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Spawning induction, and larval and juvenile rearing of the giant clam, Tridacna gigas
Aquaculture, 58 (1986) 281-295 Elsevier Science Publishers B.V., Amsterdam
Technical
281 -
Printed
in The Netherlands
Paper
Spawning Induction, and Larval and Juvenile Rearing of the Giant Clam, Triducna gigus C.M. CRAWFORD’,
W.J. NASH and J.S. LUCAS
Zoology Department, James Cook University, Townsville, Queensland, 4811 (Australia) ‘Present address: Fisheries Laboratories, Department of Sea Fisheries, Taroona, Tasmania, 7006 (Australia) (Accepted
9 June 1986)
ABSTRACT Crawford, C.M., Nash, W.J. and Lucas, J.S., 1986. Spawning induction, rearing of the giant clam, Tridacna gigas. Aquaculture, 58: 281-295.
and larval and juvenile
Specimens of Tridacna gigas (L.) with mature gonads were selected for broodstock using a biopsy technique and induced to spawn using intragonadal injections of serotonin. Some larvae were reared using conventional bivalve rearing techniques in 500-l tanks, with up to 40% survival to settlement after 7-8 days. Other larvae were reared extensively from eggs added directly to raceways. There was heavy mortality at or soon after metamorphosis in all groups of juvenile clams and survival of juveniles from intensive larval rearing was less than 1% to harvesting at 5 months. Similar numbers of juvenile clams were harvested from raceways to which 4 x lo5 intensively reared pediveligers or 2 x lo7 eggs (extensive culture) were added initially, yielding a total of ca. 6000 juvenile clams. Some juvenile clams were fed cultured microalgae for several months to supplement the nutrients obtained from their symbiotic zooxanthellae. These clams showed poorer survival than unfed clams, apparently because they were more overgrown by benthic algae. The distribution of juveniles in the raceways at harvesting was not influenced by water turbulence or shading, but growth rates were reduced by shading. Growth rates and densities of juveniles were much higher on textured than smooth substrates. We have shown that the mass rearing methods used for bivalve mollusc larvae are appropriate to T. gigas larvae; however, the typical methods for bivalve nursery rearing are not readily applicable to giant clam juveniles. Further research is required on these methods and on factors affecting juvenile survival and growth.
INTRODUCTION
The mariculture of giant clams (Family Tridacnidae) is currently receiving considerable attention for several reasons. Populations of giant clams, particularly the larger species, have declined precipitously in many areas because of over-exploitation (Wells et al., 1983). Their biology and ecology also would suit them for cultivation (see Munro and Heslinga, 1983). Of prime interest for mariculture is the largest species, Triducna gigm, which also has the fastest
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282
growth rate (Munro and Heslinga, 1983). This species has suffered the greatest over-exploitation and has recently become extinct in Fiji ( A.D. Lewis, personal communication, 1985) and four Micronesian Islands - Guam, Truk, Ponape and Kosrae (Heslinga et al., 1984). It is on the verge of extinction in the Philippines (A. Alcala, personal communication, 1985) and regions of Indonesia (Usher, 1984; Brown and Muskanofola, 1985). Substantial advances in the techniques for mass culture of giant clams have been made at the Micronesian Mariculture Demonstration Center in Palau and these are summarized by Heslinga and Watson (1985). The research at Palau, however, has concentrated on T. derusa, with little work being conducted on T. gigus. Several constraints to the mariculture of T. gigus have been outlined by Munro and Gwyther (1981) , Munro and Heslinga (1983) and Wells et al. (1983 ) . T. gigus, unlike other tridacnids, has not been successfully induced to spawn eggs by either chemical stimuli or the addition of gonadal extract. Only spontaneous spawnings have occurred and these are too unreliable for maricultural purposes. Moreover, although T. gigus spawns spontaneously throughout the year in Palau (Heslinga et al., 1984)) Braley (1984) reported that T. gigus has a restricted spawning period in the Great Barrier Reef region. Other investigators of the biology of giant clams have reported very high mortality during the larval stages ( LaBarbera, 1975; Jameson, 1976; Beckvar, 1981; Fitt and Trench, 1981; Gwyther and Munro, 1981). There have, however, been no attempts to rear tridacnid larvae using conventional bivalve hatchery techniques such as those used for oysters, Ostrea edulis and Crussostrea gigus, and the hard clam Mercenaria mercenuriu, e.g. Walne (1974) and Castagna and Kraeuter (1981) . This paper describes methods for spawning induction and collection of eggs and sperm to ensure cross-fertilization of T. gigus. Survival and growth rates of T. gigus larvae and early juveniles, reared using methods similar to those of Heslinga and Watson (1985) and using standard bivalve techniques, are reported. Some factors influencing juvenile survival and growth are discussed. METHODS
Broodstock specimens of T. gigus, 60-100 cm shell length, were collected from fringing reefs and transported to onshore holding tanks at Orpheus Island Research Station (18’ 40’S 146’ 30’E), where they were maintained in flowing seawater for up to 6 months. Reproductive condition of broodstock clams was determined by inserting a human biopsy needle (15.2 cm cannula, 20 mm specimen notch) through the mantle and into the gonad to collect a small sample of gonad tissue. Induction of spawning was attempted in February, March and June 1985 using macerated gonad tissue or the chemical serotonin (5-hydroxytryptamine creatine sulfate complex). Serotonin was used because it induces spawning in
283
the scallop Patinopecten yessoensis ( Matsutani and Nomura, 1982) and in six other bivalve species (Gibbons and Castagna, 1984). Only clams with mature gonads, as indicated by biopsy samples, were selected for spawning induction. Gonad extract ( fresh, or frozen for about 1 month) was squirted into the inhalent aperture, or l-2 ml of 1 mA4 seroronin in filtered seawater were injected directly into the gonad. Clams responded by producing sperm a few minutes after induction and samples were collected from several individuals. When eggs were released they were collected by placing a large plastic bag (90 x 60 cm) over the exhalent siphon just prior to a spawning contraction; the clam then expelled its eggs directly into the bag. The eggs were fertilized using a small quantity of sperm from another animal. This procedure greatly reduced the risk of polyspermy and self fertilization. The zygotes were then rinsed through sieves to remove gonadal debris and excess sperm, and subsampled to estimate numbers and percent fertilized. Larval rearing Fertilized eggs from a spawning two larval rearing systems:
induction
in February
were transferred
to
Intensive rearing. Larvae were reared in two 500-l tanks containing l-pm filtered seawater with light aeration and at 27°C. Initial egg densities were ca. 10 ml-’ in one tank and 2 ml-’ in the other. From the veliger stage (day 2 ) the water was changed daily by draining through an 80-pm sieve, and the larvae were fed unicellular algae, Isochrysis gulbana (Tahitian strain), at a concentration of lo-100 cells PI- ‘. Survival rates were estimated daily and at least 50 preserved larvae were measured along their anteroposterior axis. When most larvae were pediveligers (day 8-9) they were transferred to three 500~80x50 cm outdoor raceways at a density of 10 larvae cm-’ of bottom area. The raceways contained lo-pm filtered seawater and were covered with a canopy excluding 50% of incident light. The seawater was static, except for a daily 4-h flushing (30-40% water replacement) when an 80-pm screen was attached to the overflow pipe. I. galbana was added every second day at a concentration of l-10 cells ~1~‘. The bottoms of two raceways (A and B) were each randomly lined with asbestos sheet and fibreglass plates of approximate dimensions 29 x 13.5 cm. Another raceway ( E) contained various materials, asbestos sheet, fibreglass, plastic fabric, small coral pieces, plastic mesh and polyethylene sheet, placed randomly on the bottom to test for substrate preference. Extensive rearing. Fertilized eggs were added to two outside raceways (C and D ) 500 x 80 x 50 cm containing static lO+m filtered seawater to achieve densities of ca. 10 ml-‘. On day 2, dense bacterial growth was evident in the water
284
