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Larval biology of tridacnid clams
181
Aquaculture, (1984) 181~-195 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
LARVAL BIOLOGY
OF TRfDACNID
CLAMS
WiI_LIAM K. FITT, CHARLES R. FISHER, and ROBERT K. TRENCH Department of Biological Sciences and Marine Science Institute, University of California, Santa Barbara, CA 93106 (U.S.A.)
ABSTRACT Fitt. W.K., Fisher, Aqua&we.
CR.,
and
Trench,
R.K.,
1984.
Larval
biology
of
tridacnid
clams.
39: I8 l-195.
Previous attempts at the mariculture of tridacnid bivalves have emphasized parameters such as growth rates of metamorphosed spat and adults. Many of these studies agree that larval mortality in these clams is high, but there have been few attempts made to define culture conditions that would enhance larval survival. We have attempted to address aspects of the larval biology of Hippopus hippopus and Tridacnu gigas with an aim toward improving larval survival. We emphasize the effects of larval density and exogenous food (dissolved and particulate) supply on survival and growth of tridacnid larvae. The brief trochophore stage showed high mortality, but optimal survival was found at densities between 0.2 and 5 larvae/ml. Survival and growth rate of veligers were found to be optimal at densities of 0.2-10 larvae/ml. Control (unfed) veligers demonstrated high growth rates during the first half of the veliger stage, after which growth rates declined to zero, and mortality increased markedly. In contrast, veligers provided with particulate food (Z~oc~~sjs g~l~n~, Tahitian strain) had significantly higher growth rates (p < ,051 and lower mortality than the controls. Phaeoductylumtricornatum is not an effective food for tridacnid larvae. Optimal growth and survival of veligers fed I. gulbana occurred with algal densities of 104-l@ cells/ml. Dissolved organic nutrients in the form of vitamins and yeast extract significantly enhanced veliger growth and survival. Although all adult tridacnids possess symbiotic algae which contribute significantly to their nutrition, these algae are not passed from one clam generation to another. Under experimental conditions, veiigers take all strains of ~yrnbi~jffi~rnmier~driutic~m provided into their stomachs, but symbiosis is not established until after me~morphosis. A larval history of adequate nutrition may well enhance veliger growth, shorten the developmental time to metamorphosis, and improve growth after the establishment of symbiosis. INTRODUCTION
Tridacnid bivalves are currently the subject of mariculture efforts in the Indo-Pacific, mostly from the point of view of offsetting potential extinction as well as for their use as food (Jameson, 1976; Yamaguchi, 1977; Beckvar, 1981; Gwyther and Mum-o, 1981; Fitt and Trench, 1981; Heslinga er al., this volume). The commercial potential of the larger species is relativefy high because clam meat, especially the large adductor muscle, is considered a 0044-%4~6/84~$~3.00
0 1984 Elsevier Science Publishers B.V.
