in many cases, are synchronized to diel distribution patterns. Inaddition, environmental conditions differ between sea and laboratory;therefore, feeding rhythms occurring under the natural conditionsshould differ greatly from those in hatcheries. A number of studies havereported diel feeding rhythms under laboratory conditions (Kitajimaet al., 1976; Okauchi et al., 1980; Yamamoto, 1996; Dou et al., 2000;Yamamoto et al., 2003, 2005; Teruya et al., 2008). For successfullarviculture, feeding rhythms need to be considered (Spieler, 1977;Parker, 1984). We have investigated the diel feeding rhythm of some
fish species cultured in Japan, Japanese flounder P. olivaceus, red seabream P. major, devil stinger Iminicus japonicas and ocellate pufferTakifugu rubripes. In the following, wewill discuss our results in relationto literature.
2.1. Japanese flounder
The Japanese flounder is an important aquaculture species inJapan, China and Korea. In the wild, Japanese flounder larvae andjuveniles feed during daytime (Minami, 1982; Koshiishi et al., 1982;Hirota et al., 1990). Feeding peaks have also been reported at dusk injuveniles; nevertheless, the relative intensity between dawn and duskis inconclusive (Koshiishi et al., 1982; Hirota et al., 1990; Katayamaet al., 2007). There are no reports on the feeding rhythm of larvaeunder the natural conditions. Under laboratory condition, Dou et al.(2000) observed that during the larval development of Japaneseflounder there was a major feeding peak in the morning followed by asecondary lower peak. On the other hand, their recording of gutcontents at intervals of 2–4 h every five days may be too long toascertain the exact feeding peak. Therefore, we observed the gutcontents of larvae at intervals of 30 min in every 5 day.
2.1.1. MethodsLarval rearing was performed under laboratory conditions in a 1-m3
black polycarbonate cylindrical tank. Aeration, provided through fourceramic air-stones hung ~10 cm above the bottom, was increased withlarval growth and ensured circulation of water within the culture tanks.Seawaterwas sterilizedwithUV light andwas exchanged at a daily ratioof 30 to 300%.Water was drained by siphon through a 30-mmdiameterof flexible vinyl hose, screened through a polyethylene mesh fitted to ametal frame (14×15×55 cm) fixed at the center of the tank. The meshsize was changed from 0.2 to 1.5 mm with larval growth. Watertemperaturewas controlled at 18 °C using a 1 kWtitanium stick-heater.Lightingwasmaintained automatically on a 12-h light/dark cycle lights-on at 07:00 h and off at 19:00 h. Light intensity was 3000 lx duringdaytime. Eggs were spawned naturally, fertilized and 22,000 fertilizedeggs were stocked per rearing tank.
Rotifers were enriched nutritionally with concentrated Nanno-chloropsis oculata (Chlorella Industry Co., Ltd., Kurume, Japan) for24 h. Enriched rotifers were fed to the larvae from 2 to 25 days afterhatching (DAH). Artemia nauplii were enriched with Marine Omega(Nisshin Marine Tech Co., Ltd., Yokohama, Japan) for 24 h. To observethe feeding rhythm of larvae, a constant stocking density of live feedwas maintained in the rearing water; the rotifer density was kept at5 individuals/ml. Artemia nauplii were fed from 20 to 35 DAH and thedensity was increased with larval growth from 1 to 5 individuals/ml.Larvae were sampled at intervals of 30 min in every 5 days from 5 to35 DAH. Sampled larvae were placed on a slide glass, mashed using acoverslip and the number of rotifer mastax or Artemia nauplius headsin the gut observed and counted using an optical microscope. Feedingwas carried out at 07:00, 10:00 and 15:00 h.
2.1.2. Results and discussionsThe variations in gut content at each observation are showed in
Fig. 1. Throughout the experimental rearing period, gut contents wereonly observed in the daytime. Several observations were performed atnight, but we failed to ascertain when the gut emptied. However,since gut contents decreased after lights-out (Fig. 1), it appears that
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the larvae did not feed but evacuated only at nighttime. For eachobservation, gut contents increased after lights-on and the feedingamount depended on the growth increment.
