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Larval release and settlement of the marine sponge Hymeniacidon perlevis (Porifera, Demospongiae) under controlled laboratory conditions
Aquaculture 290 (2009) 132–139
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Larval release and settlement of the marine sponge Hymeniacidon perlevis (Porifera, Demospongiae) under controlled laboratory conditions Lingyun Xue a,b, Xichang Zhang a,b, Wei Zhang a,c,⁎ a b c
Marine Bioproducts Engineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China Graduate School of the Chinese Academy of Sciences, Beijing 10089, China Molecular Bioprocessing and Bioproducts Laboratory, Department of Medical Biotechnology, School of Medicine, Flinders University, Adelaide, SA 5042 Australia
a r t i c l e
i n f o
Article history: Received 20 April 2008 Received in revised form 9 January 2009 Accepted 22 January 2009 Keywords: Porifera Sponge larvae Hymeniacidon perlevis Release Settlement
a b s t r a c t The insufficient supply of wild sponge biomass, i.e., “the supply problem,” critically limits the development of sponge-derived bioactive natural products and other applications. Intensive aquaculture of sponges through artificial seed rearing may provide an alternative sustainable supply of sponge biomass. To develop the technology of sponge aquaculture, protocols for artificial seed production need to be established. To understand larval release and settlement under artificial controlled environments, a model marine sponge Hymeniacidon perlevis was investigated under controlled laboratory conditions. The larval release of H. perlevis is an asynchronous event in the laboratory-controlled environment. Sponge explants attached on substrata release 5 times more larvae than unattached sponge explants. Over the course of 12 days of release, the mean release rate was 7.2 larvae g− 1 wet sponge day− 1 for attached sponges. Over the course of 7 days of release for unattached sponges, the mean release rate was 2.6 larvae g− 1 wet sponge day− 1. Light (6000 lx) stimulated the sponges to release more larvae than did dark incubation. The highest number of sponge larvae (195.8 larvae g− 1 wet sponge) was released at 18 °C, while only 48 and 51.7 larvae g− 1 wet sponge were released at 14 °C and 25 °C, respectively. Larval settlement was favored in dark condition. The highest percentage of larvae settled at 22 °C, among all temperatures tested. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Sponges are by far the richest sources of bioactive natural products among marine invertebrates (Duckworth and Battershill, 2003). Due to the insufficient supply of wild sponge biomass, the progress and development of sponge-derived drugs have been hindered at the preclinical phase, preventing further investigation (Pomponi, 1999). Cultivation of sponge fragments under controlled conditions could be an effective option to resolve this problem; however, mass cultivation in bioreactors has not been successful (Osinga et al., 1999; Belarbi et al., 2003; Hausmanna et al., 2006). This may be due to the use of sponge fragments that come from wild harvested sponges. As an alternative, the cultivation of eggs, reduction bodies and larvae may represent a better approach (Osinga et al., 1999). Intensive aquaculture of economical marine animals through seed (eggs or larvae) rearing, including seed production and rearing of juveniles, has achieved great success for biomass production. Extensive studies have focused on improving the production and survivorship of seeds and juveniles, in
⁎ Corresponding author. Marine Bioproducts Engineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. Tel./fax: +86 411 84379069. E-mail address: [email protected] (W. Zhang). 0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.01.037
fishes (Chakraborty and Mirza, 2007), abalone (Najmudeen and Victor, 2004), sea cucumber (Asha and Muthiah, 2005) and bivalves (Southgate and Lee, 1998; Liu et al., 2002; Muthiah et al., 2002). These rearing successes indicate that artificial seed rearing of sponges provides a sustainable supply of seeding sponges for sponge aquaculture, and it can also minimize the ecological and environmental impacts of wild sponge harvest. To establish the sponge seed-rearing technology, the seed production protocols must be established based on knowledge of the sponge reproductive cycle; methods for artificially inducing larval or egg and sperm release; optimal conditions for egg hatching and larval settlement; and metamorphosis to the juvenile stage. By far, the reproductive cycles of the following sponges are the most well understood: Hymeniacidon perleve (Stone, 1970), Halisarca dujardini (Ereskovsky and Gonobobleva, 2000), Geodia cydonium (Jameson 1811) (Mercurio et al., 2007), and Corticium candelabrum (Riesgo et al., 2007). It was reported that larval release can be triggered by adjusting the water flow, light, and temperature (Maldonado, 2006). Maternal sponges of Haliclona tubifera and Halichondria magniconulosa can release larvae following intense light irradiation after 12–20 h of incubation in the dark (Maldonado and Young, 1996). In the demosponge H. (G.) Slgmadocia caerulea, larvae release can be triggered by exposure to the air lasting for only a few seconds (Maldonado and Young, 1996). Carefully tearing mature Crambe crambe individuals can also cause larvae release (Uriz et al.,
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1998; Caralt et al., 2007). The rate of larvae release varies among different species and individual sponges. In addition, limited available data reveal that the duration of larval release is usually short in laboratory conditions. For example, for an individual sponge, the encrusting sponge Mycale fistulifera released larvae at a rate of 500 larvae day− 1 for 5 days in laboratory conditions (Meroz and Ilan, 1995). Ophlitaspongia seriata released larvae at a rate of 4–5 larvae min− 1 for an individual sponge (Bergquist and Sinclair, 1968). The settlement rate was faster, and the mortality was higher, when adults were incubated at lower temperature (10 and 15 °C) as compared to higher temperature (20 and 25 °C) (Maldonado and Young, 1996). Light was thought to be the cue for larval settlement. Larvae that show negative phototaxis prefer to settle onto shaded places for physical refuge (Woollacott, 1993; Maldonado and Young, 1996; Maldonado et al., 1997; Maldonado and Uriz, 1998). Many studies have reported sponge larval release and settlement, but to our knowledge, there are no other systematic quantitative studies on sponge seed production under controlled conditions. The life cycle of Marine sponge H. perleve in Langstone Harbour, Hampshire has previously been studied (Stone, 1970). H. perleve can produce larvae; the small red-amber granule-like embryos of H. perlevis develop in sponge tissues and are visible to the naked eye prior to release (Stone, 1970). H. perlevis is a demosponge exhibiting broad distribution throughout the coastal waters of the China Yellow Sea around Dalian City and displays an encrusting shape (Zhang et al., 2003). The growth cycle of H. perlevis in Dalian is similar to that observed for H. perlevis in Hampshire (data not published). In this paper, H. perlevis was selected as a model species to investigate the dynamics of quantitative larval release and settlement under laboratory-controlled conditions, in order to determine the feasibility of developing methods for artificial seed production (larval and juvenile sponges).
