Effects of salinity, aeration and light intensity on oil

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spawns readily in concrete tanks and floating net ... them copepod nauplii during the early larval ... Larvae were exposed to different levels of aeration (0mL/min per L, ... 24 ppt, 32 ppt, or 40 ppt); and light intensity (0 lx, 120 lx, 230 lx, 500 lx, ...
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2002; 68: 478–483

Effects of salinity, aeration and light intensity on oil globule absorption, feeding incidence, growth and survival of early-stage grouper Epinephelus coioides larvae Joebert Delfin TOLEDO,1* Nora Blanca CABEROY,1 Gerald Fontelera QUINITIO,1 Casiano Hermopia CHORESCA Jr1 AND Heisuke NAKAGAWA2 1

Aquaculture Department, Southeast Asian Fisheries Development Center, Tigbauan, Iloilo 5021, Philippines and 2Faculty of Applied Biological Science, Hiroshima University, Higashi-hiroshima, Hiroshima 739-8528, Japan ABSTRACT: A series of experiments were conducted to examine the effects of salinity, aeration and light intensity on oil globule absorption, feeding incidence, and growth and survival of early-stage Epinephelus coioides larvae. Newly hatched larvae were transferred to 40-L aquaria at a density of 1500 individuals/aquarium. Larvae were exposed to different levels of aeration (0 mL/min per L, 0.62 mL/min per L, 1.25 mL/min per L, 2.50 mL/min per L, or 3.75 mL/min per L); salinity (8 ppt, 16 ppt, 24 ppt, 32 ppt, or 40 ppt); and light intensity (0 lx, 120 lx, 230 lx, 500 lx, or 700 lx) for 4–6 days. Twenty larvae were sampled daily at 11:00 hours to measure for total length (TL), oil globule volume, and feeding incidence. Survival rates were determined by counting the total number of larvae remaining in each aquarium at the end of the experiment. Significantly higher survival rates (P < 0.05) were observed at aeration levels of 0.62 mL/min per L and 1.25 mL/min per L, at salinity levels of 16 ppt and 24 ppt, and at light intensities of 500 lx and 700 lx. The influence of aeration level, salinity and light intensity on oil globule absorption, feeding incidence, and growth and survival of early-stage grouper larvae are discussed. KEY WORDS:

aeration, feeding incidence, first-feeding, grouper larvae, light, salinity.

INTRODUCTION The orange-spotted grouper Epinephelus coioides spawns readily in concrete tanks and floating net cages during its natural spawning season.1 Based on a series of studies, a protocol for the intensive hatchery production of E. coioides has been proposed by Duray et al.2 The growth and survival of early-stage larvae was further improved by feeding them copepod nauplii during the early larval stages.3–5 Larval rearing in the hatchery can be shortened by accelerating metamorphosis of the larvae with the use of thyroid hormones.6 Despite these recent developments, the survival of earlystage larvae, particularly before or during the onset of feeding, is still low.5 Low survival rates during the

early larval stage are caused by the low quality of spawned eggs, the size and nutritional value of prey, and the environmental conditions during the incubation of fertilized eggs and newly hatched larvae. A lack of knowledge of the optimal environmental conditions and feeding behavior during the early life stages of larvae are among the various obstacles to the development of techniques for larval rearing.7 In the present study, the effects of salinity, aeration, and light intensity on oil globule absorption, feeding incidence, and growth and survival of early stage E. coioides larvae were examined.

MATERIALS AND METHODS *Corresponding author: Tel: 33-336-2937/33-336-2965. Fax: 33-511-9070/33-335-1008. Email: [email protected] Received 15 May 2001. Accepted 5 November 2001.

Fertilized eggs were obtained from spontaneous spawnings of E. coioides reared in 5 m ¥ 5 m ¥ 2 m

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concrete tanks. Broodstock management and protocol for the collection of spawned eggs were followed after Toledo et al.1 Collected eggs were incubated in 200-L transparent, plastic conical tanks with 35–36 ppt seawater and moderate aeration.

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of ± 1 ppt. Aeration was adjusted to 0.62 mL/min per L during the experiment. Stocking and sampling of larvae, measurement of TL, oil globule volume, and determination of feeding incidence were conducted following the procedure in the preceding experiment. Larval survival was determined on day 4 by counting the remaining larvae in each of the 15 aquaria.