182
delicacy by the Japanese, Chinese, and Taiwanese (Gwyther and Munro, 19811, and now there are restaurants on the continental United States serving Tridacna meat. Tridacnid clams also form a significant portion of the protein in the diet of some populations in Micronesia and Polynesia (Maclean, 1975; Weingarten and Tuxson, 1977). In some areas of the Indo-Pacific, the larger species of tridacnids have been rendered virtually extinct (Hester and Jones, 1974; Bryan and McConnell, 1976; Pearson, 1977; Hirshberger, 1980). Tridacnid clams are an attractive mariculture subject because the juveniles and adults contain the symbiotic dinoflagellate Sym~~odin~umm~croadr~uticum, which contributes significantiy to the nutrition of the clam (Muscatine, 1967; Trench et al., 1981). Until reaching a size where they can be transpl~ted onto the reef, juvenile and small adult clams can be maintained in large tanks in natural illumination where they grow without food additions (Beckvar, 1981; Heslinga et al., this volume). As adult tridacnids do not pass their symbionts directly to their young (Stephenson, 1934; LaBarbera, 1975; Jameson, 1976; Fitt and Trench, 1981) the larvae or juveniles must acquire their complement of symbionts from the environment. The veliger larvae of Triducna squamosa are able to ingest S. microadriuticum into their stomachs, but a permanent symbiosis with this alga is not established until after metamorphosis when symbionts in juveniles somehow pass through the digestive gland and into the developing hemal sinuses of the hypertrophied siphon tissues (Fitt and Trench, 1981). Because the symbiotic algae do not appear to be the major source of nutrition of the larval forms, as they are in adults, basic questions concerning the nutrition of the larval clams become important, especially with respect to techniques for raising clams for mariculture purposes. Possible nutritional sources in nature include natural assemblages of phytoplankton (as the veligers are planktotrophic), suspended particles, and dissolved organic material (see Stephens and Manahan, this volume). Several studies have recorded very high mortality of the larval stages of tridacnid clams (LaBarbera, 1975; Jameson, 1976; Beckvar, 1981; Fitt and Trench, 1981, Gwyther and Munro, 19811, and many investigators place the emphasis in their mariculture attempts on the growth rates and survival of the metamorphosed juveniles (see Heslinga et al., this volume). Hence, very little is known about the nutritional requirements of the larval stages, In this study we have attempted to define optimum conditions for the culture of tridacnid larvae. We find that optimal densities of larvae and appropriate nutrition enhance the survival and growth rates of tridacnid larvae and may also have profound influence on growth of the metamorphosed juveniles. MATERIALS AND METHODS
Collection and maintenance of clams Adult specimens of Hippopus hippopus were collected from their habitats on
backreefs in the Republic of Belau, Western Caroline Islands and transported in containers of seawater to the Micronesia Mariculture Demonstration Center (~MDC~ on the island of Malakal where they were maint~ned in
183
12004iter concrete raceways under ambient conditions of temperature and insolation. The specimens of Tridacna gigas used in this study were the same large adults maintained by Heslinga et al. (this volume). Spa wnirlg oj’ clams For I-I. hippopus,
the spawning procedure began by stopping the water flow to the raceway and adjusting the water level to a height just above the top of the clams. When the temperature had risen about 2-3°C above ambient (2-3 h), fresh or frozen macerated gonad tissue was dispersed in the water as previously described by Fitt and Trench (1981). Spawning usually began within 5 min after the addition of gonads and followed the pattern described by Wada (1954) and Fitt and Trench (1981). About 50 ml of water containing sperm from one individual, collected early in the spawning period, was mixed in a 35-liter aquarium containing eggs later spawned by another individual. The fertilized eggs were sorted into containers of 0.45-,um filtered seawater (FSW). T. gigas spawned spontaneously. Trochophores and veligers were siphoned from raceways and sorted into containers of FSW. All experiments were conducted under laboratory conditions of 27 -t l”C, and a 14:10 h (light:dark) photoperiod. Photon flux density was 100 pE/m2/sec. ,C@cts oj‘density
on survival
and growth
of larvae
The effect of density on growth and survival of trochophores and veligers was tested for the larvae of H. h~~~o~~s. Six replicates of densities ranging from 0.2 to 30 aninlals/mi, and at least 2 replicates of 50 and 100 animaIs/ml were established in plastic petri dishes, Zsochrysis galbana (5 x 104/ml) was used as food for veligers. Survival between days 1 and 2 after fertilization was determined for trochophores with no food additions by counting the number of veligers in the cultures on day 2. Veliger survival was determined after 4 days at the experimental densities (on day 6) and after a water change and addition of I. ga~ba~a (5 x 104/ml} again on day 11. No aeration or agitation was provided in any of the experimental conditions. The size distribution of veligers on day 8 was determined. l$$x~s of‘ particulate and dissolved organic supplements
on larval
survival
and grow&
Various particulate and dissolved organic substances were added separately to culture media containing trochophores of H. h~~~o~z~sand T. gigas at densities of 3 animals/ml. Particulate material included 1. galbana (at concentrations of lo3 to lo6 cells/ml), and live yeast (5 x 104/ml). Dissolved organic substances provided were a vitamin mix (described by McLaughlin and Zahl, 1957, for the culture of S. microadriaticffm, but with putrescine omitted) and yeast extract (1 pglml). Combinations of vitamins, yeast extract, and I. ga~ba~a (the latter at 5 x 104/ml) were also employed. Controi larvae were maintained in FSW only. Survival of trochophores was assayed by the number of live and dead veligers observed in six replicate cultures after 36 h, each original1.y containing 30 trochophore larvae in 10 ml of medium.