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Fig. 1. Schematic drawing of the location of adult sponges in the culture tank. a: Culture tank; b: air stone; c: glass substrate; d: adult sponge.
water bath at 22 ± 0.5 °C and in dark conditions (light intensity of 0– 30 lx). Larvae were collected using a plastic pipette from each tank and checked under light inverted microscopy (COIC IBE). When larvae were observed, photos were taken by digital camera (Olympus 3020). The number of larvae released in each tank was counted daily. The total number of larvae released by sponges in each tank during the whole experiment was calculated by summing the daily counts. The rate of larval release was calculated as the number of larvae released per gram wet sponge per day by dividing the number of larvae collected each day by the total wet weight of three sponge specimens in each tank. The mean number of larvae released per gram wet sponge during the whole experiment was calculated by dividing the total number of larvae collected from each tank throughout the entire experiment by the total wet weight of three sponge specimens. Larval release was followed until no larvae were released in the water for three consecutive days.
2. Materials and methods 2.3. Larval release under continuous illumination and dark incubation 2.1. Sponge collection, preparation and larval collection Ripe H. perlevis were collected from the Lingshui Bay, Dalian, China Yellow Sea during September and October 2006 and 2007, with average temperature and salinity of around 22 °C and 32%, respectively. Sponge specimens were collected from rocky substrates and immediately placed into plastic buckets filled with natural seawater. After transportation to the laboratory, sponge specimens with similar color, thickness and distribution of orange granules (as visible in sponge tissue) were chosen for the experiments. Each sponge specimen was attached onto a 4 × 4 cm2 glass slide with sewing threads after the wet weight was measured using an electronic balance (BS210S, Sartorius). Three sponge specimens of 2–3 g wet weight each were placed into a 1.2 l glass tank filled with 1 l natural seawater at a temperature of 22 ± 0.5 °C and salinity of approximately 32% (Fig. 1). An aquarium heater (H708) and seawater cooler (HXLS 1000I) were used to control the temperature of the water bath in all experiments. In each tank, an air pump was used to supply air through an air stone at a rate of 1.0 vvm (air volume per water volume per minute) and to generate water flow with air bubbles. These tanks of sponges were used in the release experiments. The seawater was exchanged at a rate of 100% daily. Natural seawater used in these experiments was sand-filtered after being collected at a depth of 15 m from the China Yellow Sea, near Dalian City. Algae Isochrysis galbana was fed to maternal sponges at a concentration of 2 × 105 cells ml− 1 once daily. 2.2. Larval release under attached and unattached conditions To investigate the effect of sponge attachment on larval release, two tanks of attached sponges (on glass slides) and two tanks of unattached sponges were used. Tanks were placed into thermostatic
To study the effect of light on larval release, three levels of light conditions were investigated: continuous dark incubation (D); 6000 lx artificial illumination at 12 h light:12 h darkness (L12); and continuous 6000 lx artificial illumination (L24). Three tanks of attached sponges were incubated at each light level and 22 ± 0.5 °C. Counts of released larvae in each tank were performed at 8:00am and 20:00pm each day. Larval production was calculated as described in Section 2.2. The light intensity was controlled by a set of fluorescent lamps (30 W each). 2.4. Larval release under different temperatures To investigate the effect of temperature on larval release, attached sponges were incubated at four different temperatures (14 ± 0.5, 18 ± 0.5, 22 ± 0.5 and 25 ± 0.5 °C) in the dark (0–30 lx); two tanks of sponge specimens were used at each temperature level. Counts of larvae released in each tank were performed daily. 2.5. Larval settlement under different light intensities Larval settlement was investigated under four different light intensities (0–30, 800–1100, 1800–2000, and 4000–5000 lx). We used 500 ml flasks containing 400 ml of seawater. This set of experiments was performed twice. In the first experiment, 40 larvae (20 larvae swimming in water and 20 larvae swimming or crawling on the bottom of the beaker) were placed in one flask at each light intensity; the experiment lasted for 68 h. In the second experiment, 20 larvae (swimming in water) were placed into one flask at each light intensity; the experiment lasted for 64 h. All larvae used in each experiment were collected from the same batch under darkness at 22 °C. A 50% water exchange was conducted every 12 h. The
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Table 1 Shape and size of swimming and settled larvae. Larvae
Swimming (1 h old)
Shape
Sphere
Oval
Circle
Ellipse
n = 22
n = 30
n = 39
n = 16
203 ± 33.1
273.5 ± 34.7 166.7 ± 20.1
350 ± 41.8
412.2 ± 52.0 318.8 ± 69.2
Size (mean ± SD) (µm)
D/L W
Settled (1 day old)
D the diameter of larvae; L the length of larvae; W the width of larvae.
percentages of swimming, crawling, bottom-settled and dead larvae were recorded over time. 2.6. Larvae settlement under different temperatures Larval settlement was investigated at four different temperatures (14 ± 0.5, 18 ± 0.5, 22 ± 0.5 and 25 ± 0.5 °C). This set of experiments was performed twice. In the first experiment, a total of 80 larvae (swimming in water) were placed into a 2 l beaker filled with 800 ml seawater at each temperature under the dark condition. In the second experiment, a total of 40 larvae were placed into 500 ml flaks filled with 400 ml of seawater at each temperature under the dark condition. Larvae used in each experiment were collected from the same batch at 18 °C and dark incubation. A 50% water exchange was conducted every 12 h. The first experiment lasted for 118 h, and the second lasted for 64 h. The percentages of swimming, crawling, bottom-settled and dead larvae were recorded over time.
Fig. 3. Number of released larvae under attached and unattached conditions. A: Larval release dynamics under fixed and unfixed conditions. B: Total number of released larvae under fixed and unfixed conditions. The curves and charts in A and B comprise the average values for larvae release, for two tanks.
3. Results 3.1. Larval shape and behavior Larvae of H. perlevis are non-tufted parenchymella with a spherical or oval shape (Table 1, Fig. 2a, b). Larvae are uniformly covered with short cilia of 10–13 µm, as visible when examined under light inverted microscopy. After release, larvae swam immediately to the water surface and then moved to the tank bottom, exhibiting exploratory behavior.
