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Effective use of plastic sheet as substrate in enhancing tropical oyster (Crassostrea iredalei Faustino) larvae settlement in the hatchery
Aquaculture 212 (2002) 277 – 287 www.elsevier.com/locate/aqua-online
Effective use of plastic sheet as substrate in enhancing tropical oyster (Crassostrea iredalei Faustino) larvae settlement in the hatchery M.N. Devakie a,*, A.B. Ali b a Fisheries Research Institute, Batu Maung, 11960 Penang, Malaysia School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia
b
Received 5 February 2002; received in revised form 9 May 2002; accepted 3 June 2002
Abstract Various methods were employed to exploit plastic sheet as substrate for enhancing tropical oyster (Crassostrea iredalei) larval settlement in the hatchery. Plastic texture (rough and smooth surfaces) and condition (with and without biofilm) showed a significant difference ( P < 0.05) on the larval setting rate. The highest setting rate was achieved for rough plastic without biofilm (37.6 F 2.2%) followed by rough plastic with biofilm (27.1 F 1.8%), smooth plastic with biofilm (22.0 F 2.1%) and the least recorded was on the control or smooth plastic without biofilm (11.0 F 1.2%), all of which differed significantly ( P < 0.05). Plastic substrate also appeared attractive to setting larvae by presoaking it in tissue extracts of several bivalve species. Plastic sheets presoaked in tissue extracts of C. iredalei and Crassostrea belcheri promoted better setting rates (28.3 F 3.9% and 26.1 F 5.5%, respectively) as compared to those soaked in tissue extracts from other bivalve species such as the green mussel, Perna viridis (15.7 F 1.4%) and the rock oyster, Saccostrea cucullata (12.9 F 3.6%). The setting rate on the control was also low (15.3 F 2.7%) and was not significantly different ( P > 0.05) from plastic with tissue extracts of the green mussel and the rock oyster. Exhalent water (used as media) from the above mentioned bivalve species were also found to increase oyster larval settlement on plastic substrate as compared to the control (without exhalent water). Higher setting rate was obtained for plastic with exhalent water from S. cucullata (48.9 F 3.5%) followed by C. belcheri (32.0 F 2.3%), P. viridis (30.4 F 4.3%) and C. iredalei (27.0 F 1.7%) which did not vary significantly ( P>0.05), while lower setting rate was recorded for the control (18.9 F 5.9%). D 2002 Published by Elsevier Science B.V. Keywords: Oyster larvae; Plastic sheet condition; Tissue extract; Exhalent water; Setting rate
*
Corresponding author. Tel.: +60-4626-3925; fax: +60-4626-2210. E-mail address: [email protected] (M.N. Devakie).
0044-8486/02/$ - see front matter D 2002 Published by Elsevier Science B.V. PII: S 0 0 4 4 - 8 4 8 6 ( 0 2 ) 0 0 2 7 0 - 3
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1. Introduction In the wild, oyster spat can be found attached on almost any hard substratum like submerged rocks, coarse sand particles, roots of mangrove trees, concrete pillars supporting overhead bridges or on any other permanent structures. In the absence of suitable substrates, they may even choose to set on plastic drums which are used to support cages or rafts, glass pieces, seaweeds or on calcareous materials such as mollusc shells (Quayle and Newkirk, 1989). In the tropics, the most common and traditionally used cultch materials for the collection of the oyster (Crassostrea iredalei, Crassostrea belcheri, Saccostrea spp.) spat are bamboo poles, coconut shells, oyster shells, asbestos and motorcycle tyres (Devakie, 1993). In 1989, the Department of Fisheries Malaysia, under the technical and financial support of the Bay of Bengal Program, has carried out several oyster spat collection trials in the wild using netlon material which were dipped in cement–sand – lime mixture (ratio of 5:2:1) and were allowed to dry and leach before being hung out in the waters for spat collection (Devakie et al., 1993; Mohd. Yatim, 1993). This material proved to be effective for the collection of cultchless or single spat as compared to other cultches such as oyster shells, coconut shells and inverted motorcycle tyres that often gather clumps of oysters which are usually shucked for their meat. Oyster spat attached on the netlon materials are easily flicked off as they tend to detach themselves together with the cement follicles, whereas those collected on shells and tyres would have to be on-grown and culled from time to time in order to avoid overcrowding until they attain marketable size. Spat shells are usually brittle and tend to break off easily during their removal from these hard cultches resulting in high mortality of the oyster spat. In Malaysia, C. iredalei has been identified as the commercially potential species which is preferred for its sweet flavour and creamy coloured meat (Devakie et al., 1993). This species is commonly served shell-on in local restaurants and hotels but due to its inconsistent supply from the wild, oysters are presently being imported from other countries like France and Thailand to cater for the local demand. In view of the short supply of oyster spat for the local farms, the Fisheries Research Institute, Penang embarked on the hatchery propagation of C. iredalei (Ng, 1992). A preliminary experiment for the production of cultchless spat was carried out in 2-ton fibreglass tanks using cultch materials such as plastic (high density polyethylene) sheet, marble chips, and netlon pieces (1 1 m) dipped in cement –sand mixture (Devakie, 1992). Observations showed that the setting rate was better on plastic sheet as compared to marble chips or netlon material. Spat settlement obtained for plastic substrate was 15% as compared to about 10% on marble chips. This was due to the larvae getting buried under the marble chips as a result of continuous aeration and water change. In addition, it was more difficult to siphon out waste from tanks that contained marble chips than those lined with plain plastic. Another problem was to separate the chips with spat from those without the spat. Another advantage of using plastic sheet was that there was no necessity to cull the spats as they would eventually be removed at about 5 mm in size for grow-out at the culture site. As for the netlon pieces, it was not possible to do an initial spat density count due to the colour (grey) of the spat which was almost similar to the cement coat on the netlon. Using netlon also tends to foul the water as the detached cement follicles fall to the bottom of the tank
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and get mixed up with the spat thus rendering problem to distinguish the spat during water change. The use of netlon was recommended for use in open waters as the detached cement follicles would be swept away by the current. Though plastic material proved to be the best among other substrates for cultchless spat, the larval setting rate was low. It is the objective of this experiment to determine the ways of maximising spat settlement by treating the plastic substrate, using different attractants such as biofilm coverage, calcium deposits of previously settled spats and exploiting their gregarious behaviour in response to waterborne inducers such as tissue extracts and exhalent water from different bivalves.