At 5 DAH (3.5 mm total length (TL)), when the larval mouthformed and functioned, gut contents increased until midday butdecreased again until lights-out. At 11 DAH (5.9 mmTL), when the gut
looped, gut contents increased from the beginning of observation,stabilized around midday and increased from the afternoon untillights-out. At 15 DAH (7.2 mm TL), larvae had the onset of notochordflexion, gut contents increased quickly until midday, then stabilizedand increased gradually again from afternoon to lights-out. At 20 DAH(8.3 mm TL), with the onset of eye migration, the larvae were fed
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Fig. 1. Diel variation in gut contents of Japanese flounder and changes with growth. Solid circles: average rotifer number in gut (n=10); open squares: average Artemia naupliinumber (n=10). Each vertical line indicates 95% confidence interval. Solid arrows indicate the feeding time of rotifers; open arrows indicate the feeding time of Artemia nauplii.Number in parenthesis under the number of days after hatching (DAH) indicates the average total length (TL).
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Artemia nauplii and the feeding rhythm of Artemia nauplii appeared.The number of rotifers in the gut increased from the beginning ofobservation with a peak around midday, but decreased rapidly afterthe peak. Thereafter, it increased again gradually and had a new peak1 h before lights-out, coinciding to the decrease in gut content.Although fish were fed Artemia nauplii, only a few nauplii were foundin the gut. At 25 DAH (9.9 mm TL), when the eye migrated near themedian plane, Artemia nauplii were found more abundantly thanrotifer in gut contents. The number of Artemia nauplii in the gutincreased from the beginning of observation. It peaked before noon,decreased in the afternoon, increased again and peaked 3 h beforelights-out, then it decreased again. On the other hand, rotifers in thegut peaked in the afternoon and few nauplii were found. At 30 DAH(13.2 mm TL), rotifer feeding was stopped. Large numbers of Artemianauplii were already observed in the gut at the beginning ofobservation, decreasing till noon when a new peak appeared.Numbers decreased again to 14:00 h with a third smaller peak at14:00–15:00 h, decreasing again until lights-out. At 35 DAH (15.9 mmTL), the trend was similar to 20 and 25 DAH. The number of Artemianauplii in the gut increased from the beginning of observation, peakedaround noon and then decreased rapidly. It increased again gradually,had a second peak 1 h before lights-out and decreased againthereafter.
In all observations, the gut contents increased with lights-on, thendecreased with lights-out. This indicates that feeding in Japaneseflounder larvae depends on light conditions and that they alreadyhave a well-developed photosensitive organ at 5 DAH. However, theremarkable variation in feeding rhythm, as appeared in the gutcontent between dawn and dusk, was not observed until 15 DAH.From 20 DAH, the changes in gut contents could be observed indaytime. The onset of looping of the gut occurred around 11 DAH,with gut development started at this time.
After 20 DAH, gut contents tended to decrease 1–2 h before noonand increased 1–2 h after noon. A similar tendency was observed at20, 25 and 35 DAH, suggesting a characteristic feeding rhythm ofJapanese flounder larvae.
2.2. Red sea bream
In Japan, aquaculture of red sea bream is dependent on hatchery-reared juveniles, with numerous studies focusing on the biologicalcharacteristics and rearing conditions of larvae. However, little isknownof the feeding rhythmof red sea bream. In thewild, juveniles oryoung fish actively feed during daytime, especially at dawn and dusk.Kitajima et al. (1976) reported a similar trend in feeding rhythmbetween cultured andwild red sea bream larvae (Okada, 1965). On thecontrary, the closely related crimson sea bream Evynnis japonicashowed active feeding between dawn and dusk, in the wild(Tomiyama et al., 1985).Moreover blackhead seabreamAcanthopagrusschlegelii, a well-known sparid species in Japan, showed nighttimefeeding during the early developmental stages, but shifted to diurnalfeeding when grown under hatchery condition (Okauchi et al., 1980).Thus, feeding rhythms vary among sparid species and little is knownabout the feeding rhythm of red sea bream larvae, especially underhatchery conditions. To address this issue, we observed the gutcontents of red sea bream larvae every 30 min over a 25-h period.