Larvae showed two kinds of behavior: swimming or crawling forward and moving in circles. Whenever larvae swam forward or in circles, they rotated along a longitudinal axis. Larvae exhibited a drilling motion when they swam, as described in Maldonado (2006). Later, larvae settled onto substrates and became sessile juveniles (Fig. 2e), with disclike or elliptical shapes (Table 1, Fig. 2c, d).
Fig. 2. Larvae and juvenile sponge of H. perlevis under light microscopy. a, b — Swimming larvae, a — spherical shape, b — oval shape; c, d — settled larvae, c — round shape, d — ellipse shape; e — juvenile sponge with spicule and ostium, spicule (s), ostium (o) Scale bars 44 µm for a and b; 60 µm for c and d, 100 µm for e.
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larvae, over two times the levels released at 22 °C and four times the levels released at 14 and 25 °C (Fig. 5B). 3.5. Larvae settlement favors dim artificial illumination
Fig. 4. Number of released larvae under light and dark conditions. A: Larval release dynamics under light and dark conditions. B: Total number of released larvae under light and dark conditions. The curves and charts in A and B comprise the mean number of released larvae for three tanks.
The duration of larvae swimming in water was longer in dim artificial illumination (0–30 lx and 800–1000 lx) than in strong artificial illumination (1800–2000 lx and 4000–5000 lx) (Fig. 6A, B). In the first experiment, 35% and 22.5% of larvae were swimming in water under dim artificial illumination (0–30 lx and 800–1000 lx); there were 17.5% and 7.5% swimming under strong artificial illumination (1800–2000 lx and 4000–5000 lx) (Fig. 6A) at 9 h. In the second experiment, 10% and 7.5% of larvae were swimming at 24 h in dim artificial illumination; no larvae were swimming in water under strong artificial illumination at 16 h (Fig. 6B). Though the period of larval swimming was prolonged under dim artificial illumination, the rate of larval settlement was reduced. At 43 h in the first experiment, 95%, 30%, 37.5% and 25% of larvae were settled on the bottom of the beaker at 0–30, 800–1100, 1800–2000 and 4000–5000 lx, respectively (Fig. 6A); at 54 h in the second experiment, 70%, 42.5%, 30% and 25% of larvae were settled (Fig. 6B). Larvae mortality was higher under the strong artificial illumination condition than under dim artificial illumination (Fig. 6A, B). At the end of the first experiment (68 h), the mortality of larvae was 5% and 25% under light intensity of 0–30 lx and 4000–5000 lx, respectively, and 2.5% and 7.5% (64 h) at the end of the second experiment. 3.6. Effects of temperature on larvae settlement The duration of larval swimming was longer at 18 °C than at 14, 22 and 25 °C (Fig. 7A, B). In the first experiment, 70% of larvae were still
3.2. Attached sponges release more larvae than unattached sponges Larval release of H. perlevis was an asynchronous event under both attached and unattached conditions (Fig. 3A). The mean larval release rate and release duration of attached and unattached sponges were 7.2 larvae g− 1 wet sponge day− 1 and 12 days; and 2.6 larvae g− 1 wet sponge day− 1 and 7 days, respectively. Overall, attached sponges released 5 times more larvae than unattached sponges (Fig. 3B). 3.3. Continuous illumination stimulates sponge larvae release Sponge H. perlevis can release larvae under both continuous illumination and dark conditions, but 6000 lx light illumination stimulated larval release in this experiment (Fig. 4A). The total numbers of larvae released in L24, L12 and D conditions were 411, 289 and 154 larvae g− 1 wet sponge, respectively (Fig. 4B). Among the 289 larvae per gram wet sponges released by H. perlevis in the L12 condition, 238 larvae were released during light illumination; only 51 larvae were released in darkness. Light not only increased the total number of larvae released by H. perlevis but also shortened the duration of larval release. The duration of larval release was 11 days and 12 days in L24 and L12 conditions, respectively, but 23 days in the dark condition. As a result, the mean release rate was 37.4 and 24.1 larvae g− 1 wet sponge day− 1 for L24 and L12 conditions, but 6.7 larvae g− 1 wet sponge day− 1 in dark incubation. 3.4. Effect of temperature on larvae release Maternal sponge can release larvae at temperatures ranging from 14 °C to 25 °C. The mean larval release rates of sponge incubated at 14, 18, 22 and 25 °C were 4.0, 8.9, 7.2 and 8.7 larvae g− 1 wet sponge day− 1 with a duration of 12, 22, 12 and 6 days, respectively (Fig. 5A, B). In the first 2 days, sponges incubated at higher temperature (25 °C) released more larvae when compared to other temperatures (Fig. 5A, B). Overall, sponges incubated at 18 °C released the highest number of
Fig. 5. Number of released larvae under different incubation temperatures. A: Larval release dynamics under different incubation temperatures. B: Total number of released larvae under different incubation temperatures. The curves and charts in A and B comprise the mean number of released larvae for two tanks.
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Fig. 6. Cumulative percentage of free-swimming larvae, crawling larvae, settlers and mortality over time under four light intensities. A: First experiment; B: Second experiment. Siw: larvae swimming in water; Sab: larvae swimming or crawling on the bottom of the beaker; Settlers: Larvae that settled on the bottom of the beaker.
swimming at 72 h at 18 °C, but 22%, 6% and 0% were still swimming at 14, 22 and 25 °C, respectively (Fig. 7A). In the second experiment, there was 50% larvae swimming at 64 h at 18 °C, but 7.5%, 15% and 0% were still swimming at 14, 22 and 25 °C, respectively (Fig. 7B). The rate of larval settlement was slower at 18 °C than at other temperatures. In the first experiment, 26% of larvae had settled at 72 h at 18 °C, but 70%, 84% and 88% of larvae had settled at 14, 22 and 25 °C, respectively (Fig. 7A). In the second experiment, 50% of larvae had settled at 64 h at 18 °C, but 90%, 83% and 93% of larvae had settled at 14, 22 and 25 °C, respectively (Fig. 7B). The rate of larval settlement was higher at 14 °C and 25 °C, but the mortality of larvae was higher at the end of these
experiments (Fig. 7A, B). As a result, 22 °C appeared to be the optimal temperature for larval settlement. 4. Discussion 4.1. Larval release kinetics of H. perlevis under artificial conditions Larval release of H. perlevis under laboratory-controlled conditions was an asynchronous event (Figs. 3A, 4A and 5A). There were some mass releases during experiments, similar to those reported for tropical shallow water sponges Tedania ignis and H. tubifera (Maldonado and
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Fig. 7. Cumulative percentage of free-swimming larvae, crawling larvae, settlers and mortality over time under four temperature incubations. A: First experiment in 2 l beaker; B: Second experiment in 500 ml flask. Siw: larvae swimming in water; Sab: larvae swimming or crawling on the bottom of the beaker; Settlers: Larvae that settled on the bottom of the beaker.