Aeration intensity experiment Newly hatched larvae (day of hatching = day 0) were randomly distributed in 15 units of 40-L aquaria containing 35 ppt seawater at a density of 1500 larvae/aquarium. Five aeration levels were tested: 0 (no aeration); 0.62 mL/min per L, 1.25 mL/min per L, 2.50 mL/min per L, and 3.75 mL/min per L. There were three replicates for each aeration level. A single airstone was positioned at the bottom center of each aquarium. The aeration level in each tank was adjusted by using a flow meter (Gilmont Instruments Inc., Lincolnshire, IL, USA). Twenty larvae in each aquarium were sampled daily at 11:00 hours from day 1 to day 5. To inhibit larval development, sampled larvae were placed in 5 mL cell wells and immediately refrigerated at 4∞C. The total length (TL) and oil globule diameter of each larva were measured using an Image Analysis System (Carl Zeiss Pte. Ltd, Singapore) equipped with an Image-Pro Plus Imaging Software for Windows (Media Cybernetics Inc., Silver Spring, MD, USA). Oil globule volume (V) was calculated using the equation: V = 4 3 pab 2

(1)

in which a is half of the maximum length of the oil globule and b is half of the maximum width of the oil globule. Because the yolk sac of E. coioides is almost fully absorbed by day 3,8,9 only the remaining oil globule was measured in each larva. Larvae were then preserved in 5% formalin–seawater for gut content analysis. Larval survival was determined on day 6 by counting the remaining larvae in each tank.

Salinity experiment Newly hatched larvae were distributed randomly in 15 units of 40-L aquaria containing seawater of different salinities. Five salinity levels (8 ppt, 16 ppt, 24 ppt, 32 ppt, and 40 ppt) were tested with three replicates per test. The desired salinity levels were adjusted by adding fresh tapwater or brine solution. Salinity was measured using a salinity refractometer (Atago Inc., Japan) with an accuracy

Light intensity experiment Five light intensities (0 lx, 120 lx, 230 lx, 500 lx, and 700 lx) were randomly assigned to five enclosed chambers. Three 40-L aquaria containing 22 ppt seawater were placed in each chamber. The desired light intensities were obtained by adjusting the configuration of 21–100 W florescent bulbs (Phillips Electronics and Lighting Inc., Makati, Philippines) in each chamber. Light intensity was measured using the probe of a light meter (LI192SA Underwater Quantum Sensor; LI-COR Inc., Lincoln, NE, USA) positioned at the middle layer of the rearing water. Chambers with light intensities of 500 lx and 700 lx were provided with a recirculating water bath to keep temperature in the aquaria closely similar to those at lower light intensities. Except for the dark chamber (0 lx), the photoperiod cycle was maintained at 12 h light/12 h dark (12L : 12 D). All aquaria were provided with aeration at 0.62 mL/min per L. Larval stocking and sampling for the examination of TL, oil globule volume, feeding incidence, and survival of larvae followed the procedures of the aeration intensity experiment described previously.

Feeding and water management Water of the same salinity was added, if necessary, to each aquarium daily to replace the volume of water taken during sampling. Starting on day 2, the larvae were fed rotifers, Brachinus rotundiformis, at an initial density of 30 individuals/mL. Thereafter, the density of rotifer in each aquarium was maintained to at least 30 ind/mL by adding the needed amount every morning. Tetraselmis sp. were added every morning to obtain a density of 105 cells/mL in the rearing water.

Statistical analysis anova (SAS) was used to test the effects of various aeration, salinity and light intensity levels on TL, oil globule absorption, feeding incidence, and survival of early-stage E. coioides larvae. Percentage

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values for feeding incidence and survival were arcsine-transformed prior to analysis. Significant differences between means were determined by Duncan’s multiple range test at P < 0.05.

RESULTS Aeration intensity experiment The intensity of aeration affected the rate of oil globule absorption, feeding incidence, and the growth and survival of early-stage grouper larvae (Fig. 1). The size of the oil globule remaining on day 2 was significantly larger in larvae reared at gentler aeration rates of 0.62 mL/min per L (43.8 mm3) and 1.25 mL/min per L (25.5 mm3) compared with those reared at higher aeration levels (12.0–12.9 mm3). There was no significant difference between aeration levels in the percentage of larvae with food in their gut on days 3 (20–35%) and 4 (45–65%) (data not shown). On day 5, however, larvae reared at 0.62 mL/min per L and 1.25 mL/min per L aeration (a)

JD Toledo et al.

levels had a significantly higher feeding incidence (65–82%) than those reared at 2.50 mL/min per L and 3.75 mL/min per L (12–25%). On day 2, mean TL was significantly longer in larvae reared at the 0.62 mL/min per L (2.25 mm) and 1.25 mL/min per L aeration rates (2.24 mm) compared with those reared at the higher aeration rates (2.20 mm). All larvae died under static conditions (no aeration) on day 1. No larvae survived at aeration levels of 2.50 mL/min per L and 3.75 mL/min/L on day 6, whereas 1.9–2.4% survived at aeration levels of 0.62 mL/min per L and 1.25 mL/min/L. Average daily temperature during the experiment ranged from 25.5∞C to 26.8∞C.