2.2.1. MethodsThe rearing methods were basically as described above for
Japanese flounder larvae. Rotifers were enriched with DHA ProteinSELCO (INVE Technologies NV, Dendermonde, Belgium) and Artemianauplii were enriched with DC DHA SELCO (INVE Technologies NV,).Lights were on from 07:30 to 19:30 h. Rotifers were fed on larvae from3 to 21 DAH. Artemia nauplii were fed from 15 DAH. Watertemperature was controlled at 20 °C. Rotifer feeding was performedat 08:30 and 14:30 h in the single feeding, and at 09:30 and 15:30 h
co-fed with Artemia nauplii. Artemia nauplii feeding was performed at08:30, 14:30 and 17:30 h either co-fed with rotifer or as a singlefeeding.
2.2.2. Results and discussionsThe variation in gut contents throughout the experimental period
is shown in Fig. 2. Food was confirmed in the gut during daytime,while it was empty at night. As with Japanese flounder larvae, gutcontents decreased after lights-out (Fig. 2), indicating that the larvaedid not feed and evacuated only at night. The amount of food wasdependent on larval growth.
Similar to Japanese flounder larvae, in all observations, gutcontents increased after lights-on and decreased after lights-off,indicating that feeding in red sea bream larvae dependent on lightconditions and they already have well-developed photosensitiveorgan at 3 DAH (3.4 mm TL). However, significant feeding rhythmswere only observed after lights-on and lights-off. Slight increaseswere not observed in daytime until 10 DAH (4.9 mm TL), which maybe due to the inability of larvae to store feed in their guts and/orevacuate well.
From 15 DAH (6.2 mm TL), changes in gut contents were observedin daytime. The larvae were 6.2 mm TL and, according to Fukuhara(1985), looping of the gut should have already begun, which seems tohave been achieved based on the ability to store feed and evacuate.From 15 to 20 DAH (6.2–9.9 mm TL), gut contents increased from thebeginning of the observation to noon; thereafter, they decreased 4–5 hbefore lights-out and then increased again. Although rotifer contentsin the gut changed little at 20 DAH, total gut contents showed a similartendency (Fig. 2). At 20 DAH (9.9 mm TL), red sea bream becomesjuveniles (Fukuhara, 1985).
At 22 and 24 DAH (~11 mm TL), gut contents showed a similartendency as previously. After an increase after noon, there was nolarge variation. On the other hand, the gut did not empty soon afterlights-out but decreased gradually.
Overall observations confirmed red sea bream as daytime feederswith slight variations in feeding rhythmwith development of the gut.The similarity in feeding rhythm between wild and hatchery larvaesuggests that this species has a characteristic feeding rhythmregardless of habitat (natural and laboratory conditions).
2.3. Devil stinger
The devil stinger is also a commercially important fish in Japan.However, low growth rate and highmortality during larviculture havehindered intensive aquaculture of this species. In the wild, adult fishshow nocturnal behavior feeding on small fishes or crustacean. On theother hand, there is little knowledge of their larval feeding habitats inthe wild (Sudo and Kajihara, 2008). In Japan, government hatcherieshave been involved in developing the larviculture technology of devilstinger and have reported many successes (Gorie, 1994; Mutsutani,1997; Ichikawa, 1997; Kondo and Sugino, 1998); however, thefeeding rhythm of the larvae has never been investigated.
2.3.1. MethodsThe fundamental rearing methods were the same as described
above for Japanese flounder and red sea bream. A total of 17,000 eggswere stocked in a1000-l rearing tank. Water temperature was notcontrolled but light was turned on from 06:30 to 18:50 h. Rotiferswere enriched nutritionally with concentrated Nannochloropsisoculata (Chlorella Industry Co., Ltd.) for 24 h and were fed to thelarvae from 2 to 11 DAH. Artemia nauplii were enriched with MarineOmega (Nisshin Marine Tech Co., Ltd.) for 24 h and fed from 5 DAH.Rotifer feeding was performed at 07:30, and 15:00 h in a singlefeeding, and at 08:30 and 16:00 h in a mixed feeding with Artemianauplii. The Artemia nauplii feeding period was at 07:30 and 15:00 hin a mixed feeding with rotifer and single feeding, respectively.
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Fig. 2. Diel variation in gut contents of red sea bream and changes with growth. Solid circles: average rotifer number in gut (n=10); open squares: average Artemia nauplii number(n=10). Each vertical line indicates 95% confidence interval. Solid arrows indicate the feeding time of rotifers; open arrows indicate the feeding time of Artemia nauplii. Number inparenthesis under the number of days after hatching (DAH) indicates the average total length (TL).