Young, 1996). This phenomenon could be the result of an asynchrony of gametogenesis (Ilan and Loya, 1990) and fertilization (Maldonado and Riesgo, 2008) in viviparous sponges, representing an effort to avoid predators and decrease mortality under adverse environmental conditions. The duration of larval release in our experiment was longer than observed for the encrusting sponge M. fistulifera (5 days) (Meroz and Ilan, 1995), lasting over 20 days for maternal sponges of H. perlevis collected in October 2006. H. perlevis larvae were photonegative, similar to other reported sponge larvae (Amano, 1986; Leys and Degnan, 2001; Leys et al., 2002; Maldonado et al., 2003). Larvae escaped from a light source of diameter ~ 5 cm within 10 min when the light density increased to over 1000 lx. It has been reported that larval release can be triggered
by strong illumination and by exposing ripe adult sponges to air for a few seconds (Amano, 1986; Maldonado and Young, 1996). However, these conditions can be very stressful for sponges because many sponges are sensitive to ultraviolet radiation and air exposure (Osinga et al., 1999). Ripe adults of H. perlevis can release larvae under both light and dark incubations. However, continuous artificial illumination significantly stimulated larval release of H. perlevis in our experiment. The results could be explained by larval photo-negativity and faster maternal sponge disorganization under the light condition. As reported earlier, the structure of sponge tissue was disorganized, permitting larvae to emerge before they were released (Maldonado and Uriz, 1999). In our experiments, more larvae tended to emerge from the adult sponges under light illumination, which led to faster sponge
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disorganization, the formation of more osculum pipes, and tissue decline (data not shown). Negative phototaxis allows larvae to escape faster from the declined tissues. Photonegativity also has an important effect on larval settlement in the natural intertidal environment. Larvae probably use the photonegative response to settle in small shaded microhabitats, where they will be protected from desiccation during low tide and exposure to high-intensity sunlight. Increased temperature was also found to be a factor favoring larval release in temperate sponges (Uriz, 1982). In our experiments, a similar result was observed for H. perlevis. However, an optimal temperature of 18 °C was found to yield the greatest larval release for a longer duration. H. perlevis in Langstone Harbour (Stone, 1970) were ripe in August, when the temperature is at its peak. Larvae that develop during the high-temperature period exhibit lower mortality (Maldonado and Young, 1996). Maternal sponges expel more larvae when they sense a decrease in water temperature. Fewer larvae were released at the lower incubation temperature (14 °C), which is a suboptimal living temperature for H. perlevis (unpublished). This is probably due to the disturbance of normal physiological activity in adult sponges at lower temperatures. Fewer larvae were released by unattached sponges as compared to attached sponges in our experiments. This effect may be partially due to the water flow and the sessile living habits of H. perlevis. The unattached sponge specimens moved freely and were not subjected to the same water flow forces as encountered when attached on substrata (similar to their attachment on natural rocks in the sea). It is documented in the literature that larval release may be affected by water flow. Calm water has been reported to enhance larval release among temperate sponges (Bergquist et al., 1970). But for H. perlevis, very few larvae were released without water flow in our experiment (not published). Furthermore, no free-swimming larvae were ever observed in the tidal pools inhabited by the sponges used for this study. It is likely that natural larval release takes places during high tide and that most larvae are washed off from the parental habitat with the outgoing tidal wave. 4.2. Larval swimming and settlement The free-swimming life of sponge H. perlevis larvae after release can last from 3 h to 10 days in laboratory-controlled conditions, in contrast to the 3 days observed for larvae of the same sponge living in Hauraki Gulf, Auckland, New Zealand (Bergquist and Sinclair, 1973). Swimming and settlement of larvae were affected by temperature levels (Maldonado and Young, 1996; Maldonado, 2006), light levels (Maldonado and Uriz, 1998) and types of substrates (Woollacott, 1993; Maldonado et al., 1997). The swimming and settlement of H. perlevis larvae were also affected by light illumination in the laboratory. However, the effects contrasted with those reported in an earlier work that found that illumination could not dislodge larvae of H. perlevis from the water surface (Bergquist and Sinclair, 1973). Larval behavior was affected by illumination, possibly because larvae use light cues to find shaded places to settle. Lévi (1950) reported that larvae of sponge H. perlevis at Roscoff were released only at daybreak. Light after daybreak may be the cue that causes larvae to sink to the water bottom after a short period of pelagic life, avoiding visual predators and ultraviolet light (Lindquist et al., 1997). In our experiments, more larvae settled under higher and lower temperatures as compared to moderate temperature. The effect of lower temperature on larval settlement was similar to that reported for sponge H. magniconulosa (Maldonado and Young, 1996). 5. Conclusion Large quantities of larvae and early juveniles of an inter-tidal marine sponge H. perlevis could be obtained by subjecting ripe sponges collected during the larval-releasing season to optimal
controlled artificial conditions. The results indicate great potential for sponge seed production under artificial conditions in the context of sponge aquaculture. Whether the optimal conditions for larval release and settlement of H. perlevis are applicable to other sponge species remains to be demonstrated. Nonetheless, the current study provides important information for the future development of seed-rearing technology. Acknowledgements The authors wish to thank the “Hi-Tech Research and Development Program of China” (2006AA09Z435), “Innovation Fund” from the Dalian Institute of Chemical Physics and the “Innovative Key Project of the Chinese Academy of Sciences” (KZCX2-YW-209) for financial support. The authors would also like to thank Dr. K. Manmadhan, Dr. Li-Ming Sun, Dr. Xu-Peng Cao and Dr. Wan-Tao Fu for their useful comments on the manuscript and Mr. Yuan-Ling Liu for constructing the cooling and heating systems. References Amano, S., 2006. 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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Larval release and settlement of the marine sponge Hymeniacidon perlevis (Porifera, Demospongiae) under controlled laboratory conditions Lingyun Xue a,b, Xichang Zhang a,b, Wei Zhang a,c,⁎ a b c