and the larvae were transferred, by siphoning onto sieves, to new raceways containing fresh filtered seawater. The raceways were flushed (30-40% turnover) for about 4 h daily from day 5 until the majority of larvae had metamorphosed. I. galbana was added on day 3 at a concentration of 50 cells ~1~‘, and by day 5 had formed a dense culture. Algae were not added again until day 11. With this system it was impractical to determine larval survival rates. Zooxanthellae from adult T. gigas were distributed among the larval and juvenile tanks every 2-3 days for 10 days from day 7. The zooxanthellae were collected by scraping a small piece of excised adult mantle tissue to release them into suspension and then rinsed through a 25-pm mesh. Juvenile rearing From day 12 when almost all larvae in the raceways had metamorphosed, 5-lo-pm filtered seawater was supplied continuously to each raceway at a flow of 8 1 min-‘. Feeding, by trickling I. galbana in with the inflowing seawater for 4 h every l-2 days, was continued for 2 weeks until zooxanthellae were clearly visible in the juveniles’ mantle tissue. For the following 2 months, I. galbana or Choreh sp. were added every second day to two raceways (B and D ) , one with intensively and the other with extensively reared larvae. Anteroposterior shell lengths of about 50 juveniles from each raceway, including various substrate types, were measured periodically. As the juveniles were quite motile for several months, it was not possible to determine survival rates or densities per unit area of substrate during this time. The second spawning induction in March also resulted in fertilized eggs. These were maintained using both the intensive and extensive larval culture methods, as described for the February spawning. Juvenile rearing was similar, with the following exception. Some pediveligers from the intensive larval culture system were transferred to plastic boxes 32 cm square, with 125~pm mesh on the base. The boxes contained larvae at densities of l-20 cm-’ of bottom area and were positioned in a small outside tank with water 10 cm deep and seawater flowing in from above. After the larvae had metamorphosed, the boxes were converted to upwelling systems, whereby seawater entered up through the mesh boxes. Harvesting of juveniles Juvenile clams were harvested at 5 months by siphoning or scraping them off the various substrates. The effect of environmental factors on the numbers and sizes of juveniles in raceways A, B, C and D was investigated in the following manner. Each raceway was divided longitudinally into two halves (sides) because the raceways were oriented in an east-west direction and therefore the northern and southern sides received different levels of shading. Each side
then was divided into four sectors because the water turbulence, observed from dye testing, was higher at the inflow and outflow ends of the raceway than in the middle. The inflow and outflow sectors measured 40 x 90 cm and the middle sectors 40 x 160 cm. In each sector of raceways A and B the number of juveniles on at least nine settlement plates (chosen at random) was counted and the density (number cm-‘) was calculated. In raceways C and D total counts were made of the juveniles on the bottom in each sector. Some clams were harvested from the side walls, but were not included in the analysis. In raceway E the density of juveniles on each substrate type was calculated from total counts. All clams, to a maximum of 50 on each plate or to 121 on the tank bottom in each sector, were measured to the nearest 0.1 mm. After removal from the raceways, the juveniles were placed on small pieces of gravel or coral rubble in plastic trays (53 x 28 x 8 cm) at densities of 0.2-0.3 cm-2. These trays were placed in tanks with flowing unfiltered seawater. Samples of 50-100 juveniles originating from each raceway were then measured once a month. Where appropriate, the results of harvesting the juveniles from the raceways were subjected to analysis of variance (ANOVA) (Zar, 1984) to examine the effects of position in the raceway, i.e. sides and sectors, and raceway treatments on juvenile densities and on shell lengths. If the ANOVA gave a significant result for a factor, but non-significant interaction, then the specific levels of the factor between which differences occurred were determined using Tukey’s Test (Zar, 1984) at P=O.O5 level of significance. RESULTS Spawning induction A simple classification of maturity stages for T. gigas broodstock was developed from the gonad biopsies. If immature, spent or resting, no gonadal tissue could be collected in the biopsy needle. Ova in maturing gonads were irregularly shaped with a large perivitelline space, and those in mature gonads were predominantly spherical, with small to no perivitelline space and diameter greater than 100 pm. Many active sperm were present in mature gonads. The introduction of macerated gonad into the inhalent siphon of mature clams on several occasions resulted in some sperm release, but never eggs. In February, the first injection of serotonin into five of eleven clams in a tank resulted in sperm production, and the release of a small quantity of eggs by two clams. A second injection 2 days later produced copious quantities of sperm from most animals, low numbers of eggs from three clams, and the spawning of all eggs from a fourth clam. A small sample from this clam’s spawning yielded 125 million eggs. After this spawning, the eleven clams were distributed between five tanks, and over the next 2 weeks at least five clams, some of which had
not been injected with serotonin, spawned spontaneously. One isolated clam spawned more than 500 million eggs at once. Many of these eggs were fertilized and some developed to the veliger stage, proving that self-fertilization can occur, although none of these larvae survived to metamorphosis. All spontaneous spawnings occurred in mid-late afternoon and generally followed the pattern described by Wada (1954). A spawning induction attempted in March did not result in any eggs after two injections of serotonin. A third injection one week later produced sperm from four clams and eggs from one. In June, only one injection of serotonin was necessary to induce egg release in one mature clam. Viable gametes were produced from the spawning inductions. Many eggs were fertilized and developed normally. The larvae from the June spawning, however, developed very slowly and did not survive to metamorphosis, probably because the water temperature was too low. It was 225°C at spawning and decreased to 19°C overnight. Larval rearing Embryological and larval development to metamorphosis of T. gigas were similar to these processes in other tridacnids (see LaBarbera, 1975; Jameson, 1976). Larvae began feeding on day 3, settling occurred on days 7-8 and metamorphosis between days 8 and 12. Zooxanthellae were visible in the mantle tissue l-2 weeks postmetamorphosis. These developmental times differ only slightly from the observations of Heslinga et al. (1984) for T. gigas where larvae settled on days 5-6, more than 50% had metamorphosed by days 7-8 and zooxanthellae were visible by day 11. Intensive rearing. Survival rates of larvae to pediveligers were higher in tank 2 (40.2% ) at a density of 2 ml-’ than in tank 1 (26.6% ) at 10 larvae ml-’ (Fig. 1A). At settlement, however, there were 1.3 x lo6 larvae surviving in tank 1 compared with 0.4 x lo6 in tank 2. Growth rates of larvae were 5.8 pm day- ’ in tank 1 and 5.3 pm day-’ in tank 2 (Fig. 1B). Algae were visible in the guts from day 3, but the concentration of algae in the culture water altered little, implying that only a small quantity of food was eaten. Bacterial growth was obvious in the tanks, but apparently did not affect the larvae. Extensive rearing. The transfer of larvae to clean raceways on day 2 resulted in the mortality of many larvae. The total number in each raceway on day 3 was approximately 7.25 x 105, a decrease from 20 x lo6 eggs. Mean length of larvae on day 5 was 173.7 ? 8.1 pm (c.f. 186 ,um in larval tank 1) and on day 9 was 188.15 8.1 pm.
281
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200 .y: .----:A
2 2 =
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.
175-
F f
F 2’
150 I 1
I
I
I
I
2
3
4
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6
7
6
Days from fertilization
Fig. 1. Survival rates (A) and growth rates (B) of 2’. gigas larvae in tank 1 at 10 larvae ml-’ (circles), and in tank 2 at 2 larvae ml-’ (squares).
Juvenile rearing Heavy mortality was observed amongst the metamorphosing larvae and newly-metamorphosed juveniles in all raceways. Of 12 samples of juvenile clams taken randomly during the first week after larvae had been transferred from intensive culture tanks to raceways, an average of 71% were dead, size 195-215 pm. A sample taken 3 weeks after settlement from a raceway with extensivelyreared larvae yielded 20% live clams of average length 422 ,um. Ninety-one percent of the dead shells collected were in the size range MO-208 pm, the
/
0
_*__.+-W+ 0
l
’
40
60
120
age
160
200
(days)
Fig. 2. Mean lengths and standard deviations harvesting.
of T. gigas juveniles from settlement
to post-
approximate size of pediveligers, and thus mortality occurred at metamorphosis or soon thereafter. Individual growth rates varied markedly and after 5 months shell lengths ranged between 1 and 16 mm. The growth of juveniles from intensively-reared larvae and unfed raceways only is shown in Fig. 2. Initial growth rates were low and for the first 6 weeks from settlement averaged 14 pm day-‘. Growth rates were higher over the next 5 months, from April to September, and approximately linear at 61 pm day-‘. A major problem encountered during the juvenile rearing period was the overgrowth of juveniles by benthic, filamentous algae. This was most apparent in the raceways which received unicellular algae every second day for 2 months. There also was a progressive increase in the abundance of invertebrates, including various crustaceans, polychaetes and gastropods, in the raceways. These probably entered as eggs or larvae and completed their development in the raceways. Although no evidence of predation on juvenile clams was obtained, it is suspected that these invertebrates caused some mortalities. Although eggs and larvae from the second spawning in March were treated in a similar manner to those from the first spawning, mortalities at all stages were much higher. The survival rate of intensively-reared larvae to the pediveliger stage on day 9 post-fertilization was 8.5% and the average growth rate was 4.25 pm day-‘. Survival and growth rates of juveniles also were lower.