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2.3.2. Results and discussionsVariations in gut contents are shown in Fig. 3. Throughout the
experimental rearing period, gut contents were observed in thedaytime. When observations were performed at night, we couldascertain when the gut emptied except 2–3 DAH (4.1 mm TL). At thisage, gut contents increased steadily until 2–3 h after lights-out, thendecreased. This infers that 2–3 DAH larvae have not yet developed thephotosensitive organs to acquire a clear circadian or feeding rhythm.
On the other hand, since the gut contents increased after lights-on at3 DAH, it is presumed that photosensitive organs were well-developed after 3 DAH.
At 4 DAH (4.9 mm TL), a change in gut contents was observed,indicating that a feeding rhythm was already established at this time.Gut contents changed slightly until the afternoon, when the increasewas more pronounced until 1–2 h before lights-out. This was thetendency until 10 DAH (7.5 mm TL). At 13 DAH (8.9 mm TL), gut
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Fig. 3. Diel variation in gut contents of devil stinger and changes with growth. Solid circles: average rotifer number in gut (n=10); open squares: average Artemia nauplii number(n=10). Each vertical line indicates 95% confidence interval. Solid arrows indicate the feeding time of rotifers; open arrows indicate the feeding time of Artemia nauplii. Number inparenthesis under the number of days after hatching (DAH) indicates the average total length (TL).
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contents increased until around noon and then decreased or onlychanged slightly.
As this study shows, the feeding habits of devil stinger larvae arediurnal but, as adults show nocturnal habits, it is inferred that feedinghabits shift after 14 DAH.
2.4. Ocellate puffer
The ocellate puffer is another commercially important fish inJapan, Korea and China, with larviculture being widespread andintensively practiced in private hatcheries. In the wild, juvenilesingest small benthic crustaceans (Takita and Intong, 1991); however,little is known of their feeding habits, especially at the larval stage.Under stressful conditions, such as high stocking density, Kotani et al.(2009) observed cannibalistic behavior in ocellate puffer larvae,which is reported to be due to shortage of food (Fox, 1975; Smith andReay, 1991). In the case of ocellate puffer larvae, cannibalisticbehavior was observed when feeding of rotifers or Artemia naupliiwas inappropriately timed, and was probably due to food shortage ora decline of their nutritional values. Therefore, to select the correctfeeding schedule, the feeding rhythm of the larva of this species needsto be clarified.
2.4.1. MethodsThe fundamental rearing methods were the same as described
above for the other three species. Bloodstock of ocellate puffer wascollected from the Mekariseto Channel (Hiroshima, Japan). Eggs werespawned and fertilized at Bio Ehime Co., Ltd. They were transported toFukuyama University and incubated at 16.5 °C in a 500 l tank for1 week until hatching. Since eggs of puffer are demersal and adhesive,water exchange was performed at 300%/day (35 ml/s) with intensiveaeration to prevent eggs from adhering to the tank wall and bottom.Eggs hatched after 7 days; larvae were counted and 5000 larvae werestocked initially in a 1-m3 rearing tank. Water temperature was notcontrolled and lights were turned on from 06:30 to 18:50 h. Rotiferswere enriched nutritionally with concentrated Nannochloropsisoculata (Chlorella Industry Co., Ltd.) for 24 h and fed to the larvaefrom 4 to 26 DAH. Artemia nauplii were enriched with Marine Omega(Nisshin Marine Tech Co., Ltd.) for 24 h and fed from 13 DAH. Rotiferfeeding was performed at 07:30 and 13:00 h in a single feeding, and at8:30 and 14:00 h in mixed feeding with Artemia nauplii. Artemianauplii feedings were performed at 07:30 and 15:00 h in mixedfeeding with rotifer and at 07:30 and 13:00 h in a single feeding.