184
The effect of food concentration on veliger growth and survival was investigated using only 1. gu~~~~~. Stationary phase I. gff~~~~?ffwere added to 2-day-old (day 4) cultures of H. ~~~~0~~sveligers (3 veligerslml) in FSW in IO-ml plastic petri dishes. Densities of algae tested were 103-lo6 ceils/ml. Controls were maintained in FSW. Survival and growth were monitored and new FSW and food added at approximately 3-day intervals. Survival was measured as number of animals alive out of the original 30 present on day 4, and expressed as the mean percentage of six replicates. Growth was calculated as the mean daily increment from a minimum of I8 measurements made on animals from the six replicate dishes. Survival and growth of veligers fed the three phytoplankton species 1. tricornatum and all subcordijtirmis, Phaeodactylum galbana, Plaiymonas combinations of these species was investigated using If. ~~~~0~~~larvae. Six replicate IO-ml plastic petri dishes, each containii~g 30 veligers in FSW and fed a total concentration of 5 x lo4 algae/ml were monitored for each condition. Controls were maintained in FSW. Water changes, new food additions, and measurements were made at 2- to 3-day intervals. The effect of vitamins and yeast extract (1 yglml) on growth and survival of veligers was tested in the same set of experiments. Acquisirion qf symbiofic algae Cultured S. microadriaticum, isolated from the anemones Aiptasia tagetes (strain A), Zoanthus sociatus (strain Z), Heteractis lucida (strain H), Cassiopeia xamachana (strain C), T. gigas (strain T), and H. hippopus (unidentified strain) were added to petri dishes containing pediveligers of H. h~ppopusor T. gigas. In addition motile cells of A~hjd~n~urn klebsii isolated from a flatworm tentatively identified as Amphiscolops lungerhansi, found in Flatworm Lake on the Island of Koror in Belau, were introduced to cultures of pediveligers of H. hippopus. Microscopic observation of the veligers and metamorphosed juveniles was done periodically. RESULTS
Spawning of adult clams and behavior of larvae During the period July 10 to September 15, 1982, H. hippopus spawned sperm on 10 different occasions when exposed to macerated gonads, but only spawned eggs twice. Both egg-spawning events occurred in the late afternoon (1500-1900 h), within a week after the new moon. These observations are consistent with the summer spawning for this species observed by Stephenson (19341, Jameson (19761, Yamaguchi (19771, Beckvar (1981), and Gwyther and Munro (1981). Tridacna gigus spontaneously spawned both eggs and sperm in the late afternoon about a week after a new moon on July 27-28 and August 26-27 (see Heslinga et al., this volume). These observations compare favorably with assays of gonad condition and observations of natural spawning of T. gigas on the Great Barrier Reef, suggesting a summer breeding season for this species (R.D. Braley, personal communication, 1982).