Marine Bioproducts Engineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China Graduate School of the Chinese Academy of Sciences, Beijing 10089, China Molecular Bioprocessing and Bioproducts Laboratory, Department of Medical Biotechnology, School of Medicine, Flinders University, Adelaide, SA 5042 Australia
a r t i c l e
i n f o
Article history: Received 20 April 2008 Received in revised form 9 January 2009 Accepted 22 January 2009 Keywords: Porifera Sponge larvae Hymeniacidon perlevis Release Settlement
a b s t r a c t The insufficient supply of wild sponge biomass, i.e., “the supply problem,” critically limits the development of sponge-derived bioactive natural products and other applications. Intensive aquaculture of sponges through artificial seed rearing may provide an alternative sustainable supply of sponge biomass. To develop the technology of sponge aquaculture, protocols for artificial seed production need to be established. To understand larval release and settlement under artificial controlled environments, a model marine sponge Hymeniacidon perlevis was investigated under controlled laboratory conditions. The larval release of H. perlevis is an asynchronous event in the laboratory-controlled environment. Sponge explants attached on substrata release 5 times more larvae than unattached sponge explants. Over the course of 12 days of release, the mean release rate was 7.2 larvae g− 1 wet sponge day− 1 for attached sponges. Over the course of 7 days of release for unattached sponges, the mean release rate was 2.6 larvae g− 1 wet sponge day− 1. Light (6000 lx) stimulated the sponges to release more larvae than did dark incubation. The highest number of sponge larvae (195.8 larvae g− 1 wet sponge) was released at 18 °C, while only 48 and 51.7 larvae g− 1 wet sponge were released at 14 °C and 25 °C, respectively. Larval settlement was favored in dark condition. The highest percentage of larvae settled at 22 °C, among all temperatures tested. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Sponges are by far the richest sources of bioactive natural products among marine invertebrates (Duckworth and Battershill, 2003). Due to the insufficient supply of wild sponge biomass, the progress and development of sponge-derived drugs have been hindered at the preclinical phase, preventing further investigation (Pomponi, 1999). Cultivation of sponge fragments under controlled conditions could be an effective option to resolve this problem; however, mass cultivation in bioreactors has not been successful (Osinga et al., 1999; Belarbi et al., 2003; Hausmanna et al., 2006). This may be due to the use of sponge fragments that come from wild harvested sponges. As an alternative, the cultivation of eggs, reduction bodies and larvae may represent a better approach (Osinga et al., 1999). Intensive aquaculture of economical marine animals through seed (eggs or larvae) rearing, including seed production and rearing of juveniles, has achieved great success for biomass production. Extensive studies have focused on improving the production and survivorship of seeds and juveniles, in
⁎ Corresponding author. Marine Bioproducts Engineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. Tel./fax: +86 411 84379069. E-mail address: [email protected] (W. Zhang). 0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.01.037
fishes (Chakraborty and Mirza, 2007), abalone (Najmudeen and Victor, 2004), sea cucumber (Asha and Muthiah, 2005) and bivalves (Southgate and Lee, 1998; Liu et al., 2002; Muthiah et al., 2002). These rearing successes indicate that artificial seed rearing of sponges provides a sustainable supply of seeding sponges for sponge aquaculture, and it can also minimize the ecological and environmental impacts of wild sponge harvest. To establish the sponge seed-rearing technology, the seed production protocols must be established based on knowledge of the sponge reproductive cycle; methods for artificially inducing larval or egg and sperm release; optimal conditions for egg hatching and larval settlement; and metamorphosis to the juvenile stage. By far, the reproductive cycles of the following sponges are the most well understood: Hymeniacidon perleve (Stone, 1970), Halisarca dujardini (Ereskovsky and Gonobobleva, 2000), Geodia cydonium (Jameson 1811) (Mercurio et al., 2007), and Corticium candelabrum (Riesgo et al., 2007). It was reported that larval release can be triggered by adjusting the water flow, light, and temperature (Maldonado, 2006). Maternal sponges of Haliclona tubifera and Halichondria magniconulosa can release larvae following intense light irradiation after 12–20 h of incubation in the dark (Maldonado and Young, 1996). In the demosponge H. (G.) Slgmadocia caerulea, larvae release can be triggered by exposure to the air lasting for only a few seconds (Maldonado and Young, 1996). Carefully tearing mature Crambe crambe individuals can also cause larvae release (Uriz et al.,
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1998; Caralt et al., 2007). The rate of larvae release varies among different species and individual sponges. In addition, limited available data reveal that the duration of larval release is usually short in laboratory conditions. For example, for an individual sponge, the encrusting sponge Mycale fistulifera released larvae at a rate of 500 larvae day− 1 for 5 days in laboratory conditions (Meroz and Ilan, 1995). Ophlitaspongia seriata released larvae at a rate of 4–5 larvae min− 1 for an individual sponge (Bergquist and Sinclair, 1968). The settlement rate was faster, and the mortality was higher, when adults were incubated at lower temperature (10 and 15 °C) as compared to higher temperature (20 and 25 °C) (Maldonado and Young, 1996). Light was thought to be the cue for larval settlement. Larvae that show negative phototaxis prefer to settle onto shaded places for physical refuge (Woollacott, 1993; Maldonado and Young, 1996; Maldonado et al., 1997; Maldonado and Uriz, 1998). Many studies have reported sponge larval release and settlement, but to our knowledge, there are no other systematic quantitative studies on sponge seed production under controlled conditions. The life cycle of Marine sponge H. perleve in Langstone Harbour, Hampshire has previously been studied (Stone, 1970). H. perleve can produce larvae; the small red-amber granule-like embryos of H. perlevis develop in sponge tissues and are visible to the naked eye prior to release (Stone, 1970). H. perlevis is a demosponge exhibiting broad distribution throughout the coastal waters of the China Yellow Sea around Dalian City and displays an encrusting shape (Zhang et al., 2003). The growth cycle of H. perlevis in Dalian is similar to that observed for H. perlevis in Hampshire (data not published). In this paper, H. perlevis was selected as a model species to investigate the dynamics of quantitative larval release and settlement under laboratory-controlled conditions, in order to determine the feasibility of developing methods for artificial seed production (larval and juvenile sponges).