2.4.2. Results and discussionsVariations in gut contents are shown in Fig. 4. In the earlier stages,
ocellate puffer larvae showed a different tendency from the otherthree species, i.e. a decrease in gut contents immediately after lights-out, to empty in 2–3 h and feeding again after lights-on (Figs. 1–3). Inocellate puffer larvae, on the other hand, gut contents had notemptied until midnight 9 DAH. Moreover, ocellate puffer larvae beganfeeding before lights-on during the first 22 DAH. Accumulation of gutcontents until midnight seems to be related to the fact that theyalready have a well-developed gut. On the other hand, since larvae didnot receive light because the rearing experiments were performedindoor and the room was shaded, it is unclear why they start feedingbefore lights-on. To understand the feeding activity before lights-on, adevelopmental and histological analysis of larvae is necessary.
At 4–5 DAH (3.5 mm TL), an increase in gut content in theafternoon was followed by a decrease 2–3 h after noon to lights-out.During subsequent days, large fluctuations between the increase ingut content in the morning and the decrease after lights-out were notdetected. At 25–26 DAH (9.2 mm TL), a large increase in gut contentwas observed 4 h before lights-out. A similar trend was also observedat 35–36 DAH (12.7 mm TL).
At 13 DAH (5.5 mm TL), Artemia nauplii feeding started, and thediel amount fed was similar to that of rotifer. At 17–18 DAH (6.2 mmTL), larvae ingested more Artemia nauplii than rotifers. Thereafter,almost no rotifers were ingested, except in some specimen thatingested rotifers around the time of lights-out, when on a fewexperimental days only, larvae ingested Artemia nauplii intensivelyfrom afternoon to lights-out,. Therefore, those observations canexplain the ingestion for rotifers.
2.5. Inclusive discussions
Previous studies have reported the feeding rhythm of larvae ofsome fish species based on two methods. The first, followed in thisstudy, consists of the observation of the gut contents under amicroscope after the larvae were mashed (Yamamoto, 1996;Yamamoto et al., 2003, 2005). The second approach estimates theingested amount of food from the decrease in live feed in the rearingwater (Kitajima et al., 1976; Okauchi et al., 1980). The first methodobserves the gut contents directly; nevertheless, mashing of larvaewith the rotifers and Artemia nauplii in the gut poses some technicaldifficulties. The second method is easier because it counts thenumbers of live feed in the rearing water over time. However, thereduction in density due to water exchange and increases byreproduction, especially of rotifers, needs to be considered. We,therefore, adopted the first method and, although previous studiesobserved gut contents every 2–3 h, we believe this interval is too longto clarify exactly the trends in larval ingestion. In this study, variationsin gut contents were observed frequently, every 1–2 h, which clearlydetected rapid trends, as, for example, the increase just after lights-onin each species, the change in rotifer content of red sea bream larvae at20–21 DAH (Fig. 2), the decrease of rotifer contents of devil stingerlarvae just before lights-out at 8–9 DAH (Fig. 3), the change in Artemianauplii content of ocellate puffer larvae at 30–31 DAH (Fig. 4). To tracesuch rapid changes, observations at 2–3 h interval are inadequate,thus, our shortening of the period between each observation to30 min.
Larvae of three fish species in this study, Japanese flounder, red seabream and devil stinger, began ingestion just after lights-on, while theirgut contents decreasedat lights-out (Figs. 1–3). This suggests that a lightstimulus may be the exogenous factor acting as the cue for ingestion inthe larvae of these three fish species. To confirm this hypothesis, it isnecessary to perform larval rearing experiments under constant light ordark conditions (Boujard and Leatherland, 1992), aspreviously reported(Barlow et al., 1995; Yoseda et al., 2003; Teruya et al., 2008). Underconstant light, diel changes in gut contents demonstrated the existenceof small time periods of no content in the gut (Barlow et al., 1995;Yoseda et al., 2003; Teruya et al., 2008). Therefore, it can be concludedthat the feeding rhythm of larvae is due to endogenous factors. On theother hand, Teruya et al. (2008) reported that grouper larvae did notingest food under constant dark conditions. In the present study, larvaeof Japanese flounder, red sea bream and devil stinger started ingestionjust after lights-on (Figs. 1–3), as reported in other studies usingalternative light/dark photoperiod (Barlow et al., 1995; Yamamoto,1996; Dou et al., 2000; Yamamoto et al., 2003, 2005; Yoseda et al., 2003;Teruya et al., 2008). Thus, it can be concluded that the food ingestion infinfish larvae is light-induced. In the case of ocellate puffer larvae, thegut contents started to decrease at lights-out (Fig. 4); therefore,darkness may be the cue of cessation of the feeding in this species.However, they started ingestion before lights-on; therefore, it seemsthat light is not the only factor inducing ingestion. To clarify suchbehavior, it is necessary to perform the same experimental methodol-ogy under constant dark conditions.