Salinity experiment Variation in salinity influenced the rate of oil globule absorption, feeding incidence, and growth and survival of early-stage larvae (Fig. 2). On day 1 and day 2, the oil globule volume of larvae was similar among the various salinities tested (37.9–45.9 µm3 and 14.2–17.8 µm3, respectively;

(c) (c)

(b)

Total length (mm)

Oil globule (µm3)

Oil globule (µm3)

Total length (mm)

(a)

(d) (d)

Aeration (mL/min per L)

Fig. 1 Effects of various aeration levels on: (a) oil globule absorption; (b) feeding incidence; (c) total length; and (d) survival of Epinephelus coioides larvae. Each bar represents the mean ± SE of three replicates (n = 20). Means with different letters are significantly different (P < 0.05). *All larvae died on day 6.

Survival (%)

Feeding incidence (%)

Survival (%)

Feeding incidence (%)

(b)

Salinity (ppt)

Fig. 2 Effects of various salinity levels on: (a) oil globule absorption; (b) feeding incidence; (c) total length; and (d) survival of Epinephelus coioides larvae. Each bar represents the mean ± SE of three replicates (n = 20). Means with different letters are significantly different (P < 0.05). *All larvae died on day 3.

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Light intensity experiment The amount of oil globule left on day 3 in larvae at 0 lx and 120 lx (18.1–21.8 mm3) were significantly larger than larvae reared at 230–700 lx (4.1–5.3 mm3) (Fig. 3). Except for larvae reared in dark conditions, larvae exposed to light intensities ranging between 120 lx and 700 lx started to feed on day 3. Feeding incidence was markedly higher in larvae reared at 500 lx (60%) and 700 lx (63%) compared with those reared at 120 lx (30%). There were no significant differences in the feeding incidence of larvae between the 120 lx and 230 lx (39–50%) experiments and also between the 230 lx and 700 lx (50–63%) experiments. On day 3, larvae reared in dark conditions (0 lx) had the shortest TL (2.31 mm). No significant difference in TL of larvae was observed between light intensities of 120 lx and 700 lx (2.40–2.43 mm). All larvae at 0 lx and 120 lx died by day 4 and day 6, respectively. On day 6, survival was significantly lower at a light intensity of 230 lx (1.1%) than at 500 lx (6.5%) and 700 lx (6.2%).

DISCUSSION The present study demonstrated the effects of salinity, aeration, and light intensity on oil globule absorption, feeding incidence, and growth and survival of E. coioides early-stage larvae. Early-stage grouper larvae survived much better at gentle aeration rates of 0.62 mL/min per L and 1.25 mL/min per L compared with those under

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(a)

(c)

Total length (mm)

Oil globule (µm3)

(b)

(d)

Survival (%)

data not shown). On day 3, larvae at 40 ppt had a significantly larger oil globule left (7.7 µm3) than those reared at lower salinities (2.6–5.0 µm3). The percentage of larvae with rotifer in the gut on day 3 was significantly higher at 16 ppt and 24 ppt (62–69%) than at 32 ppt (53%) and 40 ppt (12%). On day 1, the mean TL of larvae reared at 8 ppt and 16 ppt (2.46–2.71 mm) was significantly longer than those reared at higher salinities (2.28–2.34 mm). On day 2, larvae reared at 8 ppt (2.49 mm) were significantly longer than those reared between 16 ppt and 40 ppt (2.36–2.37 mm). At the onset of feeding (day 3), average TL of larvae reared at 16 ppt and 24 ppt (2.37 mm) were significantly longer than those reared at 32 ppt (2.30 mm) and 40 ppt (2.16 mm). Average survival on day 4 was significantly higher at 16 ppt (71%) and 24 ppt (59%) compared with those reared at 32 ppt (32%) and 40 ppt (11%). All larvae exposed to 8 ppt died by day 3. The daily average temperature ranged from 25.5∞C to 26.8∞C.