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Fig. 1. Schematic drawing of the location of adult sponges in the culture tank. a: Culture tank; b: air stone; c: glass substrate; d: adult sponge.
water bath at 22 ± 0.5 °C and in dark conditions (light intensity of 0– 30 lx). Larvae were collected using a plastic pipette from each tank and checked under light inverted microscopy (COIC IBE). When larvae were observed, photos were taken by digital camera (Olympus 3020). The number of larvae released in each tank was counted daily. The total number of larvae released by sponges in each tank during the whole experiment was calculated by summing the daily counts. The rate of larval release was calculated as the number of larvae released per gram wet sponge per day by dividing the number of larvae collected each day by the total wet weight of three sponge specimens in each tank. The mean number of larvae released per gram wet sponge during the whole experiment was calculated by dividing the total number of larvae collected from each tank throughout the entire experiment by the total wet weight of three sponge specimens. Larval release was followed until no larvae were released in the water for three consecutive days.
2. Materials and methods 2.3. Larval release under continuous illumination and dark incubation 2.1. Sponge collection, preparation and larval collection Ripe H. perlevis were collected from the Lingshui Bay, Dalian, China Yellow Sea during September and October 2006 and 2007, with average temperature and salinity of around 22 °C and 32%, respectively. Sponge specimens were collected from rocky substrates and immediately placed into plastic buckets filled with natural seawater. After transportation to the laboratory, sponge specimens with similar color, thickness and distribution of orange granules (as visible in sponge tissue) were chosen for the experiments. Each sponge specimen was attached onto a 4 × 4 cm2 glass slide with sewing threads after the wet weight was measured using an electronic balance (BS210S, Sartorius). Three sponge specimens of 2–3 g wet weight each were placed into a 1.2 l glass tank filled with 1 l natural seawater at a temperature of 22 ± 0.5 °C and salinity of approximately 32% (Fig. 1). An aquarium heater (H708) and seawater cooler (HXLS 1000I) were used to control the temperature of the water bath in all experiments. In each tank, an air pump was used to supply air through an air stone at a rate of 1.0 vvm (air volume per water volume per minute) and to generate water flow with air bubbles. These tanks of sponges were used in the release experiments. The seawater was exchanged at a rate of 100% daily. Natural seawater used in these experiments was sand-filtered after being collected at a depth of 15 m from the China Yellow Sea, near Dalian City. Algae Isochrysis galbana was fed to maternal sponges at a concentration of 2 × 105 cells ml− 1 once daily. 2.2. Larval release under attached and unattached conditions To investigate the effect of sponge attachment on larval release, two tanks of attached sponges (on glass slides) and two tanks of unattached sponges were used. Tanks were placed into thermostatic
To study the effect of light on larval release, three levels of light conditions were investigated: continuous dark incubation (D); 6000 lx artificial illumination at 12 h light:12 h darkness (L12); and continuous 6000 lx artificial illumination (L24). Three tanks of attached sponges were incubated at each light level and 22 ± 0.5 °C. Counts of released larvae in each tank were performed at 8:00am and 20:00pm each day. Larval production was calculated as described in Section 2.2. The light intensity was controlled by a set of fluorescent lamps (30 W each). 2.4. Larval release under different temperatures To investigate the effect of temperature on larval release, attached sponges were incubated at four different temperatures (14 ± 0.5, 18 ± 0.5, 22 ± 0.5 and 25 ± 0.5 °C) in the dark (0–30 lx); two tanks of sponge specimens were used at each temperature level. Counts of larvae released in each tank were performed daily. 2.5. Larval settlement under different light intensities Larval settlement was investigated under four different light intensities (0–30, 800–1100, 1800–2000, and 4000–5000 lx). We used 500 ml flasks containing 400 ml of seawater. This set of experiments was performed twice. In the first experiment, 40 larvae (20 larvae swimming in water and 20 larvae swimming or crawling on the bottom of the beaker) were placed in one flask at each light intensity; the experiment lasted for 68 h. In the second experiment, 20 larvae (swimming in water) were placed into one flask at each light intensity; the experiment lasted for 64 h. All larvae used in each experiment were collected from the same batch under darkness at 22 °C. A 50% water exchange was conducted every 12 h. The
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Table 1 Shape and size of swimming and settled larvae. Larvae
Swimming (1 h old)
Shape
Sphere
Oval
Circle
Ellipse
n = 22
n = 30
n = 39
n = 16
203 ± 33.1
273.5 ± 34.7 166.7 ± 20.1
350 ± 41.8
412.2 ± 52.0 318.8 ± 69.2
Size (mean ± SD) (µm)
D/L W
Settled (1 day old)
D the diameter of larvae; L the length of larvae; W the width of larvae.
percentages of swimming, crawling, bottom-settled and dead larvae were recorded over time. 2.6. Larvae settlement under different temperatures Larval settlement was investigated at four different temperatures (14 ± 0.5, 18 ± 0.5, 22 ± 0.5 and 25 ± 0.5 °C). This set of experiments was performed twice. In the first experiment, a total of 80 larvae (swimming in water) were placed into a 2 l beaker filled with 800 ml seawater at each temperature under the dark condition. In the second experiment, a total of 40 larvae were placed into 500 ml flaks filled with 400 ml of seawater at each temperature under the dark condition. Larvae used in each experiment were collected from the same batch at 18 °C and dark incubation. A 50% water exchange was conducted every 12 h. The first experiment lasted for 118 h, and the second lasted for 64 h. The percentages of swimming, crawling, bottom-settled and dead larvae were recorded over time.
Fig. 3. Number of released larvae under attached and unattached conditions. A: Larval release dynamics under fixed and unfixed conditions. B: Total number of released larvae under fixed and unfixed conditions. The curves and charts in A and B comprise the average values for larvae release, for two tanks.
3. Results 3.1. Larval shape and behavior Larvae of H. perlevis are non-tufted parenchymella with a spherical or oval shape (Table 1, Fig. 2a, b). Larvae are uniformly covered with short cilia of 10–13 µm, as visible when examined under light inverted microscopy. After release, larvae swam immediately to the water surface and then moved to the tank bottom, exhibiting exploratory behavior.