In this study, feeding schedules differed between fish speciesbecause, based on our experience, we assumed the interval in which aclear reduction in rotifers and/or Artemia nauplii might be observed inthe rearing water. However the feeding schedules in this study were
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(6.2mm)
0
50
100
0
40
80
Fig. 4. Diel variation in gut contents of ocellate puffer and changes with growth. Solid circles; average rotifer number in gut (n=10); open squares: average Artemia nauplii number(n=10). Each vertical line indicates 95% confidence interval. Solid arrows indicate the feeding time of rotifers; open arrows indicate the feeding time of Artemia nauplii. Number inparenthesis under the number of days after hatching (DAH) indicates the average total length (TL).
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similar to general methodology in hatcheries. On the other hand, thedifferences in feeding schedule could affect the feeding rhythm ofeach species. Nevertheless, ingestion induced by light and activeingestion after noon were common to Japanese flounder, red seabream and devil stinger larvae in this study. Therefore we concludedthat the feeding schedule for each species did not affect thesetendencies.
Even if the timing of feeding affected other diurnal feeding rhythms,theymay be reflex actions to feeding and not potential feeding rhythms.To clarify the influence of feeding schedule on the rhythmof ingestion, itis necessary tomaintain the density of rotifers and Artemianauplii in therearingwater constant at all times or perform feeding experimentswithdifferent feeding schedule. Ingestion activity induced by feeding onlydoes not have to be considered in determining the feeding schedule.
3. Feeding regime and schedule
The optimum diel time or schedule for feeding live food to finfishlarvae has not been a subject of discussion. This is because rotifers orArtemia nauplii are periodically added to the rearing water tomaintain the numbers/density determined beforehand as the opti-mum food requirement for the larvae of that particular fish species(Yamamoto, 1996; Sawada et al., 2000; Yamamoto et al., 2003, 2005).As live food is used, the diel feeding time or schedule is not regardedas important. As evidenced in this study, however, each species has adefinite and characteristic feeding rhythm. In all four species studiedexcept ocellate puffer, larvae began ingestion just after lights-on. Gutcontents before the start of ingestion were empty and the subsequentincrease was rapid in many cases. In hatcheries, finfish larvae are notgenerally fed at night but rearingwater is always exchanged. Thus, thedensity of rotifers or Artemia nauplii decreases during nighttime.Moreover, the effect of enrichment on live food, such as rotifers andArtemia nauplii, declines with time (Olsen et al., 1993; Furuita et al.,1996a,b; Øie et al., 1997; Navarro et al., 1999), suggesting that it isimportant to feed on larvae just after lights-on. Additional feedingsare necessary before the larvae show active ingestions or the gutcontents increase. Since larvae ingest a lot of live food in the morningand again in the afternoon, feeding around noon or in the afternoon isnecessary. The water exchange rate is normally low in the periodwhen rotifers only are being fed on larvae. Therefore, one feed in theafternoon seems sufficient. Another characteristic of all species is thatgut contents decreased beginning at lights-out and then emptied,suggesting that larvae only evacuate after dark. Therefore, feeding justbefore lights-out is not important. On the other hand, the waterexchange rate is greater after the onset of Artemia nauplii feeding andtheir nutritional value declines progressively with time, as mentionedbefore. Thus, Artemia nauplii should be fed once more before lights-out.
In this study, ocellate puffer larvae started ingesting food beforelights-on (Fig. 4). Larvae had already been actively ingesting at thetime of lights-on. Therefore, larvae should be fed before lights-on, atmidnight, for example. However, such feeding is impractical in thehatcheries and, in this case, it is better to perform feeding just beforelights-out. This gives plenty of times until larvae start active ingestion,but makes the decline in nutritional value of live food, especiallyArtemia nauplii.