2. Materials and methods Oyster broodstock (C. iredalei) was procured from a nearby farm and oyster larvae for the experiment were produced in the hatchery at the Fisheries Research Institute, Penang. Eyed larvae >220 Am in length were used in the experiments. Experiments were carried out in 1 l Pyrex beakers. High density polyethylene (HDPE) colourless plastic sheet of 1 mm thickness was used to line the beakers for larval settlement. The preparation of various conditions of plastic material have been described in the respective experiments. All experiments were carried out in replicates of six at room temperature. Salinity for the experiment was reduced to 22 ppt., a level that has been routinely used for oyster larvae production in the hatchery (Devakie et al., unpublished data). Duration allowed for larval settlement was 4 days based on experiments by Tan (1993). Larval settlement rate (%) was assessed based on the number of larvae set on the plastic lining while unset larvae were considered not competent and discarded after 4 days. The larval density used was 1000 larvae l 1 and algal mixture of Isochrysis galbana and Chaetoceros calcitrans was given daily at a density of 70,000 cells ml 1 (Wong, 1990; Tan, 1993) with complete water change after 48 h. Slight aeration was provided to all beakers from a portable aerator. 2.1. Effect of plastic texture and condition on oyster larvae settlement In this experiment, texture (rough and smooth) and condition (with and without biofilm) represented the independent variables while larval settlement rate was the dependent variable. A rough textured surface was obtained from plastic which had calcium deposits from previous spat settlements (Fig. 1). These deposits covered the whole plastic sheet, rendering vertical (sides of the beaker) and horizontal settlement (bottom of the beaker) surfaces for the larvae. The smooth surface was obtained from unused plastic, which was soaked and washed in freshwater and dried prior to use. To condition the plastic sheets, biofilm was allowed to form on the surface of the plastic as described by Parsons et al. (1993). Plastic sheets (rough and smooth) were submerged in tanks (held down in the water column by weights) containing filtered (50 Am nominal pore size cotton bags) running seawater for a 1-week period prior to use, to promote the formation of biofilm layer. Thus, four treatments namely rough plastic with biofilm, rough plastic without biofilm, smooth plastic with biofilm and smooth plastic without biofilm (control) were used in the experiment.
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Fig. 1. Calcium deposits (C) from previous oyster spat settlement on plastic sheet.
2.2. Effect of plastic sheet presoaked in tissue extracts of several bivalve species on oyster larvae settlement Tissue extracts were prepared separately for each bivalve species (C. belcheri, C. iredalei, Saccostrea cucullata and Perna viridis) as described by Lund (1971). Ten individuals of each species of shellfish were shucked. The bivalve meat was blended and made up to 1 l in a standing measuring cylinder and left overnight in a low heat incubator (Shel-Lab Model 2020) at 4 jC. The supernatant (about 100 ml of the top layer) was poured out and made up to 0.5 l and the plastic sheets were immersed in this solution for up to 24 h. The sheets were then air dried for another 24 h period prior to lining the beakers for spat settlement. Untreated plastic was soaked in freshwater and dried prior to use as the control. 2.3. Effect of exhalent water from several bivalve species on oyster larvae settlement The experimental design was also based on Lund (1971) where exhalent water from C. belcheri, C. iredalei, S. cucullata and P. viridis were prepared. Ten individuals of each species were put in separate pails containing seawater of 22 ppt. and aerated overnight. The water from the pails was then filtered using suction through 1 Am filter paper using the Buchner apparatus. The filtered water was then used as the water media for spat settlement in beakers lined with plastic sheet. For the control, 1 Am filtered seawater (22 ppt.) was used. 2.4. Statistical analyses Data from the experiment were analysed using the ANOVA from the Analytical Software Statistix 4.0 Programme (Anon., 1992). Where significant differences were
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Table 1 Analysis of variance of the effects of substrate texture and condition on the settlement of oyster (C. iredalei) larvae, using arcsine-transformed percentage data Source
df
ss
F
P
Condition Texture Condition texture Error Total
1 1 1 20 23
7.53 724.90 333.31 31.27 1097.01
4.81 463.65 213.19
0.0402 0.0000 0.0000
observed, a further analysis using the Tukey’s (HSD) test was used to determine the pairwise comparison of their means. Percentage values were transformed to arcsine values prior to analysis (Snedecor and Cochran, 1989).