185
Fertilized eggs of H. hippopus remained on the bottom of containers through the ciliated gastrula stage. Trochophore larvae became free-swimming 16-20 h after fertilization, and actively swam at the surface. They did not appear to orient to light. Veligers first appeared 24 h after fertilization, and within 48 h all larvae had reached the veliger stage of development. Two- to three-day-old veiigers actively swam throughout the medium, whereas 4- to 9-day-old veligers were observed most often at the surface, their velums sometimes flush with the surface film. This behavior was not a function of larval density (lack of oxygen), as larvae at the lowest densities demonstrated this behavior. Pediveligers developed 9-14 days after fertilization and alternately crawled on the bottom by extension and retraction of the foot, or swam just off the bottom. Metamorphosis to juveniles occurred 9-13 days after fertilization in the first batch (day 4 veliger size = 187.5 pm f 6.3 sd., n = 201, and 13-18 days after fertilization in the second batch of spawned eggs (day 2 veliger size = 170.5 pm & 4.3 s.d., n = 15). Influence of’ larval density on survival
and growth
Fig. 1 shows that trochophore survival was greater at low densities (0.2-5 larvae/ml) than at higher (lo-100 larvae/ml) densities. Similarly, veliger survival was greater at low densities. Measurement of the size distribution of 8-day-old veligers showed (Table I) that animals maintained at low densities were significantly (p < .05) larger than those grown at higher densities. Influence
qf’added
particulate
and dissolved
organic
nutrients
on swvival
and growth
of larvae Survival
of trochophore
larvae
The addition of particulate or dissolved organic nutrients to cultures of trochophores of either H. hippopus or T. gigas had no obvious effect on the enhancement of survival (Table II). All treatments, with the exception of high densities of I. galbana, resulted in survival values that were insignificantly different from untreated controls. Trochophores of T. gigas exposed to high densities of I. galbana demonstrated significantly lower survival than controls. Microscopic observations of the trochophores showed no evidence that the algae were being consumed. Survival
and ,rrowtll
qf’ tleliger larvae
To determine the influence of particulate food on survival and growth, veligers of H. hippopus were provided with I. galbana at increasing densities ranging from lo3 to lo6 cells/ml. Control veligers were maintained without food. Table III shows that the highest survival of veligers through metamorphosis (day 13 in this experiment) was found among those fed I. galbana at densities between lo3 and lo5 cells/ml. During the early veliger stage (days 4-9), growth was optimal at densities of 1. galbana of 104-10’ cells/ml. During the later veliger stages, optimum growth was observed at lo5 cells/ml. Veligers characteristically demonstrated higher growth rates during
Fig. 1. Effect of density on survival of trochophores (Cl), day 6 veligers (e), and day II veligers (0) of H. hippoptis. Bars represent 95% confidence intervals. The number of replicates is indicated next to each point. See text for details. TABLE I Effect of density of larvae on growth of veligers of ~~~0~~~~~iip~~pu~ Density (veligers/mt) 0.2 1 5 10 30 50 100
Size km> 191.9 z!z4.3 195.8 i 4.2 192.2 -c 3.9
(101 (IOf (10)
190.9 185.4 185.6 184.1
(14) (14) (20) (20)
* f + k
4.2 2.7 3.7 2.8
“Numbers are means f 95% confidence intervals of &day-old veligers
the early stages, growth becoming progressively metamorphosis. In another experiment using a different batch hippopus at densities of 3 animals/ml were provided subcordiformis, and Phaeodactylumtricornatumsingly,
slower with approaching of spawn, veligers of H. with I. galbana, Plalymonas in pairs, or all together.
187
TABLE II Effect of additions
of various dissolved
and particulate
“foods” on survival and development
of
into veligers for Tridacna gigas and Hippopus hippop&
trochophores
Survival
Addition 1. galbana
Hippopus hippoprrs
Triducna gigas
1.3 f
103/ml
104/ml
(VI)
3.3
9.3 Ik 5.2
11.7 + 3.0
9.0 f
7.0
5x104/ml
6.8 f
1.8
lO’/ml
6.3 f
2.4
17.3 +
9.8
106/ml
5.3 + 1.6b
16.6 f
8.1
Vitamin and yeast extract
8.3 f
11.0 f
3.5
Vitamin
5.8 k 3.3
11.7 +- 4.9
Yeast extract
5.8 -t 2.7
15.0 +
17.3 + 10.2
1.1
9.1
Live yeast
6.7 + 1.5
12.3 f
8.1
Vitamin and yeast extract + 1. gulbuna (5x104/ml)
6.5 f
2.2
6.7 k
6.3
Control
9.0 * 2.2
9.0 f
4.2
aValues are the mean percentage of trochophores (k 95% confidence intervals) reaching the veliger stage from six replicate samples, 2.5 days after fertilization. See text for details. bSignificantly (p < .05) different from controls.