Feeding incidence (%)

Survival of early grouper larvae

Light intensity (lx)

Fig. 3 Effects of various light intensities on: (a) oil globule absorption; (b) feeding incidence; (c) total length; and (d) survival of Epinephelus coioides larvae. Each bar represents the mean ± SE of three replicates (n = 20). Means with different letters are significantly different (P < 0.05). *†All larvae died on days 4 and 6, respectively.

stronger aeration conditions of 2.50–3.75 mL/min per L and under static conditions (Fig. 1). Newly hatched larvae of E. coioides are positively buoyant at salinities above 30 ppt and most of the larvae accumulated and become trapped at the water surface (Toledo JD, pers. obs., 2000). Kaji et al. have shown that Epinephelus akaara larvae develop mucous cells on the body surface between 2 and 4 days after hatching.10 When trapped at the water surface, the mucous cells from E. akaara larvae function as a glue for the floating larvae, causing them to attach to one another.11 Static conditions could also have inhibited respiration because gas exchange in yolk-sac larvae occurs at the integument and may be facilitated by water movement or by swimming.12,13 In the present study, larvae at higher aeration levels had smaller remaining oil globules and shorter TL on day 2. It is likely that the higher aeration levels demanded increased energy to maintain the larvae’s swimming position. The low feeding incidence of larvae on day 5 at the higher aeration rates would also suggest that the larvae

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found it difficult to catch prey. Ellis et al. have suggested that high water turbulence (150– 300 mL/min) might increase contact between Epinephelus striatus larvae and the aquarium walls, causing injury.14 Post-metamorphic juveniles of orange-spotted grouper are caught in the wild using fish-aggregating devices such as shelters distributed along estuaries.8,15,16 Peak season for the collection of 2–4 cm juveniles occurs during or immediately after the rainy season. The results of the present study indicate that E. coioides larvae have a strong tolerance to a wide range of salinities. In the present study, newly hatched larvae survived the abrupt transfer from the spawning and hatching salinity of 35 ppt to salinities ranging between 8 ppt and 40 ppt (Fig. 2). Immediately after stocking, larvae at 8–24 ppt were observed to sink to the bottom of the aquarium. On day 1, larvae at 16 ppt and 24 ppt swam to the middle or upper portion of the water column, whereas larvae reared at 8 ppt remained clustered at the bottom of the aquarium, although they did not show floating death as described by Yamaoka et al. for E. akaara.11 Total mortality of larvae reared at 8 ppt on day 3 and the very low survival rate at 40 ppt on day 4 indicates that early larval stages of E. coioides had difficulty in adapting to these salinities. Based on the activity of the enzyme Na+,K+-ATPase and the morphological changes of gill chloride cells, Caberoy and Quinitio have concluded that E. coioides larvae and juveniles are efficient osmoregulators over a wide range of salinities, even those lower than normal seawater.17 Larvae exposed to the lower salinities (i.e. 16 ppt and 24 ppt) in the present study had significantly longer TL and a smaller remaining oil globule. Similar works on striped bass (Morone sexatilis), weakfish (Cynoscion regalis), and black porgy (Acanthopagrus schegeli) have shown maximum growth efficiency at lower levels of salinity because of their relatively lower metabolic rates at the lower salinities.18–20 In the present study, the higher feeding incidence, and higher growth and survival of larvae exposed to salinity levels between 16 ppt and 24 ppt could be a reflection of the better growth conditions. Light is generally required for the optimum growth of larvae.21 Duray and Kohno have reported 1000 lx as the optimum light intensity for rabbitfish (Siganus guttatus), a tropical marine species.22 European sea bass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata), which are both temperate marine species, requires 600 lx and 600–1300 lx, respectively, for optimal growth.23,24 In the present study, larval survival was significantly higher at 500 lx and 700 lx than at lower light inten-

JD Toledo et al.

sities (Fig. 3). The lowest growth and biggest remaining oil globule volume observed at 0 lx conditions was probably due to the reduced activity of the larvae in total darkness. Although no significant difference in growth was observed between the 120 lx and 700 lx light conditions, a higher percentage of larvae started to ingest feed at higher light intensities at the onset of feeding. Total mortality of larvae at 0 lx and 120 lx and the very low survival rate at 230 lx suggested that a light intensity higher than 500 lx is appropriate for firstfeeding E. coioides larvae. Light intensity higher than 700 lx should be tested to determine the optimal light intensity range for E. coioides earlystage larvae. Yamaoka et al. have suggested that the light intensity for the seed production of E. akaara must be lower than 1000 lx at the water surface level of rearing tanks until 10 days after hatching.11 Based on the results of the present study, a gentle aeration of 0.62–1.25 mL/min per L, rearing water at 16–24 ppt salinity, and a light intensity of 500–700 lx is recommended to maximize the survival of early-stage E. coioides larvae in the hatchery.

ACKNOWLEDGMENTS This study was financially supported by SEAFDEC/ AQD (Nr-06-F99T) and the Australian Center for International Agricultural Research (FIS/97/73). JDT is grateful to the Japan Society for the Promotion of Science for being awarded a Ronpaku PhD scholarship grant on grouper seed production.

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