3. Results 3.1. Effect of plastic texture and condition on oyster larvae settlement A significant interaction ( P < 0.05) was observed between the substrate texture and substrate condition on the oyster larval settlement, using the two-way ANOVA (Table 1). It was observed that the rough surfaced plastics had higher oyster (C. iredalei) larval setting rates as compared to the smooth surfaced plastics. At the same time, setting rates
Fig. 2. Oyster (C. iredalei) larvae setting rates (% F S.D.) for various plastic substrate texture and condition.
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for both the rough and smooth surfaced plastics, which were conditioned, were higher than the smooth but unconditioned plastic (control). A one-way ANOVA indicated that larval settlement rates among the four treatments were significantly different ( P < 0.05). Further comparison of their means using the Tukey’s (HSD) test indicated that the best setting rate was obtained for rough plastic without biofilm (37.6 F 2.2%) followed by rough plastic with biofilm (27.1 F 1.8%). Smooth surface plastic with biofilm layer contributed a setting rate of 22.0 F 2.1% while that without biofilm (control) contributed only 11.0 F 1.2% (Fig. 2). 3.2. Effect of plastic sheet presoaked in tissue extracts of several bivalve species on oyster larvae settlement A one-way ANOVA of the effect of plastic substrates presoaked in different tissue extracts showed a significant difference ( P < 0.05) on the setting rate of oyster (C. iredalei) larvae. Further comparison of their means using the Tukey’s (HSD) test, showed that tissue extracts from the Crassostrea spp. promoted better setting rates as compared to tissue extracts from other bivalves and the control. It was observed that the setting rate obtained for the C. iredalei tissue extract was not significantly different ( P>0.05) from that of C. belcheri which was 28.3 F 3.9% and 26.1 F 5.5%, respectively (Fig. 3). As for tissue
Fig. 3. Oyster (C. iredalei) larvae setting rates (% F S.D.) for substrates treated with various bivalve tissue extracts.
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Fig. 4. Oyster (C. iredalei) larvae setting rates (% F S.D.) for exhalent water from various bivalves.
extracts from other bivalves, setting rates obtained for P. viridis, S. cucullata and the control were 15.7 F 1.4%, 15.3 F 2.7% and 12.9 F 3.6%, respectively, which were not significantly different ( P>0.05) from each other. Larval setting rates were thus found to be higher for plastics presoaked in tissue extracts from the same genus of oysters. 3.3. Effect of exhalent water from several bivalve species on oyster larvae settlement A one-way ANOVA of the use of exhalent waters from various bivalves to enhance larval setting on plastic substrate showed a significant difference ( P < 0.05). Comparison of means using the Tukey’s (HSD) test showed that the highest setting rate was achieved for exhalent water from S. cucullata (48.9 F 3.5%) which was significantly different ( P < 0.05) from C. belcheri (32.0 F 2.3%), P. viridis (30.4 F 4.3%) and C. iredalei (27.0 F 1.7%). The control contributed the lowest setting rate (19.2 F 5.9%) and was significantly different ( P < 0.05) from the others (Fig. 4).