289 TABLE 1 Mean densities of juvenile clams (number cm- ‘) in each w&r of raceways A-D at harvesting. Mean densities in raceways A and B are for replicate samples with standard deviations (values in brackets), and in raceways C and D for whole sectors Raceway A
sector
1 2 3 4
Total raceway: Total number Mean density
Raceway B
Raceway c
Raceway D
side 1
side 2
side 1
side 2
side 1
side 2
side 1
side 2
0.056 (0.048) 0.051 (0.038) 0.032 (0.022) 0.082 ( 0.095 )
0.052 ( 0.046 ) 0.048 (0.037) 0.036 (0.040) 0.062 (0.087)
0.007 ( 0.007
0.005 (0.006) 0.001 (0.002 ) 0.011 (0.020) 0.011 (0.017)
0.008
0.013
o.cmO3
0.0003
0.041
0.041
0.0009
0.0005
0.033
0.045
0.006
0.001
0.049
0.076
0.019
0.002
2009 0.050
) 0.008 (0.011) 0.005 (0.004) 0.020 (0.019)
303 0.008
1573 0.039
133 0.003
Raceways: A, B, intensive larval culture; C, D, extensive culture; A, C. juveniles unfed, B, D, juveniles fed with microalgae. Sectors are numbered along raceways from 1 =inflow to 4=outltlow. Sides 1 and 2 are north (more shaded) and south (less shaded), respectively.
Mean shell lengths of 0.39 mm and 0.83 mm after 7 and 10 weeks, respectively, in the upwelling system were significantly lower (P 0.05). The two-way and three-way interactions also were not significant.
290 TABLE 2 Mean shell length harvesting
(mm)of juvenile clams and standard deviations Raceway A
sector
1
2
3
4
Total raceway
Raceway B
(values in brackets) in each sector of raceways A-D at
Raceway D
Raceway C
side 1
side 2
side 1
side 2
side 1
side 2
side 1
side 2
4.82 (2.39) n=221 4.26 (1.78) 11~284 3.75 (1.72) n=182 3.45 (1.36) II=272
5.00 (2.22) n=269 4.64 (1.98) !I=270 4.11 (2.09) n=225 4.01 (1.60) n=196
5.55 (2.25) n=26 4.84 (1.91) n=40 4.25 (2.17) r&=20 5.60 (2.38) nz80
5.49 (2.43) n=20 6.38
3.75 (0.70) n=29 5.09 (1.67) n=92 4.40 (1.20) n=105 3.92 (1.09) n=99
4.22 (0.94) n=45 5.20 (1.69) n=lOl 4.63 (1.25) n=121 4.37 (1.32) n=114
4.48
6.20
4.27 (1.98) n= 1919
5.43 (2.34) n=28a
II=1 5.40 (2.57) n=45 5.95 (2.36) n=56
4.52 (1.39) n=706
ll=l 3.06 (0.36) n=6 4.44 (1.53) n=36 4.93 (1.70) n=70
n=l 2.97
(0.74) II=3 4.06 (1.07) II=8 2.75 (0.76) n=8
4.49 (1.64) n=133
See Table 1 for explanation of raceway treatments, sectors and sides. n=number sampled.
The shell length data also showed significant heterogeneity of variances (Cochran’s test P < 0.001). The results of analysis of variance of lengths, therefore, must be treated with caution. A one-way ANOVA of shell lengths in raceways A-D showed that there was a highly significant difference between raceways (P-c 0.001) . Lengths in raceways A, C and D were not significantly different, but they were significantly lower than in raceway B (Tukey’s multiple range test). A three-way analysis of variance to examine the effects of position in raceway on lengths was performed on the results from raceways A and C only (sample sizes in several sectors of raceways B and D were too low for reliable analysis). There was a significant difference in lengths between raceways (P < 0.05 ) and between sides and sectors (P < 0.001) . The interaction of raceway by sector also was significant (P < 0.001) , but the other interactions were not. The mean lengths in all sectors of side 2 (more sunlight) were higher than those from the same sectors of side 1 for both raceways. In raceway A the mean lengths decreased from sectors 1 to 4, whereas in raceway C they were higher in the middle than end sectors. This explains the significant interaction of raceway by sector. In raceway E where various substrate types were placed randomly on the bottom, the total mean density was similar to that in raceways A and C (Table 3) and the mean length was substantially higher than in all the other raceways (Table 4). Densities were highest in the coral pieces and woven plastic fabric, intermediate on the asbestos sheet and fibreglass plates, and lowest on the plastic mesh and polyethylene sheet. Mean lengths were highest on the woven
291 TABLE 3 Mean densities
Substrate
of juvenile clams (number
1 2 3 4 5 6
cm-‘)
on different
types of substrate
in raceway E
Mean
SD.
n
Min. density
Max. density
0.037 0.032 0.101 0.116 0.015 0.004
0.091 0.042 0.096 0.137 0.011 0.003
16 32 4 11 3 3
0 0 0.014 0 0.002 0
0.374 0.126 0.233 0.417 0.022 0.006
Total raceway 0.046 Total clams collected from raceway=
1831
Substrate: 1 = asbestos sheeting; 2 = fibreglass sheeting; 3 = woven plastic fabric; 4 = coral pieces; 5 = plastic mesh; 6 = polyethylene sheet. SD. = standard deviation. n = number of replicates.
plastic fabric, followed by coral pieces, plastic mesh and fibreglass. They were much lower on the polyethylene and asbestos sheet. DISCUSSION
Fitt and Trench (1981) referred to the confusion in the literature surrounding the use of the term “spawning” for giant clams. “Spawning” has been used to describe the release of eggs or sperm or both, and often no distinction is made between these. Although egg release by giant clams is almost always accompanied by sperm release, either by the same or another animal (Fitt and TABLE 4 Mean lengths of juvenile clams (mm) on different
types of substrates
in raceway E
Mean
S.D.
nl
n2
Min. size
Max. size
Substrate
5.62 6.92 8.81 7.27 7.16 5.7
2.16 2.27 2.35 2.56 3.08 1.45
193 1032 299 130 89 5
16 32 4 11 3 3
1.8 1.8 1.7 1.7 2.1 3.6
13.7 16.0 16.2 15.1 15.5 7.3
Total raceway
7.42
2.52
1748
S.D. = standard deviation; nl = number of clams; n2 = number of replicates of each substrate Key for substrate types as in Table 3.
type.