TABLE III Effect of different densities of lsoch~wis ga//xwa on growth and survival of veligers of Hppopus I
Density
”
Growth
(pm/day)
days 4-9
(cells/ml)
/~q~popusl
Survival (‘XI)
days 9- 13
day 4
day 6
day 9
day 13
0 (control)
20
100
19.2 f 26.2
60. I k36.2
lX.lk23.6
I03
22
0
0.13kO.17
100
R2.3k 18.9
69.2-1- 17.0
565t336b
104
1x
0.60k0.13b
0.13+-0.17
100
83.0*
9.x
62.2t31.9
42.St
67 Rt 10.6b
0.22kO.14
0
IO5
1R
0.8X*O.l4b
0.50+-o 19b
100
73.0*
11.2
70.0t
106
18
0.76+0.14b
0.05+0.15
100
17.3k
5.1
42.5-+33.2
“Numbers represent means + 9% confidence intervals; data are from six replicates. See text for details. “Significantly (p < .05) different from controls.
Dissolved
extract.
organic
nutrients
In this experiment,
n. number
97
of clams measured
14.0b
38.X-+35 2 for growth
Surwval
provided in the form of vitamins and yeast metamorphosis was complete after 18 days.
were
expressed as pm/day
2.64-t0.29 (22)
Control
Srowth
3.42~0.20
Vitamin + yeast extract
-
(14) (121 (19) (24)
0.58kO.18 0.57t0.23 0.7720.16 1.13+0.16
(19) (28) (12)
0.4720.14 0.90+0.13 0
1.02+0.15 (18)
(201
(t-t)
1.03t0.19
Days 8-13
& 95% confidence intervals.
(45)
2.56-+0.20 (201
(25) (241 (23) (28)
z.g. + P.S. + P.t.
P.S. + P.t.
1.16kO.22 1.76kO.23 0.80+0.22 1.28+0.19
2.50&0.26 (26) 1.90+0.21 (28)
I. galbana I? subcordifarmis P. tricornatum I.g. + P.S. I.g. + P.t.
(n)
Days 3-8
Food
0
0.36+0.16
0
0 0.46kO.26 0 0 0.70+0.15 0.2OkO.27
(4)
(20)
(6) (16) (151 (15)
(18) (9)
Days 13-18 (t-t)
(n>
180.9
194.8
1.62 ~~~0.03(20) 0.69-eO.09 (4)
186.7
186.6 185.4 177.8 183.9 184.0 184.0
Mean Iength (pm, day 18)
1.08+-0.04 (11)
1.07+-0.07 (18) 0.991to.04 (91 0.49kO.05 (6) 0.89z!zO.O4(16) 0.90-r-0.05 (15) 0.9OrtO.06 (15)
Days 3-18
Effect of phytoplankton “food” species and dissolved nutrients on growth of veligers of Hippopus hippop&
TABLE IV
189
Regardless of treatment, veligers grew most rapidly during the early stages, growth rates declining through the middle veliger stage and the onset of metamorphosis (Table IV). Unfed control veligers ceased growing after the first 8 days, and only 5% of the veligers survived to metamorphosis. In contrast, Table IV shows that all of the veligers provided with either particulate or dissolved organic matter, with the exception of those provided with P. tri~,o~r~a~L~nl~ showed enhanced growth and survival. Maximum sizes of metamorphosed juveniles were found among those that had been provided with vitamins and yeast extract, and with 1. ~~lb~~ff. Veliger survival (Fig. Za,b) was approximately five times higher than controls when provided with vitamins and yeast extract or with I. ~Q/~Q~IQ,alone or in combination with either P. subcordiformis or P. fricorrzatrm~. Veliger survival was lower when veligers were provided with either P. sabcord(ftirmis or P. tricornatum alone. Light-microscopic observations of veligers provided with the three algal species showed that i. galbana and P. sabcord0vri.s were consumed, that is, the algal cells were observed in the digestive tract. In contrast, P. tt+cornanrmwere not consumed.