4. Discussion The selection of a suitable and most readily available substrate is a pre-requisite for oyster spat collection in the wild or hatchery. Amongst most collectors, plastic is
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considered to be the least attractive for oyster spat collection (Quayle and Newkirk, 1989). Besides, using chemical induction methods such as L-DOPA, epinephrine and norepinephrine (Billington, 1991), various biological stimulants such as exhalent waters, bacterial supernatants, tissue extracts, oyster glycogen and mucus trails from bivalves of the same genus or even other species can be applied to exploit the gregarious behaviour of the eyed oyster larvae to enhance setting rates on unattractive substrates. Among bivalve researchers, it is widely accepted that the existence of biofilm layer which comprises of bacteria, microalgae and detritus (Hudon and Bourget, 1981) on a substrate serves as an attraction for bivalve larvae settlement (Bracanto and Woollacott, 1982; Maki et al., 1988; Weiner et al., 1989; Fitt et al., 1990; Slattery, 1992; Tritar et al., 1992; Parsons et al., 1993). Research in the temperate countries since several decades ago has also shown that rough surfaced substrates attract more bivalve spat as compared to smooth surfaces (Bayne, 1969; Bonar et al., 1990; Beiras and Widdows, 1995). As for observations pertaining to the effect of biofilm layers, Cole and Knight-Jones (1949) compared Ostrea edulis larvae setting on oyster shells covered with biofilm (1264 spat) to those without bioflim (821 spat). The same researchers confirmed that the setting rate of polychaete (Spirorbis borealis) larvae was higher in beakers with biofilm layers (91%) as compared to those without (15%). Walne (1966) found that the setting rate of O. edulis larvae could be increased (rate not mentioned) by presoaking plastic substrates in seawater prior to use. Biofilms of periphytic, marine bacterium Alteromonas colwelliana on glass, polystyrene and mylar surfaces have also shown that oyster (Crassostrea gigas and Crassostrea virginica) larvae set three to eight times more frequently ( P < 0.05) on filmed surfaces than on the controls (Weiner et al., 1989). In terms of substrate surface texture, Butler (1954) in his experiment had observed that cement coated wooden planks (46%) attracted more oyster larvae as compared to oyster shells (28%), glass (15%) and plexiglas (4– 7%). Observations by Keck et al. (1974) on other bivalve species (Merceenaria mercenaria) had also shown that the larval setting rate was higher on sandy substrates (2083 spat) as compared to muddy substrates (781 spat), meaning that the clam larvae preferred gritty rough surfaces. In this experiment, the effect of plastic condition and texture on the settlement of tropical oyster (C. iredalei) larvae revealed a tendency for the larvae to set more readily on rough substrates with calcium deposits from previous spat settlements and on those with biofilm coverage. Several researchers from the temperate countries have also reported on the use of bivalve tissue to stimulate oyster larval setting. Crisp (1967) observed that shells devoid of proteinaceous layers equally attracted oyster larvae (C. gigas) when placed in the same container with shells presoaked in tissue extracts of C. gigas. However, in a separate experiment, he found that shells with proteinaceous layers when placed in a medium without tissue extracts failed to attract the oyster larvae to set. In an experiment by Bayne (1969), slates were coated with tissue extracts from various bivalve species to stimulate the setting rate of O. edulis larvae. Similar results, as obtained in this experiment, were observed as the setting rate was found to be higher using intragenus oysters, O. edulis (19.8 spat) and Ostrea lutaria (12.7 spat) but was lower when tissues extracts from other species such as Crassostrea angulata (6 spat), C. gigas (5.8 spat), Mytilus viridis (3.7 spat) and the control (5.2 spat) were used. In a related experiment by Lund (1971) on the Pacific oyster (C. gigas), it was found that fibreglass squares coated with tissue extracts of the
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same species received more spat (26 spat) than the uncoated ones (14 spat). In this experiment, higher setting rates for C. iredalei larvae were observed when tissue extracts from C. iredalei (28.3%) and C. belcheri (26.1%) were used as compared to setting rates using tissue extracts from P. viridis (15.7%) and S. cucullata (12.9%) or the control (15.3%). Pheromones are known to be present in the body cavity fluids of oysters (fresh oyster shell liquor) and also in the exhalent waters (Scheltema, 1974; Hidu et al., 1978) and these have been used as an effective stimulant to induce the setting of intra-species or interspecies larvae. In an experiment by Hidu (1969), 2-month old oysters (C. virginica) were stocked in plankton bags to stimulate the setting of larvae, which were outside the bags. In another experiment by Lund (1971), the setting rate of C. gigas was higher (48.2%) when exhalent waters from inter-species, O. edulis were used as compared to the setting rate obtained for exhalent water from the same species (32%). In the same experiment, the setting rate observed for exhalent water from other species such as Mytilus edulis was low (22.87%) and there was no setting in the control. These results were comparable to that obtained in this experiment where oyster larval setting rates were higher for exhalent water from intra-species oysters (S. cucullata) as compared to the exhalent water from the same species (C. iredalei) or for exhalent water from other species and the control. The presence of pheromones had been related to the presence of proteinaceous-based material which has a molecular weight of 100,000 daltons which are presumably released by the bivalves to promote spat settlement (Veitch and Hidu, 1971). According to Keck et al. (1974), this material could be ether extracted through lyophilisis and the extract, if made available commercially, may be employed for oyster spat production. Another experiment on C. virginica by Tamburri et al. (1992) had revealed that the larval settlement behaviour in response to inducers liberated by the oysters themselves and biofilms are identical with a molecular weight ranging between 500 and 1000.
5. Summary Plastic can be made to be an effective substrate for the production of cultchless oyster spats when conditioned to have a rough surface or biofilm coverage, coated with tissue extracts and if exhalent water from bivalves is used in the settlement tanks. Plastic sheets are expensive but durable as compared to other cultches like oyster shells, coconut oyster shells and marble chips which can only be made use for one to two cycles. In small scale hatcheries, plastic sheet can be used to line the bottom of either cement or fibreglass tanks to avoid the spat from setting on the sides of the tanks. They can be re-used over time and they serve as a good material for the collection of cultchless oyster spat. Oyster spat can be easily removed from the plastic sheet with minimal damage to their shells by gently tabbing the sheet from the back and directing a gentle stream of seawater to flush the spat into a collecting basin. Even if the plastic is torn during the process of removing the oyster spat or because of continuous use, these can be cut into rectangular pieces as cultches to be hung vertically in the tanks, optimising the use of the water column. Remnants of calcium deposits on the plastic sheet not only produce a rough surface but pose as an attractant to the oyster larvae for gregarious setting.