292
Trench, 1981; Braley, 1984, 1985), the release of sperm alone occurs frequently. Since egg fertilization requires the release of both eggs and sperm, it is strongly recommended that the term “spawning” be used to describe only this, and that the release of sperm alone be described as such. Intragonadal injections of serotonin into mature T. gigas will induce spawning, and produce viable gametes. Braley (1985) also used serotonin to induce spawning in several species of giant clams, but he did not select for mature animals and only one of his T. gigm produced eggs; gamete viability was not examined. Thus, serotonin appears to have alleviated the maricultural constraint of depending on spontaneous spawnings in T. gigas. The results of the March spawning trial when three injections of serotonin were required, however, suggest that the broodstock must reach a certain stage of maturity for spawning inductions to be successful. Enforced spawnings may result in low embryonic and larval survival rate. Fitt et al. (1984) observed that larvae from several spawnings differed markedly in size, possibly because the eggs contained different proportions of stored nutrients. They hypothesized that eggs with smaller nutrient stores would have lower survival and growth rates. Eggs which are spawned only after multiple serotonin injections may not be fully developed and may have smaller nutrient stores than those produced easily from a single injection of serotonin. The successful induction of spawning of T. gigas in sevearal months of the year, notably in winter, is also of importance. This finding differs from that of Braley (1984 ) who reported that T. gigm gonads are in resting condition during winter, and raises doubts about his suggested early-mid summer spawning for T. gigas on the Great Barrier Reef. T. gigm may reach a peak spawning activity at this time, but it seems that at least a small proportion of the population is mature for much of the year. The total mortality of T. gigm larvae in winter implies that heated seawater may be necessary in the colder months if they are to be reared throughout the year in higher latitudes. The high survival rates of T. gigus larvae reared intensively show that they are suited to mass culture methods typically used for bivalve larvae and that the larval rearing phase presents no major problems to the mariculture of T. gigas. The culture conditions used in this study agree with the findings of Fitt et al. (1984) from small scale experiments, i.e. that larval densities of 2-10 ml- ’ and feeding with Isochrysis gulbuna at a concentration of lo-100 cells ~1~’ are conducive to larval survival and growth. The poorer survival rates of tridacnid larvae, especially trochophores, obtained by Fitt et al. (1984) and by other researchers, e.g. Beckvar (1981) and Gwyther and Munro (1981)) may be due to irregular water changes and hence bacterial contamination. The most obvious difference between raceways at harvesting of 5-monthold juveniles was that densities were significantly higher in unfed than fed raceways, irrespective of whether larvae were reared intensively or extensively (Table 1) . Of the larvae reared intensively and placed in the raceways at set-
293
tlement, 0.6% survived to harvesting in unfed raceways and 0.1% in fed raceways. The feeding with microalgae was expected to improve growth and survival rates of juveniles because zooxanthellae apparently do not provide all the metabolic carbon requirements of clams (Trench et al., 1981). The fed raceways, however, developed far more benthic algae, because of the addition of algal nutrients along with the microalgae. The benthic algae, which grew over the juveniles, probably caused higher mortalities by reducing the light intensity and competing for nutrients with the zooxanthellae. These results therefore indicate that unless algal overgrowth can be controlled, supplementary feeding of juveniles is not warranted. Although the densities at harvesting were similar for juveniles originating from intensive and extensive larval rearing in the fed and unfed raceways, the two rearing methods cannot be compared directly. The number of pediveligers (4x 105) from intensive culture placed in each raceway at settlement was much lower than the number of eggs (2 x 107)originally stocked in the extensive rearing raceways. The numbers of pediveligers were determined in relation to the bottom area of raceways available for juveniles, and a much higher rate of survival was assumed than actually occurred. It would be relatively easy to increase the number of pediveligers reared intensively and stocked in each raceway to allow for the high mortality during and just after metamorphosis. The number of eggs placed in the extensive raceways, however, is possibly about the maximum, as evidenced by the large bacterial blooms 2 days postfertilization. No conclusions can be reached on the influence of supplementary feeding, or method of larval rearing, on growth rates of newly-metamorphosed juveniles (Table 2). Reasons for the differences in mean lengths of juveniles between raceway treatments, particularly the two unfed intensive larval raceways A and E, are not clear (Tables 2 and 4). The distributions of juveniles in the raceways apparently were not influenced by the water turbulence or the degree of shading. The lengths of juveniles, however, were highest on the southern side of the raceways (Table 2). With the zenith position of the sun being in the north for most of the experiment, the southern side received more sunlight than the northern side. The lengths of juveniles in the different sectors of raceways A and C are confusing (Table 2 ) . If the concentration of inorganic nutrients in the seawater was important for growth, then the lengths would be expected to decrease from sector 1 to sector 4, as in raceway A; but there are no clear reasons for the lengths being highest in the middle sectors of raceway C. The differences in mean lengths between the sides and sectors of raceways A and C are, however, much less than the differences between the total mean lengths of these raceways (4.27 and 4.52 mm, respectively) and raceway E ( 7.42 mm). This implies that substrate type or other factors have a much greater influence on growth rates than water circulation or light intensity. Variations in densities and mean
294
lengths of juveniles on the various substrate types (Tables 3 and 4 ) indicate that textured surfaces, such as woven plastic fabric or coral pieces, are either selected for or enhance survival, and they also improve growth rates. Smooth surfaces, e.g. polyethylene sheet, are unsuitable as a settlement substrate. The growth rates of T. gigm juveniles in this study were much lower than those recorded by Munro and Heslinga (1983) and Heslinga et al. (1984) for T. gigas over a similar period of time. This is probably because the water temperatures were much lower, decreasing to 18-20” C in the raceways in midwinter, compared with the 27-30°C recorded by Heslinga et al. (1984) in Palau. Beckvar (1981) ‘reared T. gigm juveniles in 27-33°C seawater and observed growth rates similar to those in this study, but her juvenile clams were maintained in heavy shade. Thus, in areas with substantial seasonal differences in water temperature, the best rearing strategy would appear to be to spawn the adults in spring-early summer, so that the juveniles can grow through the period with highest mortalities in the shortest period of time. Yamaguchi (1977) proposed that the early juvenile stage would present a problem to the mariculture of giant clams because of difficulties in handling the small sized newly-metamorphosed juveniles and because they are capable of crawling around the substrates. Beckvar (1981) also mentioned problems with algal overgrowth, whereas Heslinga and Watson (1985) have found that this is a potential problem only after harvesting of juveniles and it is controlled effectively by the addition of juvenile Troches niloticus. Large numbers of T. deram have been reared over the last 2 years by Heslinga and Watson in Palau and they consider that the commercial cultivation of T. derasa is now feasible, although this is still to be proven. The results of this study have shown that, at least for T. gigus on the Great Barrier Reef, further research is required on the factors affecting survival and growth of juveniles after metamorphosis and on techniques for large-scale rearing of juveniles. The methods typically used in commercial bivalve nurseries, e.g. layers of juveniles in upwelling systems, are not readily applicable to giant clams because of the light requirements of their symbiotic zooxanthellae. The acquisition of zooxanthellae, inorganic nutrient requirements and the effect of algal overgrowth on the survival and growth of juvenile clams, in particular, need further investigation. ACKNOWLEDGEMENTS
This research is part of an international project to culture giant clams, which is funded by the Australian Centre for International Agricultural Research. It is being conducted in collaboration with the International Giant Clam Mariculture Project, initiated by Dr. J.L. Munro. We are grateful to Rick Williams and Geoff Charles, station managers, for their assistance with facilities at Orpheus Island Research Station and to Stephane Westmore and Peter O’Meara for technical assistance.