The process of acquisition of algal symbionts by larvae of T. yigas and Fi. hippoptis was the same as that previously described (Fitt and Trench. 1981) for T. squamosu. As in other tridacnids and another symbiotic bivalve Corcaium cardissa (Kawaguti, 19681, the symbiotic algae are not passed from one clam generation to another, and as a result, symbiotic algae were not observed in the eggs or any of the larval stages of T. gigas or H. hippopus when maintained in FSW. This is consistent with the observations of LaBarbera (19751, Jameson (19761, and Fitt and Trench (1981), but is in contrast to the report of Gwyther and Munro (1981). All strains of S. rn~croudrjar~c~rn provided were taken into the stomachs of the pediveiigers of H. hippop~s and T. gigas within 24 h of their introduction to the larval cultures (cf. Fitt and Trench, 1981). The algae remained in the stomachs of pediveligers and newly metamorphosed clams, and about 7 days after metamorphosis could be seen in rows extending from the region of the stomach and digestive gland toward the developing siphonal tissues (Fig. 3). This observation is identical to that seen in the juveniles of T. squamosa (Fitt and Trench, 1981). Although under laboratory conditions the juveniles of tridacnids may establish a symbiosis with all strains of S. microadriaticum so far tested, they do not accept another symbiotic dinoflagellate, Amphidini~m klebsii. Finally, the veligers of T. gigus and H. hippopus do metamorphose to juveniles in the absence of symbiotic algae and phytoplankton food (cf. Gwyther and Munro, 1981) as shown in Figs. 2 and 3 and Table III. DISCUSSION
In the past, most studies of the mariculture of tridacnid bivalves have emphasized aspects of growth and survival of metamorphosed juveniles
a
Age
(days)
b
Age
(days)
Fig. 2. Survival of veligers of H hippopus fed (a) vitamins and yeast extract CX), I. galbana, (0). P. subcord@rmis (Cl), P. tricornatum (AI and (b), I. gaibana plus P. tricornatum (O), I. galbana plus P. subcordlformis (X), P. subcordiformis plus P. tricornatum (U), and I. galbana, P. subcordiformis, plus P. tricornatum (A). Unfed controls (*I. Bars represent 95% confidence intervals. See text for details.
19
Fig. 3. Juveniles of H. hippopus about 2 weeks after metamorphosis. Clam on the left contains S. microadriuricum (z), but the clam on the right, maintained throughout larval development in vitamins and yeast extract does not have zooxanthellae. Dark bodies (db), found in all juveniles, are not zooxanthellae. st, Statocyst. Bar = 50pm. See text for details.