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Effective use of plastic sheet as substrate in enhancing tropical oyster (Crassostrea iredalei Faustino) larvae settlement in the hatchery M.N. Devakie a,*, A.B. Ali b a Fisheries Research Institute, Batu Maung, 11960 Penang, Malaysia School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia
b
Received 5 February 2002; received in revised form 9 May 2002; accepted 3 June 2002
Abstract Various methods were employed to exploit plastic sheet as substrate for enhancing tropical oyster (Crassostrea iredalei) larval settlement in the hatchery. Plastic texture (rough and smooth surfaces) and condition (with and without biofilm) showed a significant difference ( P < 0.05) on the larval setting rate. The highest setting rate was achieved for rough plastic without biofilm (37.6 F 2.2%) followed by rough plastic with biofilm (27.1 F 1.8%), smooth plastic with biofilm (22.0 F 2.1%) and the least recorded was on the control or smooth plastic without biofilm (11.0 F 1.2%), all of which differed significantly ( P < 0.05). Plastic substrate also appeared attractive to setting larvae by presoaking it in tissue extracts of several bivalve species. Plastic sheets presoaked in tissue extracts of C. iredalei and Crassostrea belcheri promoted better setting rates (28.3 F 3.9% and 26.1 F 5.5%, respectively) as compared to those soaked in tissue extracts from other bivalve species such as the green mussel, Perna viridis (15.7 F 1.4%) and the rock oyster, Saccostrea cucullata (12.9 F 3.6%). The setting rate on the control was also low (15.3 F 2.7%) and was not significantly different ( P > 0.05) from plastic with tissue extracts of the green mussel and the rock oyster. Exhalent water (used as media) from the above mentioned bivalve species were also found to increase oyster larval settlement on plastic substrate as compared to the control (without exhalent water). Higher setting rate was obtained for plastic with exhalent water from S. cucullata (48.9 F 3.5%) followed by C. belcheri (32.0 F 2.3%), P. viridis (30.4 F 4.3%) and C. iredalei (27.0 F 1.7%) which did not vary significantly ( P>0.05), while lower setting rate was recorded for the control (18.9 F 5.9%). D 2002 Published by Elsevier Science B.V. Keywords: Oyster larvae; Plastic sheet condition; Tissue extract; Exhalent water; Setting rate
*
Corresponding author. Tel.: +60-4626-3925; fax: +60-4626-2210. E-mail address: [email protected] (M.N. Devakie).
0044-8486/02/$ - see front matter D 2002 Published by Elsevier Science B.V. PII: S 0 0 4 4 - 8 4 8 6 ( 0 2 ) 0 0 2 7 0 - 3
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1. Introduction In the wild, oyster spat can be found attached on almost any hard substratum like submerged rocks, coarse sand particles, roots of mangrove trees, concrete pillars supporting overhead bridges or on any other permanent structures. In the absence of suitable substrates, they may even choose to set on plastic drums which are used to support cages or rafts, glass pieces, seaweeds or on calcareous materials such as mollusc shells (Quayle and Newkirk, 1989). In the tropics, the most common and traditionally used cultch materials for the collection of the oyster (Crassostrea iredalei, Crassostrea belcheri, Saccostrea spp.) spat are bamboo poles, coconut shells, oyster shells, asbestos and motorcycle tyres (Devakie, 1993). In 1989, the Department of Fisheries Malaysia, under the technical and financial support of the Bay of Bengal Program, has carried out several oyster spat collection trials in the wild using netlon material which were dipped in cement–sand – lime mixture (ratio of 5:2:1) and were allowed to dry and leach before being hung out in the waters for spat collection (Devakie et al., 1993; Mohd. Yatim, 1993). This material proved to be effective for the collection of cultchless or single spat as compared to other cultches such as oyster shells, coconut shells and inverted motorcycle tyres that often gather clumps of oysters which are usually shucked for their meat. Oyster spat attached on the netlon materials are easily flicked off as they tend to detach themselves together with the cement follicles, whereas those collected on shells and tyres would have to be on-grown and culled from time to time in order to avoid overcrowding until they attain marketable size. Spat shells are usually brittle and tend to break off easily during their removal from these hard cultches resulting in high mortality of the oyster spat. In Malaysia, C. iredalei has been identified as the commercially potential species which is preferred for its sweet flavour and creamy coloured meat (Devakie et al., 1993). This species is commonly served shell-on in local restaurants and hotels but due to its inconsistent supply from the wild, oysters are presently being imported from other countries like France and Thailand to cater for the local demand. In view of the short supply of oyster spat for the local farms, the Fisheries Research Institute, Penang embarked on the hatchery propagation of C. iredalei (Ng, 1992). A preliminary experiment for the production of cultchless spat was carried out in 2-ton fibreglass tanks using cultch materials such as plastic (high density polyethylene) sheet, marble chips, and netlon pieces (1 1 m) dipped in cement –sand mixture (Devakie, 1992). Observations showed that the setting rate was better on plastic sheet as compared to marble chips or netlon material. Spat settlement obtained for plastic substrate was 15% as compared to about 10% on marble chips. This was due to the larvae getting buried under the marble chips as a result of continuous aeration and water change. In addition, it was more difficult to siphon out waste from tanks that contained marble chips than those lined with plain plastic. Another problem was to separate the chips with spat from those without the spat. Another advantage of using plastic sheet was that there was no necessity to cull the spats as they would eventually be removed at about 5 mm in size for grow-out at the culture site. As for the netlon pieces, it was not possible to do an initial spat density count due to the colour (grey) of the spat which was almost similar to the cement coat on the netlon. Using netlon also tends to foul the water as the detached cement follicles fall to the bottom of the tank
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and get mixed up with the spat thus rendering problem to distinguish the spat during water change. The use of netlon was recommended for use in open waters as the detached cement follicles would be swept away by the current. Though plastic material proved to be the best among other substrates for cultchless spat, the larval setting rate was low. It is the objective of this experiment to determine the ways of maximising spat settlement by treating the plastic substrate, using different attractants such as biofilm coverage, calcium deposits of previously settled spats and exploiting their gregarious behaviour in response to waterborne inducers such as tissue extracts and exhalent water from different bivalves.