REFERENCES Beckvar, N., 1981. Cultivation, spawning and growth of the giant clams Tridacnagigas, T. derasa and T. squamosa in Palau, Caroline Islands. Aquaculture, 24: 21-30. Braley, R.D., 1984. Reproduction in the giant clams Tridacna gigas and T. derasa in situ on the North-Central Great Barrier Reef, Australia, and Papua New Guinea. Coral Reefs, 3: 221-227. Braley, R.D., 1985. Serotonin-induced spawning in giant clams (Bivalvia:Tridacnidae) . Aquaculture, 47: 321-325. Brown, J.H. and Muskanofola, M.R., 1985. An investigation of stocks of giant clams (family Tridacnidae) in Java and of their utilisation and potential. Aquacult. Fish. Manage., 1: 25-39. Castagna, M. and Kraeuter, J.N., 1981. Manual for growing the hard clam Mercenaria. Special Report in Applied Marine Science and Ocean Engineering No. 249, Virginia Institute of Marine Science, Gloucester Point, VA, 110 pp. Fitt, W.K. and Trench, R.K., 1981. Spawning, development and acquisition of zooxanthellae by Tridacna squamosa (Mollusca, Bivalvia). Biol. Bull., 161: 213-235. Fitt, W.K., Fisher, C.R. and Trench, R.K., 1984. Larval biology of tridacnid clams. Aquaculture, 39: 181-195. Gibbons, M.C. and Castagna, M., 1984. Serotonin as an inducer of spawning in six bivalve species. Aquaculture, 40: 189-191. Gwyther, J. and Munro, J.L., 1981. Spawning induction and rearing of larvae of tridacnid clams (Bivalvia:Tridacnidae) . Aquaculture, 24: 197-217. Heslinga, G.A. and Watson, T.C., 1985. Recent advances in giant clam mariculture. Proc. 5th Int. Coral Reef Congr., Tahiti, 5: 531-537. Heslinga, G.A., Perron, F.E. and Orak, O., 1984. Mass culture of giant clams (F. Tridacnidae) in Palau. Aquaculture, 39: 197-215. Jameson, S.C., 1976. Early life history of the giant clams Tridacna crocea Lamarck, T. maxima (Roding) and Hippopus hippopus (Linnaeus) . Pacif. Sci., 30: 219-233. LaBarbera, M., 1975. Larval and post-larval development of the giant clams, Tridacna maxima and Tridacna squamosa (Bivalvia:Tridacnidae) . Malacologia, 15: 69-79. Matsutani, T. and Nomura, T., 1982. Induction of spawning by serotonin in the scallop, Patinopecten yessoensis (Jay). Mar. Biol. Lett., 3: 353-358. Munro, J.L. and Gwyther, J., 1981. Growth rates and maricultural potential of tridacnid clams. Proc. 4th Int. Coral Reef Symp., Manila, 2: 633-636. Munro, J.L. and Heslinga, G.A., 1983. Prospects for the commercial cultivation of giant clams (Bivalvia:Tridacnidae). Proc. Gulf. Caribb. Fish. Inst., 35: 122-134. Trench, R.K., Wethey, D.S. and Porter, J.W., 1981. Observations on the symbiosis with zooxanthellae among the Tridacnidae (Mollusca, Bivalvia) . Biol. Bull., 161: 180-198. Underwood, A.J., 1981. Techniques of analysis of variance in experimental marine biology and ecology. Oceanogr. Mar. Biol. Annu. Rev., 19: 513-605. Usher, G.F., 1984. Coral reef invertebrates in Indonesia: their exploitation and conservation needs. IUCN/WWF Report No. 2, Bogor, Indonesia, 100 pp. Wada, S.K., 1954. Spawning in the tridacnid clams. Jpn. J. Zool., 11: 273-285. Walne, P.R., 1974. Culture of Bivalve Molluscs. Fishing News (Books) Ltd, Surrey, Great Britain, 173 pp. Wells, S.M., Pyle, R.M. and Collins, N.M., 1983. The IUCN Invertebrate Red Data Book. Marine Molluscs. IUCN, Gland, Switzerland, pp. 69-90. Yamaguchi, M., 1977. Conservation and cultivation of giant clams in the tropical Pacific. Biol. Conserv., 11: 13-20. Zar, J.H., 1984. Biostatistical Analysis. Prentice-Hall Inc., Englewood Cliffs, NJ, 718 pp.
Technical
281 -
Printed
in The Netherlands
Paper
Spawning Induction, and Larval and Juvenile Rearing of the Giant Clam, Triducna gigus C.M. CRAWFORD’,
W.J. NASH and J.S. LUCAS
Zoology Department, James Cook University, Townsville, Queensland, 4811 (Australia) ‘Present address: Fisheries Laboratories, Department of Sea Fisheries, Taroona, Tasmania, 7006 (Australia) (Accepted
9 June 1986)
ABSTRACT Crawford, C.M., Nash, W.J. and Lucas, J.S., 1986. Spawning induction, rearing of the giant clam, Tridacna gigas. Aquaculture, 58: 281-295.
and larval and juvenile
Specimens of Tridacna gigas (L.) with mature gonads were selected for broodstock using a biopsy technique and induced to spawn using intragonadal injections of serotonin. Some larvae were reared using conventional bivalve rearing techniques in 500-l tanks, with up to 40% survival to settlement after 7-8 days. Other larvae were reared extensively from eggs added directly to raceways. There was heavy mortality at or soon after metamorphosis in all groups of juvenile clams and survival of juveniles from intensive larval rearing was less than 1% to harvesting at 5 months. Similar numbers of juvenile clams were harvested from raceways to which 4 x lo5 intensively reared pediveligers or 2 x lo7 eggs (extensive culture) were added initially, yielding a total of ca. 6000 juvenile clams. Some juvenile clams were fed cultured microalgae for several months to supplement the nutrients obtained from their symbiotic zooxanthellae. These clams showed poorer survival than unfed clams, apparently because they were more overgrown by benthic algae. The distribution of juveniles in the raceways at harvesting was not influenced by water turbulence or shading, but growth rates were reduced by shading. Growth rates and densities of juveniles were much higher on textured than smooth substrates. We have shown that the mass rearing methods used for bivalve mollusc larvae are appropriate to T. gigas larvae; however, the typical methods for bivalve nursery rearing are not readily applicable to giant clam juveniles. Further research is required on these methods and on factors affecting juvenile survival and growth.
INTRODUCTION
The mariculture of giant clams (Family Tridacnidae) is currently receiving considerable attention for several reasons. Populations of giant clams, particularly the larger species, have declined precipitously in many areas because of over-exploitation (Wells et al., 1983). Their biology and ecology also would suit them for cultivation (see Munro and Heslinga, 1983). Of prime interest for mariculture is the largest species, Triducna gigm, which also has the fastest
0044-8486/86/$03.50
0 1986 Elsevier Science Publishers
B.V.
282
growth rate (Munro and Heslinga, 1983). This species has suffered the greatest over-exploitation and has recently become extinct in Fiji ( A.D. Lewis, personal communication, 1985) and four Micronesian Islands - Guam, Truk, Ponape and Kosrae (Heslinga et al., 1984). It is on the verge of extinction in the Philippines (A. Alcala, personal communication, 1985) and regions of Indonesia (Usher, 1984; Brown and Muskanofola, 1985). Substantial advances in the techniques for mass culture of giant clams have been made at the Micronesian Mariculture Demonstration Center in Palau and these are summarized by Heslinga and Watson (1985). The research at Palau, however, has concentrated on T. derusa, with little work being conducted on T. gigus. Several constraints to the mariculture of T. gigus have been outlined by Munro and Gwyther (1981) , Munro and Heslinga (1983) and Wells et al. (1983 ) . T. gigus, unlike other tridacnids, has not been successfully induced to spawn eggs by either chemical stimuli or the addition of gonadal extract. Only spontaneous spawnings have occurred and these are too unreliable for maricultural purposes. Moreover, although T. gigus spawns spontaneously throughout the year in Palau (Heslinga et al., 1984)) Braley (1984) reported that T. gigus has a restricted spawning period in the Great Barrier Reef region. Other investigators of the biology of giant clams have reported very high mortality during the larval stages ( LaBarbera, 1975; Jameson, 1976; Beckvar, 1981; Fitt and Trench, 1981; Gwyther and Munro, 1981). There have, however, been no attempts to rear tridacnid larvae using conventional bivalve hatchery techniques such as those used for oysters, Ostrea edulis and Crussostrea gigus, and the hard clam Mercenaria mercenuriu, e.g. Walne (1974) and Castagna and Kraeuter (1981) . This paper describes methods for spawning induction and collection of eggs and sperm to ensure cross-fertilization of T. gigus. Survival and growth rates of T. gigus larvae and early juveniles, reared using methods similar to those of Heslinga and Watson (1985) and using standard bivalve techniques, are reported. Some factors influencing juvenile survival and growth are discussed. METHODS
Broodstock specimens of T. gigus, 60-100 cm shell length, were collected from fringing reefs and transported to onshore holding tanks at Orpheus Island Research Station (18’ 40’S 146’ 30’E), where they were maintained in flowing seawater for up to 6 months. Reproductive condition of broodstock clams was determined by inserting a human biopsy needle (15.2 cm cannula, 20 mm specimen notch) through the mantle and into the gonad to collect a small sample of gonad tissue. Induction of spawning was attempted in February, March and June 1985 using macerated gonad tissue or the chemical serotonin (5-hydroxytryptamine creatine sulfate complex). Serotonin was used because it induces spawning in
283
the scallop Patinopecten yessoensis ( Matsutani and Nomura, 1982) and in six other bivalve species (Gibbons and Castagna, 1984). Only clams with mature gonads, as indicated by biopsy samples, were selected for spawning induction. Gonad extract ( fresh, or frozen for about 1 month) was squirted into the inhalent aperture, or l-2 ml of 1 mA4 seroronin in filtered seawater were injected directly into the gonad. Clams responded by producing sperm a few minutes after induction and samples were collected from several individuals. When eggs were released they were collected by placing a large plastic bag (90 x 60 cm) over the exhalent siphon just prior to a spawning contraction; the clam then expelled its eggs directly into the bag. The eggs were fertilized using a small quantity of sperm from another animal. This procedure greatly reduced the risk of polyspermy and self fertilization. The zygotes were then rinsed through sieves to remove gonadal debris and excess sperm, and subsampled to estimate numbers and percent fertilized. Larval rearing Fertilized eggs from a spawning two larval rearing systems:
induction
in February
were transferred
to
Intensive rearing. Larvae were reared in two 500-l tanks containing l-pm filtered seawater with light aeration and at 27°C. Initial egg densities were ca. 10 ml-’ in one tank and 2 ml-’ in the other. From the veliger stage (day 2 ) the water was changed daily by draining through an 80-pm sieve, and the larvae were fed unicellular algae, Isochrysis gulbana (Tahitian strain), at a concentration of lo-100 cells PI- ‘. Survival rates were estimated daily and at least 50 preserved larvae were measured along their anteroposterior axis. When most larvae were pediveligers (day 8-9) they were transferred to three 500~80x50 cm outdoor raceways at a density of 10 larvae cm-’ of bottom area. The raceways contained lo-pm filtered seawater and were covered with a canopy excluding 50% of incident light. The seawater was static, except for a daily 4-h flushing (30-40% water replacement) when an 80-pm screen was attached to the overflow pipe. I. galbana was added every second day at a concentration of l-10 cells ~1~‘. The bottoms of two raceways (A and B) were each randomly lined with asbestos sheet and fibreglass plates of approximate dimensions 29 x 13.5 cm. Another raceway ( E) contained various materials, asbestos sheet, fibreglass, plastic fabric, small coral pieces, plastic mesh and polyethylene sheet, placed randomly on the bottom to test for substrate preference. Extensive rearing. Fertilized eggs were added to two outside raceways (C and D ) 500 x 80 x 50 cm containing static lO+m filtered seawater to achieve densities of ca. 10 ml-‘. On day 2, dense bacterial growth was evident in the water
284
and the larvae were transferred, by siphoning onto sieves, to new raceways containing fresh filtered seawater. The raceways were flushed (30-40% turnover) for about 4 h daily from day 5 until the majority of larvae had metamorphosed. I. galbana was added on day 3 at a concentration of 50 cells ~1~‘, and by day 5 had formed a dense culture. Algae were not added again until day 11. With this system it was impractical to determine larval survival rates. Zooxanthellae from adult T. gigas were distributed among the larval and juvenile tanks every 2-3 days for 10 days from day 7. The zooxanthellae were collected by scraping a small piece of excised adult mantle tissue to release them into suspension and then rinsed through a 25-pm mesh. Juvenile rearing From day 12 when almost all larvae in the raceways had metamorphosed, 5-lo-pm filtered seawater was supplied continuously to each raceway at a flow of 8 1 min-‘. Feeding, by trickling I. galbana in with the inflowing seawater for 4 h every l-2 days, was continued for 2 weeks until zooxanthellae were clearly visible in the juveniles’ mantle tissue. For the following 2 months, I. galbana or Choreh sp. were added every second day to two raceways (B and D ) , one with intensively and the other with extensively reared larvae. Anteroposterior shell lengths of about 50 juveniles from each raceway, including various substrate types, were measured periodically. As the juveniles were quite motile for several months, it was not possible to determine survival rates or densities per unit area of substrate during this time. The second spawning induction in March also resulted in fertilized eggs. These were maintained using both the intensive and extensive larval culture methods, as described for the February spawning. Juvenile rearing was similar, with the following exception. Some pediveligers from the intensive larval culture system were transferred to plastic boxes 32 cm square, with 125~pm mesh on the base. The boxes contained larvae at densities of l-20 cm-’ of bottom area and were positioned in a small outside tank with water 10 cm deep and seawater flowing in from above. After the larvae had metamorphosed, the boxes were converted to upwelling systems, whereby seawater entered up through the mesh boxes. Harvesting of juveniles Juvenile clams were harvested at 5 months by siphoning or scraping them off the various substrates. The effect of environmental factors on the numbers and sizes of juveniles in raceways A, B, C and D was investigated in the following manner. Each raceway was divided longitudinally into two halves (sides) because the raceways were oriented in an east-west direction and therefore the northern and southern sides received different levels of shading. Each side
then was divided into four sectors because the water turbulence, observed from dye testing, was higher at the inflow and outflow ends of the raceway than in the middle. The inflow and outflow sectors measured 40 x 90 cm and the middle sectors 40 x 160 cm. In each sector of raceways A and B the number of juveniles on at least nine settlement plates (chosen at random) was counted and the density (number cm-‘) was calculated. In raceways C and D total counts were made of the juveniles on the bottom in each sector. Some clams were harvested from the side walls, but were not included in the analysis. In raceway E the density of juveniles on each substrate type was calculated from total counts. All clams, to a maximum of 50 on each plate or to 121 on the tank bottom in each sector, were measured to the nearest 0.1 mm. After removal from the raceways, the juveniles were placed on small pieces of gravel or coral rubble in plastic trays (53 x 28 x 8 cm) at densities of 0.2-0.3 cm-2. These trays were placed in tanks with flowing unfiltered seawater. Samples of 50-100 juveniles originating from each raceway were then measured once a month. Where appropriate, the results of harvesting the juveniles from the raceways were subjected to analysis of variance (ANOVA) (Zar, 1984) to examine the effects of position in the raceway, i.e. sides and sectors, and raceway treatments on juvenile densities and on shell lengths. If the ANOVA gave a significant result for a factor, but non-significant interaction, then the specific levels of the factor between which differences occurred were determined using Tukey’s Test (Zar, 1984) at P=O.O5 level of significance. RESULTS Spawning induction A simple classification of maturity stages for T. gigas broodstock was developed from the gonad biopsies. If immature, spent or resting, no gonadal tissue could be collected in the biopsy needle. Ova in maturing gonads were irregularly shaped with a large perivitelline space, and those in mature gonads were predominantly spherical, with small to no perivitelline space and diameter greater than 100 pm. Many active sperm were present in mature gonads. The introduction of macerated gonad into the inhalent siphon of mature clams on several occasions resulted in some sperm release, but never eggs. In February, the first injection of serotonin into five of eleven clams in a tank resulted in sperm production, and the release of a small quantity of eggs by two clams. A second injection 2 days later produced copious quantities of sperm from most animals, low numbers of eggs from three clams, and the spawning of all eggs from a fourth clam. A small sample from this clam’s spawning yielded 125 million eggs. After this spawning, the eleven clams were distributed between five tanks, and over the next 2 weeks at least five clams, some of which had
not been injected with serotonin, spawned spontaneously. One isolated clam spawned more than 500 million eggs at once. Many of these eggs were fertilized and some developed to the veliger stage, proving that self-fertilization can occur, although none of these larvae survived to metamorphosis. All spontaneous spawnings occurred in mid-late afternoon and generally followed the pattern described by Wada (1954). A spawning induction attempted in March did not result in any eggs after two injections of serotonin. A third injection one week later produced sperm from four clams and eggs from one. In June, only one injection of serotonin was necessary to induce egg release in one mature clam. Viable gametes were produced from the spawning inductions. Many eggs were fertilized and developed normally. The larvae from the June spawning, however, developed very slowly and did not survive to metamorphosis, probably because the water temperature was too low. It was 225°C at spawning and decreased to 19°C overnight. Larval rearing Embryological and larval development to metamorphosis of T. gigas were similar to these processes in other tridacnids (see LaBarbera, 1975; Jameson, 1976). Larvae began feeding on day 3, settling occurred on days 7-8 and metamorphosis between days 8 and 12. Zooxanthellae were visible in the mantle tissue l-2 weeks postmetamorphosis. These developmental times differ only slightly from the observations of Heslinga et al. (1984) for T. gigas where larvae settled on days 5-6, more than 50% had metamorphosed by days 7-8 and zooxanthellae were visible by day 11. Intensive rearing. Survival rates of larvae to pediveligers were higher in tank 2 (40.2% ) at a density of 2 ml-’ than in tank 1 (26.6% ) at 10 larvae ml-’ (Fig. 1A). At settlement, however, there were 1.3 x lo6 larvae surviving in tank 1 compared with 0.4 x lo6 in tank 2. Growth rates of larvae were 5.8 pm day- ’ in tank 1 and 5.3 pm day-’ in tank 2 (Fig. 1B). Algae were visible in the guts from day 3, but the concentration of algae in the culture water altered little, implying that only a small quantity of food was eaten. Bacterial growth was obvious in the tanks, but apparently did not affect the larvae. Extensive rearing. The transfer of larvae to clean raceways on day 2 resulted in the mortality of many larvae. The total number in each raceway on day 3 was approximately 7.25 x 105, a decrease from 20 x lo6 eggs. Mean length of larvae on day 5 was 173.7 ? 8.1 pm (c.f. 186 ,um in larval tank 1) and on day 9 was 188.15 8.1 pm.