Larvae showed two kinds of behavior: swimming or crawling forward and moving in circles. Whenever larvae swam forward or in circles, they rotated along a longitudinal axis. Larvae exhibited a drilling motion when they swam, as described in Maldonado (2006). Later, larvae settled onto substrates and became sessile juveniles (Fig. 2e), with disclike or elliptical shapes (Table 1, Fig. 2c, d).
Fig. 2. Larvae and juvenile sponge of H. perlevis under light microscopy. a, b — Swimming larvae, a — spherical shape, b — oval shape; c, d — settled larvae, c — round shape, d — ellipse shape; e — juvenile sponge with spicule and ostium, spicule (s), ostium (o) Scale bars 44 µm for a and b; 60 µm for c and d, 100 µm for e.
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larvae, over two times the levels released at 22 °C and four times the levels released at 14 and 25 °C (Fig. 5B). 3.5. Larvae settlement favors dim artificial illumination
Fig. 4. Number of released larvae under light and dark conditions. A: Larval release dynamics under light and dark conditions. B: Total number of released larvae under light and dark conditions. The curves and charts in A and B comprise the mean number of released larvae for three tanks.
The duration of larvae swimming in water was longer in dim artificial illumination (0–30 lx and 800–1000 lx) than in strong artificial illumination (1800–2000 lx and 4000–5000 lx) (Fig. 6A, B). In the first experiment, 35% and 22.5% of larvae were swimming in water under dim artificial illumination (0–30 lx and 800–1000 lx); there were 17.5% and 7.5% swimming under strong artificial illumination (1800–2000 lx and 4000–5000 lx) (Fig. 6A) at 9 h. In the second experiment, 10% and 7.5% of larvae were swimming at 24 h in dim artificial illumination; no larvae were swimming in water under strong artificial illumination at 16 h (Fig. 6B). Though the period of larval swimming was prolonged under dim artificial illumination, the rate of larval settlement was reduced. At 43 h in the first experiment, 95%, 30%, 37.5% and 25% of larvae were settled on the bottom of the beaker at 0–30, 800–1100, 1800–2000 and 4000–5000 lx, respectively (Fig. 6A); at 54 h in the second experiment, 70%, 42.5%, 30% and 25% of larvae were settled (Fig. 6B). Larvae mortality was higher under the strong artificial illumination condition than under dim artificial illumination (Fig. 6A, B). At the end of the first experiment (68 h), the mortality of larvae was 5% and 25% under light intensity of 0–30 lx and 4000–5000 lx, respectively, and 2.5% and 7.5% (64 h) at the end of the second experiment. 3.6. Effects of temperature on larvae settlement The duration of larval swimming was longer at 18 °C than at 14, 22 and 25 °C (Fig. 7A, B). In the first experiment, 70% of larvae were still
3.2. Attached sponges release more larvae than unattached sponges Larval release of H. perlevis was an asynchronous event under both attached and unattached conditions (Fig. 3A). The mean larval release rate and release duration of attached and unattached sponges were 7.2 larvae g− 1 wet sponge day− 1 and 12 days; and 2.6 larvae g− 1 wet sponge day− 1 and 7 days, respectively. Overall, attached sponges released 5 times more larvae than unattached sponges (Fig. 3B). 3.3. Continuous illumination stimulates sponge larvae release Sponge H. perlevis can release larvae under both continuous illumination and dark conditions, but 6000 lx light illumination stimulated larval release in this experiment (Fig. 4A). The total numbers of larvae released in L24, L12 and D conditions were 411, 289 and 154 larvae g− 1 wet sponge, respectively (Fig. 4B). Among the 289 larvae per gram wet sponges released by H. perlevis in the L12 condition, 238 larvae were released during light illumination; only 51 larvae were released in darkness. Light not only increased the total number of larvae released by H. perlevis but also shortened the duration of larval release. The duration of larval release was 11 days and 12 days in L24 and L12 conditions, respectively, but 23 days in the dark condition. As a result, the mean release rate was 37.4 and 24.1 larvae g− 1 wet sponge day− 1 for L24 and L12 conditions, but 6.7 larvae g− 1 wet sponge day− 1 in dark incubation. 3.4. Effect of temperature on larvae release Maternal sponge can release larvae at temperatures ranging from 14 °C to 25 °C. The mean larval release rates of sponge incubated at 14, 18, 22 and 25 °C were 4.0, 8.9, 7.2 and 8.7 larvae g− 1 wet sponge day− 1 with a duration of 12, 22, 12 and 6 days, respectively (Fig. 5A, B). In the first 2 days, sponges incubated at higher temperature (25 °C) released more larvae when compared to other temperatures (Fig. 5A, B). Overall, sponges incubated at 18 °C released the highest number of
Fig. 5. Number of released larvae under different incubation temperatures. A: Larval release dynamics under different incubation temperatures. B: Total number of released larvae under different incubation temperatures. The curves and charts in A and B comprise the mean number of released larvae for two tanks.
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Fig. 6. Cumulative percentage of free-swimming larvae, crawling larvae, settlers and mortality over time under four light intensities. A: First experiment; B: Second experiment. Siw: larvae swimming in water; Sab: larvae swimming or crawling on the bottom of the beaker; Settlers: Larvae that settled on the bottom of the beaker.
swimming at 72 h at 18 °C, but 22%, 6% and 0% were still swimming at 14, 22 and 25 °C, respectively (Fig. 7A). In the second experiment, there was 50% larvae swimming at 64 h at 18 °C, but 7.5%, 15% and 0% were still swimming at 14, 22 and 25 °C, respectively (Fig. 7B). The rate of larval settlement was slower at 18 °C than at other temperatures. In the first experiment, 26% of larvae had settled at 72 h at 18 °C, but 70%, 84% and 88% of larvae had settled at 14, 22 and 25 °C, respectively (Fig. 7A). In the second experiment, 50% of larvae had settled at 64 h at 18 °C, but 90%, 83% and 93% of larvae had settled at 14, 22 and 25 °C, respectively (Fig. 7B). The rate of larval settlement was higher at 14 °C and 25 °C, but the mortality of larvae was higher at the end of these
experiments (Fig. 7A, B). As a result, 22 °C appeared to be the optimal temperature for larval settlement. 4. Discussion 4.1. Larval release kinetics of H. perlevis under artificial conditions Larval release of H. perlevis under laboratory-controlled conditions was an asynchronous event (Figs. 3A, 4A and 5A). There were some mass releases during experiments, similar to those reported for tropical shallow water sponges Tedania ignis and H. tubifera (Maldonado and
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Fig. 7. Cumulative percentage of free-swimming larvae, crawling larvae, settlers and mortality over time under four temperature incubations. A: First experiment in 2 l beaker; B: Second experiment in 500 ml flask. Siw: larvae swimming in water; Sab: larvae swimming or crawling on the bottom of the beaker; Settlers: Larvae that settled on the bottom of the beaker.