2. Materials and methods Oyster broodstock (C. iredalei) was procured from a nearby farm and oyster larvae for the experiment were produced in the hatchery at the Fisheries Research Institute, Penang. Eyed larvae >220 Am in length were used in the experiments. Experiments were carried out in 1 l Pyrex beakers. High density polyethylene (HDPE) colourless plastic sheet of 1 mm thickness was used to line the beakers for larval settlement. The preparation of various conditions of plastic material have been described in the respective experiments. All experiments were carried out in replicates of six at room temperature. Salinity for the experiment was reduced to 22 ppt., a level that has been routinely used for oyster larvae production in the hatchery (Devakie et al., unpublished data). Duration allowed for larval settlement was 4 days based on experiments by Tan (1993). Larval settlement rate (%) was assessed based on the number of larvae set on the plastic lining while unset larvae were considered not competent and discarded after 4 days. The larval density used was 1000 larvae l 1 and algal mixture of Isochrysis galbana and Chaetoceros calcitrans was given daily at a density of 70,000 cells ml 1 (Wong, 1990; Tan, 1993) with complete water change after 48 h. Slight aeration was provided to all beakers from a portable aerator. 2.1. Effect of plastic texture and condition on oyster larvae settlement In this experiment, texture (rough and smooth) and condition (with and without biofilm) represented the independent variables while larval settlement rate was the dependent variable. A rough textured surface was obtained from plastic which had calcium deposits from previous spat settlements (Fig. 1). These deposits covered the whole plastic sheet, rendering vertical (sides of the beaker) and horizontal settlement (bottom of the beaker) surfaces for the larvae. The smooth surface was obtained from unused plastic, which was soaked and washed in freshwater and dried prior to use. To condition the plastic sheets, biofilm was allowed to form on the surface of the plastic as described by Parsons et al. (1993). Plastic sheets (rough and smooth) were submerged in tanks (held down in the water column by weights) containing filtered (50 Am nominal pore size cotton bags) running seawater for a 1-week period prior to use, to promote the formation of biofilm layer. Thus, four treatments namely rough plastic with biofilm, rough plastic without biofilm, smooth plastic with biofilm and smooth plastic without biofilm (control) were used in the experiment.
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Fig. 1. Calcium deposits (C) from previous oyster spat settlement on plastic sheet.
2.2. Effect of plastic sheet presoaked in tissue extracts of several bivalve species on oyster larvae settlement Tissue extracts were prepared separately for each bivalve species (C. belcheri, C. iredalei, Saccostrea cucullata and Perna viridis) as described by Lund (1971). Ten individuals of each species of shellfish were shucked. The bivalve meat was blended and made up to 1 l in a standing measuring cylinder and left overnight in a low heat incubator (Shel-Lab Model 2020) at 4 jC. The supernatant (about 100 ml of the top layer) was poured out and made up to 0.5 l and the plastic sheets were immersed in this solution for up to 24 h. The sheets were then air dried for another 24 h period prior to lining the beakers for spat settlement. Untreated plastic was soaked in freshwater and dried prior to use as the control. 2.3. Effect of exhalent water from several bivalve species on oyster larvae settlement The experimental design was also based on Lund (1971) where exhalent water from C. belcheri, C. iredalei, S. cucullata and P. viridis were prepared. Ten individuals of each species were put in separate pails containing seawater of 22 ppt. and aerated overnight. The water from the pails was then filtered using suction through 1 Am filter paper using the Buchner apparatus. The filtered water was then used as the water media for spat settlement in beakers lined with plastic sheet. For the control, 1 Am filtered seawater (22 ppt.) was used. 2.4. Statistical analyses Data from the experiment were analysed using the ANOVA from the Analytical Software Statistix 4.0 Programme (Anon., 1992). Where significant differences were
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Table 1 Analysis of variance of the effects of substrate texture and condition on the settlement of oyster (C. iredalei) larvae, using arcsine-transformed percentage data Source
df
ss
F
P
Condition Texture Condition texture Error Total
1 1 1 20 23
7.53 724.90 333.31 31.27 1097.01
4.81 463.65 213.19
0.0402 0.0000 0.0000
observed, a further analysis using the Tukey’s (HSD) test was used to determine the pairwise comparison of their means. Percentage values were transformed to arcsine values prior to analysis (Snedecor and Cochran, 1989).