In hatchery rearing, finfish larvae are only fed rotifers a-2 daysafter mouth opening, while rotifers and Artemia nauplii are co-fedover the next week or two. Afterwards, larvae are fed only Artemianauplii and/or artificial formulated foods for one or two weeks afterthe start of co-feeding. Generally, the timing of the start of Artemianauplii feeding is determined on the ingestion capacity of the larvae.On the other hand, the timing of the cessation of rotifer feeding hasnot been considered so far, and is normally decided based on the timelarvae stop ingesting them. However, in the present study, weobserved that the presence of rotifers in the gut fluctuated over time,
i.e. 25-26 DAH in Japanese flounder and 10–11 DAH in devil stinger.Whenever the gut contents of larvae or juveniles show oppositetendencies in rotifers and Artemia nauplii densities, rotifer feedingshould not be stopped. The fluctuations in the increase in rotifers inthe gut when Artemia nauplii decreased were wide, indicating inter-individual variations during the ingestion of food. Consequently, ifrotifer feeding is stopped when larvae show such a tendency, larvalmortalities will occur. Moreover, as gut contents are examined onlyonce a day, it is impossible to ascertain the exact feeding rhythm ortendency of ingestion. Therefore, a comprehensive diel investigationof gut contents is important.
4. Perspective
Whenever a new fish species is selected for aquaculture develop-ment, daily food rations should be estimated to construct amethodical feeding schedule. It requires some knowledge of feedingrhythms, which as similar in some species, such as Japanese flounder,red sea bream and devil stinger, though they do not completelycorrespond. On the other hand, other species show significantdifferences, as observed in ocellate puffer. In many cases, thedetermination of feeding times is not based on specified reasonsand, as this study confirmed that feeding rhythms are not uniform, itis recommended not to standardize feeding times or daily schedulesfor all species.
It is also important not to determine feeding schedules from theextrapolation of feeding rhythms observed in the wild. Naturalfeeding rhythms are influenced by not only endogenous factors butalso the distribution of prey or the diel vertical migration of fish(Yamashita et al., 1985; Boujard and Leatherland, 1992; Shoji et al.,1999). As in ocellate puffer larvae, some species show characteristicfeeding rhythms, different to other species, which is a warning on therisk of extrapolating results to other species. Moreover, feedingschedules should not be based on the expediency or convenience ofharchery operatives. Larvae of many fish species show active ingestionjust after lights-on, as observed in the three fish species above.Nevertheless, hatchery operators may need to adjust their workingtimes to the feeding rhythm of the larvae. In this regard, it is possibleto vary the light/dark cycle and, although the effects of shortened lighthours on larvae are unclear, opportunities for food ingestion willdecrease and negatively influence activity, growth and survival.
The results of the present study provide data on the feedingrhythm and schedule of four important aquaculture finfish species inJapan, which has been available to date. Previous studies ignored theinfluence of feeding, especially the delay of first ingestion, to focus ontemporary starvation or shortage of feed (Fukuhara, 1974; Laurence,1978; McGurk, 1984; Bisbal and Bengston, 1995; Peña and Dumas,2005; Yoseda et al., 2006). Improvement of larviculture, larval healthand quality requires minimizing stress (Varsamos et al., 2006) andincluding all available information on larval nutrition. To achieve this,further investigations on the feeding rhythms of larvae and juvenilesare necessary for each fish species. Finally, optimum feedingstrategies, including times, schedules and regimes, should bedetermined from detailed investigations of feeding rhythms.
Acknowledgments
We thank the following Japanese companies and organizations forproviding the fish eggs; Marua-Suisan limited company (Japaneseflounder), Bio-Ehime Co., Ltd. (red sea bream and ocellate puffer) andFisheries and Ocean Technology Center, Hiroshima PrefecturalTechnology Research Institute (devil stinger). We also would like toexpress our gratitude to Mr. N. Sato, Nagase San-Bio Co., Ltd, Japan, forhis support during the rearing experiments in this study. This studywas supported by the following students who conducted theexperimental research, Takayuki Okada, Jun Hosaka, Makoto Aoki
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(Japanese flounder), Hisaki Kanbayashi, Kensuke Sudo, Shota Eno-moto (red sea bream), Yosuke Watanabe, Keita Shioya, TomoakiTakemura, Takuhiro Nakata (devil stinger), Yoshiyuki Wakiyama,Hiromichi Yashiki, Tatsuhiro Imoto (ocellate puffer). We thank Dr.Enrique Blanco Gonzalez, Fukuyama University, for his Englishcorrection and useful advices.
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