281
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.
175-
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150 I 1
I
I
I
I
2
3
4
5
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6
7
6
Days from fertilization
Fig. 1. Survival rates (A) and growth rates (B) of 2’. gigas larvae in tank 1 at 10 larvae ml-’ (circles), and in tank 2 at 2 larvae ml-’ (squares).
Juvenile rearing Heavy mortality was observed amongst the metamorphosing larvae and newly-metamorphosed juveniles in all raceways. Of 12 samples of juvenile clams taken randomly during the first week after larvae had been transferred from intensive culture tanks to raceways, an average of 71% were dead, size 195-215 pm. A sample taken 3 weeks after settlement from a raceway with extensivelyreared larvae yielded 20% live clams of average length 422 ,um. Ninety-one percent of the dead shells collected were in the size range MO-208 pm, the
/
0
_*__.+-W+ 0
l
’
40
60
120
age
160
200
(days)
Fig. 2. Mean lengths and standard deviations harvesting.
of T. gigas juveniles from settlement
to post-
approximate size of pediveligers, and thus mortality occurred at metamorphosis or soon thereafter. Individual growth rates varied markedly and after 5 months shell lengths ranged between 1 and 16 mm. The growth of juveniles from intensively-reared larvae and unfed raceways only is shown in Fig. 2. Initial growth rates were low and for the first 6 weeks from settlement averaged 14 pm day-‘. Growth rates were higher over the next 5 months, from April to September, and approximately linear at 61 pm day-‘. A major problem encountered during the juvenile rearing period was the overgrowth of juveniles by benthic, filamentous algae. This was most apparent in the raceways which received unicellular algae every second day for 2 months. There also was a progressive increase in the abundance of invertebrates, including various crustaceans, polychaetes and gastropods, in the raceways. These probably entered as eggs or larvae and completed their development in the raceways. Although no evidence of predation on juvenile clams was obtained, it is suspected that these invertebrates caused some mortalities. Although eggs and larvae from the second spawning in March were treated in a similar manner to those from the first spawning, mortalities at all stages were much higher. The survival rate of intensively-reared larvae to the pediveliger stage on day 9 post-fertilization was 8.5% and the average growth rate was 4.25 pm day-‘. Survival and growth rates of juveniles also were lower.
289 TABLE 1 Mean densities of juvenile clams (number cm- ‘) in each w&r of raceways A-D at harvesting. Mean densities in raceways A and B are for replicate samples with standard deviations (values in brackets), and in raceways C and D for whole sectors Raceway A
sector
1 2 3 4
Total raceway: Total number Mean density
Raceway B
Raceway c
Raceway D
side 1
side 2
side 1
side 2
side 1
side 2
side 1
side 2
0.056 (0.048) 0.051 (0.038) 0.032 (0.022) 0.082 ( 0.095 )
0.052 ( 0.046 ) 0.048 (0.037) 0.036 (0.040) 0.062 (0.087)
0.007 ( 0.007
0.005 (0.006) 0.001 (0.002 ) 0.011 (0.020) 0.011 (0.017)
0.008
0.013
o.cmO3
0.0003
0.041
0.041
0.0009
0.0005
0.033
0.045
0.006
0.001
0.049
0.076
0.019
0.002
2009 0.050
) 0.008 (0.011) 0.005 (0.004) 0.020 (0.019)
303 0.008
1573 0.039
133 0.003
Raceways: A, B, intensive larval culture; C, D, extensive culture; A, C. juveniles unfed, B, D, juveniles fed with microalgae. Sectors are numbered along raceways from 1 =inflow to 4=outltlow. Sides 1 and 2 are north (more shaded) and south (less shaded), respectively.
Mean shell lengths of 0.39 mm and 0.83 mm after 7 and 10 weeks, respectively, in the upwelling system were significantly lower (P
290 TABLE 2 Mean shell length harvesting
(mm)of juvenile clams and standard deviations Raceway A
sector
1
2
3
4
Total raceway
Raceway B
(values in brackets) in each sector of raceways A-D at
Raceway D
Raceway C
side 1
side 2
side 1
side 2
side 1
side 2
side 1
side 2
4.82 (2.39) n=221 4.26 (1.78) 11~284 3.75 (1.72) n=182 3.45 (1.36) II=272
5.00 (2.22) n=269 4.64 (1.98) !I=270 4.11 (2.09) n=225 4.01 (1.60) n=196
5.55 (2.25) n=26 4.84 (1.91) n=40 4.25 (2.17) r&=20 5.60 (2.38) nz80
5.49 (2.43) n=20 6.38
3.75 (0.70) n=29 5.09 (1.67) n=92 4.40 (1.20) n=105 3.92 (1.09) n=99
4.22 (0.94) n=45 5.20 (1.69) n=lOl 4.63 (1.25) n=121 4.37 (1.32) n=114
4.48
6.20
4.27 (1.98) n= 1919
5.43 (2.34) n=28a
II=1 5.40 (2.57) n=45 5.95 (2.36) n=56
4.52 (1.39) n=706
ll=l 3.06 (0.36) n=6 4.44 (1.53) n=36 4.93 (1.70) n=70
n=l 2.97
(0.74) II=3 4.06 (1.07) II=8 2.75 (0.76) n=8
4.49 (1.64) n=133
See Table 1 for explanation of raceway treatments, sectors and sides. n=number sampled.
The shell length data also showed significant heterogeneity of variances (Cochran’s test P < 0.001). The results of analysis of variance of lengths, therefore, must be treated with caution. A one-way ANOVA of shell lengths in raceways A-D showed that there was a highly significant difference between raceways (P-c 0.001) . Lengths in raceways A, C and D were not significantly different, but they were significantly lower than in raceway B (Tukey’s multiple range test). A three-way analysis of variance to examine the effects of position in raceway on lengths was performed on the results from raceways A and C only (sample sizes in several sectors of raceways B and D were too low for reliable analysis). There was a significant difference in lengths between raceways (P < 0.05 ) and between sides and sectors (P < 0.001) . The interaction of raceway by sector also was significant (P < 0.001) , but the other interactions were not. The mean lengths in all sectors of side 2 (more sunlight) were higher than those from the same sectors of side 1 for both raceways. In raceway A the mean lengths decreased from sectors 1 to 4, whereas in raceway C they were higher in the middle than end sectors. This explains the significant interaction of raceway by sector. In raceway E where various substrate types were placed randomly on the bottom, the total mean density was similar to that in raceways A and C (Table 3) and the mean length was substantially higher than in all the other raceways (Table 4). Densities were highest in the coral pieces and woven plastic fabric, intermediate on the asbestos sheet and fibreglass plates, and lowest on the plastic mesh and polyethylene sheet. Mean lengths were highest on the woven
291 TABLE 3 Mean densities
Substrate
of juvenile clams (number
1 2 3 4 5 6
cm-‘)
on different
types of substrate
in raceway E
Mean
SD.
n
Min. density
Max. density
0.037 0.032 0.101 0.116 0.015 0.004
0.091 0.042 0.096 0.137 0.011 0.003
16 32 4 11 3 3
0 0 0.014 0 0.002 0
0.374 0.126 0.233 0.417 0.022 0.006
Total raceway 0.046 Total clams collected from raceway=
1831
Substrate: 1 = asbestos sheeting; 2 = fibreglass sheeting; 3 = woven plastic fabric; 4 = coral pieces; 5 = plastic mesh; 6 = polyethylene sheet. SD. = standard deviation. n = number of replicates.
plastic fabric, followed by coral pieces, plastic mesh and fibreglass. They were much lower on the polyethylene and asbestos sheet. DISCUSSION
Fitt and Trench (1981) referred to the confusion in the literature surrounding the use of the term “spawning” for giant clams. “Spawning” has been used to describe the release of eggs or sperm or both, and often no distinction is made between these. Although egg release by giant clams is almost always accompanied by sperm release, either by the same or another animal (Fitt and TABLE 4 Mean lengths of juvenile clams (mm) on different
types of substrates
in raceway E
Mean
S.D.
nl
n2
Min. size
Max. size
Substrate
5.62 6.92 8.81 7.27 7.16 5.7
2.16 2.27 2.35 2.56 3.08 1.45
193 1032 299 130 89 5
16 32 4 11 3 3
1.8 1.8 1.7 1.7 2.1 3.6
13.7 16.0 16.2 15.1 15.5 7.3
Total raceway
7.42
2.52
1748
S.D. = standard deviation; nl = number of clams; n2 = number of replicates of each substrate Key for substrate types as in Table 3.
type.