3. Results 3.1. Effect of plastic texture and condition on oyster larvae settlement A significant interaction ( P < 0.05) was observed between the substrate texture and substrate condition on the oyster larval settlement, using the two-way ANOVA (Table 1). It was observed that the rough surfaced plastics had higher oyster (C. iredalei) larval setting rates as compared to the smooth surfaced plastics. At the same time, setting rates
Fig. 2. Oyster (C. iredalei) larvae setting rates (% F S.D.) for various plastic substrate texture and condition.
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for both the rough and smooth surfaced plastics, which were conditioned, were higher than the smooth but unconditioned plastic (control). A one-way ANOVA indicated that larval settlement rates among the four treatments were significantly different ( P < 0.05). Further comparison of their means using the Tukey’s (HSD) test indicated that the best setting rate was obtained for rough plastic without biofilm (37.6 F 2.2%) followed by rough plastic with biofilm (27.1 F 1.8%). Smooth surface plastic with biofilm layer contributed a setting rate of 22.0 F 2.1% while that without biofilm (control) contributed only 11.0 F 1.2% (Fig. 2). 3.2. Effect of plastic sheet presoaked in tissue extracts of several bivalve species on oyster larvae settlement A one-way ANOVA of the effect of plastic substrates presoaked in different tissue extracts showed a significant difference ( P < 0.05) on the setting rate of oyster (C. iredalei) larvae. Further comparison of their means using the Tukey’s (HSD) test, showed that tissue extracts from the Crassostrea spp. promoted better setting rates as compared to tissue extracts from other bivalves and the control. It was observed that the setting rate obtained for the C. iredalei tissue extract was not significantly different ( P>0.05) from that of C. belcheri which was 28.3 F 3.9% and 26.1 F 5.5%, respectively (Fig. 3). As for tissue
Fig. 3. Oyster (C. iredalei) larvae setting rates (% F S.D.) for substrates treated with various bivalve tissue extracts.
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Fig. 4. Oyster (C. iredalei) larvae setting rates (% F S.D.) for exhalent water from various bivalves.
extracts from other bivalves, setting rates obtained for P. viridis, S. cucullata and the control were 15.7 F 1.4%, 15.3 F 2.7% and 12.9 F 3.6%, respectively, which were not significantly different ( P>0.05) from each other. Larval setting rates were thus found to be higher for plastics presoaked in tissue extracts from the same genus of oysters. 3.3. Effect of exhalent water from several bivalve species on oyster larvae settlement A one-way ANOVA of the use of exhalent waters from various bivalves to enhance larval setting on plastic substrate showed a significant difference ( P < 0.05). Comparison of means using the Tukey’s (HSD) test showed that the highest setting rate was achieved for exhalent water from S. cucullata (48.9 F 3.5%) which was significantly different ( P < 0.05) from C. belcheri (32.0 F 2.3%), P. viridis (30.4 F 4.3%) and C. iredalei (27.0 F 1.7%). The control contributed the lowest setting rate (19.2 F 5.9%) and was significantly different ( P < 0.05) from the others (Fig. 4).
4. Discussion The selection of a suitable and most readily available substrate is a pre-requisite for oyster spat collection in the wild or hatchery. Amongst most collectors, plastic is
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considered to be the least attractive for oyster spat collection (Quayle and Newkirk, 1989). Besides, using chemical induction methods such as L-DOPA, epinephrine and norepinephrine (Billington, 1991), various biological stimulants such as exhalent waters, bacterial supernatants, tissue extracts, oyster glycogen and mucus trails from bivalves of the same genus or even other species can be applied to exploit the gregarious behaviour of the eyed oyster larvae to enhance setting rates on unattractive substrates. Among bivalve researchers, it is widely accepted that the existence of biofilm layer which comprises of bacteria, microalgae and detritus (Hudon and Bourget, 1981) on a substrate serves as an attraction for bivalve larvae settlement (Bracanto and Woollacott, 1982; Maki et al., 1988; Weiner et al., 1989; Fitt et al., 1990; Slattery, 1992; Tritar et al., 1992; Parsons et al., 1993). Research in the temperate countries since several decades ago has also shown that rough surfaced substrates attract more bivalve spat as compared to smooth surfaces (Bayne, 1969; Bonar et al., 1990; Beiras and Widdows, 1995). As for observations pertaining to the effect of biofilm layers, Cole and Knight-Jones (1949) compared Ostrea edulis larvae setting on oyster shells covered with biofilm (1264 spat) to those without bioflim (821 spat). The same researchers confirmed that the setting rate of polychaete (Spirorbis borealis) larvae was higher in beakers with biofilm layers (91%) as compared to those without (15%). Walne (1966) found that the setting rate of O. edulis larvae could be increased (rate not mentioned) by presoaking plastic substrates in seawater prior to use. Biofilms of periphytic, marine bacterium Alteromonas colwelliana on glass, polystyrene and mylar surfaces have also shown that oyster (Crassostrea gigas and Crassostrea virginica) larvae set three to eight times more frequently ( P < 0.05) on filmed surfaces than on the controls (Weiner et al., 1989). In terms of substrate surface texture, Butler (1954) in his experiment had observed that cement coated wooden planks (46%) attracted more oyster larvae as compared to oyster shells (28%), glass (15%) and plexiglas (4– 7%). Observations by Keck et al. (1974) on other bivalve species (Merceenaria mercenaria) had also shown that the larval setting rate was higher on sandy substrates (2083 spat) as compared to muddy substrates (781 spat), meaning that the clam larvae preferred gritty rough surfaces. In this experiment, the effect of plastic condition and texture on the settlement of tropical oyster (C. iredalei) larvae revealed a tendency for the larvae to set more readily on rough substrates with calcium deposits from previous spat settlements and on those with biofilm coverage. Several researchers from the temperate countries have also reported on the use of bivalve tissue to stimulate oyster larval setting. Crisp (1967) observed that shells devoid of proteinaceous layers equally attracted oyster larvae (C. gigas) when placed in the same container with shells presoaked in tissue extracts of C. gigas. However, in a separate experiment, he found that shells with proteinaceous layers when placed in a medium without tissue extracts failed to attract the oyster larvae to set. In an experiment by Bayne (1969), slates were coated with tissue extracts from various bivalve species to stimulate the setting rate of O. edulis larvae. Similar results, as obtained in this experiment, were observed as the setting rate was found to be higher using intragenus oysters, O. edulis (19.8 spat) and Ostrea lutaria (12.7 spat) but was lower when tissues extracts from other species such as Crassostrea angulata (6 spat), C. gigas (5.8 spat), Mytilus viridis (3.7 spat) and the control (5.2 spat) were used. In a related experiment by Lund (1971) on the Pacific oyster (C. gigas), it was found that fibreglass squares coated with tissue extracts of the
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same species received more spat (26 spat) than the uncoated ones (14 spat). In this experiment, higher setting rates for C. iredalei larvae were observed when tissue extracts from C. iredalei (28.3%) and C. belcheri (26.1%) were used as compared to setting rates using tissue extracts from P. viridis (15.7%) and S. cucullata (12.9%) or the control (15.3%). Pheromones are known to be present in the body cavity fluids of oysters (fresh oyster shell liquor) and also in the exhalent waters (Scheltema, 1974; Hidu et al., 1978) and these have been used as an effective stimulant to induce the setting of intra-species or interspecies larvae. In an experiment by Hidu (1969), 2-month old oysters (C. virginica) were stocked in plankton bags to stimulate the setting of larvae, which were outside the bags. In another experiment by Lund (1971), the setting rate of C. gigas was higher (48.2%) when exhalent waters from inter-species, O. edulis were used as compared to the setting rate obtained for exhalent water from the same species (32%). In the same experiment, the setting rate observed for exhalent water from other species such as Mytilus edulis was low (22.87%) and there was no setting in the control. These results were comparable to that obtained in this experiment where oyster larval setting rates were higher for exhalent water from intra-species oysters (S. cucullata) as compared to the exhalent water from the same species (C. iredalei) or for exhalent water from other species and the control. The presence of pheromones had been related to the presence of proteinaceous-based material which has a molecular weight of 100,000 daltons which are presumably released by the bivalves to promote spat settlement (Veitch and Hidu, 1971). According to Keck et al. (1974), this material could be ether extracted through lyophilisis and the extract, if made available commercially, may be employed for oyster spat production. Another experiment on C. virginica by Tamburri et al. (1992) had revealed that the larval settlement behaviour in response to inducers liberated by the oysters themselves and biofilms are identical with a molecular weight ranging between 500 and 1000.
5. Summary Plastic can be made to be an effective substrate for the production of cultchless oyster spats when conditioned to have a rough surface or biofilm coverage, coated with tissue extracts and if exhalent water from bivalves is used in the settlement tanks. Plastic sheets are expensive but durable as compared to other cultches like oyster shells, coconut oyster shells and marble chips which can only be made use for one to two cycles. In small scale hatcheries, plastic sheet can be used to line the bottom of either cement or fibreglass tanks to avoid the spat from setting on the sides of the tanks. They can be re-used over time and they serve as a good material for the collection of cultchless oyster spat. Oyster spat can be easily removed from the plastic sheet with minimal damage to their shells by gently tabbing the sheet from the back and directing a gentle stream of seawater to flush the spat into a collecting basin. Even if the plastic is torn during the process of removing the oyster spat or because of continuous use, these can be cut into rectangular pieces as cultches to be hung vertically in the tanks, optimising the use of the water column. Remnants of calcium deposits on the plastic sheet not only produce a rough surface but pose as an attractant to the oyster larvae for gregarious setting.
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