Young, 1996). This phenomenon could be the result of an asynchrony of gametogenesis (Ilan and Loya, 1990) and fertilization (Maldonado and Riesgo, 2008) in viviparous sponges, representing an effort to avoid predators and decrease mortality under adverse environmental conditions. The duration of larval release in our experiment was longer than observed for the encrusting sponge M. fistulifera (5 days) (Meroz and Ilan, 1995), lasting over 20 days for maternal sponges of H. perlevis collected in October 2006. H. perlevis larvae were photonegative, similar to other reported sponge larvae (Amano, 1986; Leys and Degnan, 2001; Leys et al., 2002; Maldonado et al., 2003). Larvae escaped from a light source of diameter ~ 5 cm within 10 min when the light density increased to over 1000 lx. It has been reported that larval release can be triggered
by strong illumination and by exposing ripe adult sponges to air for a few seconds (Amano, 1986; Maldonado and Young, 1996). However, these conditions can be very stressful for sponges because many sponges are sensitive to ultraviolet radiation and air exposure (Osinga et al., 1999). Ripe adults of H. perlevis can release larvae under both light and dark incubations. However, continuous artificial illumination significantly stimulated larval release of H. perlevis in our experiment. The results could be explained by larval photo-negativity and faster maternal sponge disorganization under the light condition. As reported earlier, the structure of sponge tissue was disorganized, permitting larvae to emerge before they were released (Maldonado and Uriz, 1999). In our experiments, more larvae tended to emerge from the adult sponges under light illumination, which led to faster sponge
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disorganization, the formation of more osculum pipes, and tissue decline (data not shown). Negative phototaxis allows larvae to escape faster from the declined tissues. Photonegativity also has an important effect on larval settlement in the natural intertidal environment. Larvae probably use the photonegative response to settle in small shaded microhabitats, where they will be protected from desiccation during low tide and exposure to high-intensity sunlight. Increased temperature was also found to be a factor favoring larval release in temperate sponges (Uriz, 1982). In our experiments, a similar result was observed for H. perlevis. However, an optimal temperature of 18 °C was found to yield the greatest larval release for a longer duration. H. perlevis in Langstone Harbour (Stone, 1970) were ripe in August, when the temperature is at its peak. Larvae that develop during the high-temperature period exhibit lower mortality (Maldonado and Young, 1996). Maternal sponges expel more larvae when they sense a decrease in water temperature. Fewer larvae were released at the lower incubation temperature (14 °C), which is a suboptimal living temperature for H. perlevis (unpublished). This is probably due to the disturbance of normal physiological activity in adult sponges at lower temperatures. Fewer larvae were released by unattached sponges as compared to attached sponges in our experiments. This effect may be partially due to the water flow and the sessile living habits of H. perlevis. The unattached sponge specimens moved freely and were not subjected to the same water flow forces as encountered when attached on substrata (similar to their attachment on natural rocks in the sea). It is documented in the literature that larval release may be affected by water flow. Calm water has been reported to enhance larval release among temperate sponges (Bergquist et al., 1970). But for H. perlevis, very few larvae were released without water flow in our experiment (not published). Furthermore, no free-swimming larvae were ever observed in the tidal pools inhabited by the sponges used for this study. It is likely that natural larval release takes places during high tide and that most larvae are washed off from the parental habitat with the outgoing tidal wave. 4.2. Larval swimming and settlement The free-swimming life of sponge H. perlevis larvae after release can last from 3 h to 10 days in laboratory-controlled conditions, in contrast to the 3 days observed for larvae of the same sponge living in Hauraki Gulf, Auckland, New Zealand (Bergquist and Sinclair, 1973). Swimming and settlement of larvae were affected by temperature levels (Maldonado and Young, 1996; Maldonado, 2006), light levels (Maldonado and Uriz, 1998) and types of substrates (Woollacott, 1993; Maldonado et al., 1997). The swimming and settlement of H. perlevis larvae were also affected by light illumination in the laboratory. However, the effects contrasted with those reported in an earlier work that found that illumination could not dislodge larvae of H. perlevis from the water surface (Bergquist and Sinclair, 1973). Larval behavior was affected by illumination, possibly because larvae use light cues to find shaded places to settle. Lévi (1950) reported that larvae of sponge H. perlevis at Roscoff were released only at daybreak. Light after daybreak may be the cue that causes larvae to sink to the water bottom after a short period of pelagic life, avoiding visual predators and ultraviolet light (Lindquist et al., 1997). In our experiments, more larvae settled under higher and lower temperatures as compared to moderate temperature. The effect of lower temperature on larval settlement was similar to that reported for sponge H. magniconulosa (Maldonado and Young, 1996). 5. Conclusion Large quantities of larvae and early juveniles of an inter-tidal marine sponge H. perlevis could be obtained by subjecting ripe sponges collected during the larval-releasing season to optimal
controlled artificial conditions. The results indicate great potential for sponge seed production under artificial conditions in the context of sponge aquaculture. Whether the optimal conditions for larval release and settlement of H. perlevis are applicable to other sponge species remains to be demonstrated. Nonetheless, the current study provides important information for the future development of seed-rearing technology. Acknowledgements The authors wish to thank the “Hi-Tech Research and Development Program of China” (2006AA09Z435), “Innovation Fund” from the Dalian Institute of Chemical Physics and the “Innovative Key Project of the Chinese Academy of Sciences” (KZCX2-YW-209) for financial support. The authors would also like to thank Dr. K. Manmadhan, Dr. Li-Ming Sun, Dr. Xu-Peng Cao and Dr. Wan-Tao Fu for their useful comments on the manuscript and Mr. Yuan-Ling Liu for constructing the cooling and heating systems. References Amano, S., 2006. 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