292
Trench, 1981; Braley, 1984, 1985), the release of sperm alone occurs frequently. Since egg fertilization requires the release of both eggs and sperm, it is strongly recommended that the term “spawning” be used to describe only this, and that the release of sperm alone be described as such. Intragonadal injections of serotonin into mature T. gigas will induce spawning, and produce viable gametes. Braley (1985) also used serotonin to induce spawning in several species of giant clams, but he did not select for mature animals and only one of his T. gigm produced eggs; gamete viability was not examined. Thus, serotonin appears to have alleviated the maricultural constraint of depending on spontaneous spawnings in T. gigas. The results of the March spawning trial when three injections of serotonin were required, however, suggest that the broodstock must reach a certain stage of maturity for spawning inductions to be successful. Enforced spawnings may result in low embryonic and larval survival rate. Fitt et al. (1984) observed that larvae from several spawnings differed markedly in size, possibly because the eggs contained different proportions of stored nutrients. They hypothesized that eggs with smaller nutrient stores would have lower survival and growth rates. Eggs which are spawned only after multiple serotonin injections may not be fully developed and may have smaller nutrient stores than those produced easily from a single injection of serotonin. The successful induction of spawning of T. gigas in sevearal months of the year, notably in winter, is also of importance. This finding differs from that of Braley (1984 ) who reported that T. gigm gonads are in resting condition during winter, and raises doubts about his suggested early-mid summer spawning for T. gigas on the Great Barrier Reef. T. gigm may reach a peak spawning activity at this time, but it seems that at least a small proportion of the population is mature for much of the year. The total mortality of T. gigm larvae in winter implies that heated seawater may be necessary in the colder months if they are to be reared throughout the year in higher latitudes. The high survival rates of T. gigus larvae reared intensively show that they are suited to mass culture methods typically used for bivalve larvae and that the larval rearing phase presents no major problems to the mariculture of T. gigas. The culture conditions used in this study agree with the findings of Fitt et al. (1984) from small scale experiments, i.e. that larval densities of 2-10 ml- ’ and feeding with Isochrysis gulbuna at a concentration of lo-100 cells ~1~’ are conducive to larval survival and growth. The poorer survival rates of tridacnid larvae, especially trochophores, obtained by Fitt et al. (1984) and by other researchers, e.g. Beckvar (1981) and Gwyther and Munro (1981)) may be due to irregular water changes and hence bacterial contamination. The most obvious difference between raceways at harvesting of 5-monthold juveniles was that densities were significantly higher in unfed than fed raceways, irrespective of whether larvae were reared intensively or extensively (Table 1) . Of the larvae reared intensively and placed in the raceways at set-
293
tlement, 0.6% survived to harvesting in unfed raceways and 0.1% in fed raceways. The feeding with microalgae was expected to improve growth and survival rates of juveniles because zooxanthellae apparently do not provide all the metabolic carbon requirements of clams (Trench et al., 1981). The fed raceways, however, developed far more benthic algae, because of the addition of algal nutrients along with the microalgae. The benthic algae, which grew over the juveniles, probably caused higher mortalities by reducing the light intensity and competing for nutrients with the zooxanthellae. These results therefore indicate that unless algal overgrowth can be controlled, supplementary feeding of juveniles is not warranted. Although the densities at harvesting were similar for juveniles originating from intensive and extensive larval rearing in the fed and unfed raceways, the two rearing methods cannot be compared directly. The number of pediveligers (4x 105) from intensive culture placed in each raceway at settlement was much lower than the number of eggs (2 x 107)originally stocked in the extensive rearing raceways. The numbers of pediveligers were determined in relation to the bottom area of raceways available for juveniles, and a much higher rate of survival was assumed than actually occurred. It would be relatively easy to increase the number of pediveligers reared intensively and stocked in each raceway to allow for the high mortality during and just after metamorphosis. The number of eggs placed in the extensive raceways, however, is possibly about the maximum, as evidenced by the large bacterial blooms 2 days postfertilization. No conclusions can be reached on the influence of supplementary feeding, or method of larval rearing, on growth rates of newly-metamorphosed juveniles (Table 2). Reasons for the differences in mean lengths of juveniles between raceway treatments, particularly the two unfed intensive larval raceways A and E, are not clear (Tables 2 and 4). The distributions of juveniles in the raceways apparently were not influenced by the water turbulence or the degree of shading. The lengths of juveniles, however, were highest on the southern side of the raceways (Table 2). With the zenith position of the sun being in the north for most of the experiment, the southern side received more sunlight than the northern side. The lengths of juveniles in the different sectors of raceways A and C are confusing (Table 2 ) . If the concentration of inorganic nutrients in the seawater was important for growth, then the lengths would be expected to decrease from sector 1 to sector 4, as in raceway A; but there are no clear reasons for the lengths being highest in the middle sectors of raceway C. The differences in mean lengths between the sides and sectors of raceways A and C are, however, much less than the differences between the total mean lengths of these raceways (4.27 and 4.52 mm, respectively) and raceway E ( 7.42 mm). This implies that substrate type or other factors have a much greater influence on growth rates than water circulation or light intensity. Variations in densities and mean
294
lengths of juveniles on the various substrate types (Tables 3 and 4 ) indicate that textured surfaces, such as woven plastic fabric or coral pieces, are either selected for or enhance survival, and they also improve growth rates. Smooth surfaces, e.g. polyethylene sheet, are unsuitable as a settlement substrate. The growth rates of T. gigm juveniles in this study were much lower than those recorded by Munro and Heslinga (1983) and Heslinga et al. (1984) for T. gigas over a similar period of time. This is probably because the water temperatures were much lower, decreasing to 18-20” C in the raceways in midwinter, compared with the 27-30°C recorded by Heslinga et al. (1984) in Palau. Beckvar (1981) ‘reared T. gigm juveniles in 27-33°C seawater and observed growth rates similar to those in this study, but her juvenile clams were maintained in heavy shade. Thus, in areas with substantial seasonal differences in water temperature, the best rearing strategy would appear to be to spawn the adults in spring-early summer, so that the juveniles can grow through the period with highest mortalities in the shortest period of time. Yamaguchi (1977) proposed that the early juvenile stage would present a problem to the mariculture of giant clams because of difficulties in handling the small sized newly-metamorphosed juveniles and because they are capable of crawling around the substrates. Beckvar (1981) also mentioned problems with algal overgrowth, whereas Heslinga and Watson (1985) have found that this is a potential problem only after harvesting of juveniles and it is controlled effectively by the addition of juvenile Troches niloticus. Large numbers of T. deram have been reared over the last 2 years by Heslinga and Watson in Palau and they consider that the commercial cultivation of T. derasa is now feasible, although this is still to be proven. The results of this study have shown that, at least for T. gigus on the Great Barrier Reef, further research is required on the factors affecting survival and growth of juveniles after metamorphosis and on techniques for large-scale rearing of juveniles. The methods typically used in commercial bivalve nurseries, e.g. layers of juveniles in upwelling systems, are not readily applicable to giant clams because of the light requirements of their symbiotic zooxanthellae. The acquisition of zooxanthellae, inorganic nutrient requirements and the effect of algal overgrowth on the survival and growth of juvenile clams, in particular, need further investigation. ACKNOWLEDGEMENTS
This research is part of an international project to culture giant clams, which is funded by the Australian Centre for International Agricultural Research. It is being conducted in collaboration with the International Giant Clam Mariculture Project, initiated by Dr. J.L. Munro. We are grateful to Rick Williams and Geoff Charles, station managers, for their assistance with facilities at Orpheus Island Research Station and to Stephane Westmore and Peter O’Meara for technical assistance.
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