(Yamaguchi, 1977; Beckvar, 1981; Heslinga et at., this volume). These and other studies CLaBarbera, 1975; Jameson, 1976; Fitt and Trench, 1981) have shown that at a given spawn, tridacnids release large numbers of eggs, but less than 1% survive larval development. Therefore, from the point of view of mariculture, it seems important to determine conditions of larval rearing that
would reduce the high larval mortality that appears to be characteristic of these organisms. Compared to the life span of adults (Comfort, 1957; Bonham, 1965; McMichael, 1974; Yamaguchi, 1977), tridacnids spend a brief but critical period as larvae (Fig. 4). Development from trochophore to veliger is usually complete within 24-48 h. There is no evidence that trochophores are capable of particulate feeding. The veliger stage lasts from 1 to 3 weeks depending on the species and culture conditions, and vetigers are demonstrable planktotrophs. Veliger growth rates are highest during the early stages, growth rates becoming progressively lower with the onset of metamorphosis (see Jameson, 1976; Gwyther and Munro, 1981). It is evident that the highest mortality occurs during the transitions from trochophore to veliger and from pediveliger to juveniles. The high mortality at these transitions may partly be due to changes in the mode of acquiring nutrition. As these clams do not establish a nutritional symbiosis with Sjmbiodinium microadriaficum until after metamorphosis, their larval stages require a nutritional input from exogenous sources, and in this respect, the larval biology would trot be expected to be very different from that of nonsymbiotic clams. Thus, until trida~nid larvae are capable of feeding, they must rely on stored nutrients provided with the eggs. If, under laboratory conditions, no exogenous nutrients are provided, the clams would have to rely on stored nutrients throughout their entire larval existence. The amounts and quality of stored nutrients in the eggs of tridacnid bivalves are unknown.
192
-Id
:..*.
5-8 doys
:: :
3-8
days
<5 days
I veliger
pediveliger
metamorphosis
.a.*’, -IOh ,8&h.
Fig. 4. Representative
timetable
of the developmental
stages of H. hippopm.
The major goals of this study were to determine conditions of larval culture that would reduce the high mortality usually encountered in the mariculture of tridacnid clams (see Heslinga et al., this volume). Our results show that we have achieved a four- to fivefold increase in survival in larvae reared at low densities and fed, when compared to controls. We had little success in reducing the high mortality levels among trochophores. Despite all attempts, trochophore mortality remained at least 80%. It is possible that bacterial contamination may be responsible for the high mortality. Future judicious use of antibiotics may help to improve this situation. The optimum conditions for the culture of veligers appear to be maintenance at low densities and provision of an appropriate source of nutrition. Veligers maintained at densities less than lo/ml showed optimal survival and growth. The high mortality (80-90%) of veligers at high densities may have resulted either because of the accumulation of waste products (e.g. NH,+) or a lower quantity of food per veliger being available. That the latter explanation is less tenable gains support from our observation that control unfed veligers at low densities demonstrated only 30-400/o mortality after 6 days (see Fig. 2a). Mean growth rates for the entire veliger period were greater for all fed veligers (except those fed P. tricornatutn) than for unfed controls. The highest growth rates were found in veligers fed vitamins and yeast extract. Low growth rates of veligers provided with P. tricornatum are consistent with our observation that these diatoms were never seen in the digestive tracts of veligers and were probably not being used as food. Similarly, the largest recently metamorphosed juveniles (Table IV) were among those that had been provided with an exogenous food supply (with the same exception as mentioned earlier). However, growth increments throughout the veliger stages were not constant. Our data are consistent with those of Jameson (1976) and Gwyther and Munro (1981) showing a gradual decrease in daily growth rates, such that under most conditions, clams undergoing metamorphosis grow very little if at all. Our data on the apparent depressed growth rates of veligers fed P. tricornatutn and P. cubcordiformis (either singly or in combination), as compared to controls, in the early veliger stages, are difficult to interpret. Toxic and growth-inhibiting substances released from sotne species of algae have been
193
suggested as being responsible for similar phenomena in cultures of veligers of other bivalves (see Loosanoff and Davis, 1963). Apparently, P. tricornatutn may occur in four morphologically distinct forms (Walne, 19631, and some forms have been found to be inadequate food for other bivalves as well (Epifanio, 1975). The data we have presented show that the veligers of H. hippopus survived and grew best when provided with vitamins and yeast extract. This would imply that the veligers may be able to utilize dissolved organic nutrients (see Stephens and Manahan, this volume), although our cultures were not axenic. Because we employed both of these substances in combination, it would be important to determine which one produced the observed effects. Studies of other bivalve larvae (Loosanoff and Davis, 1963) have shown that when parameters such as feeding regimes or temperature are manipulated, growth rates and developmental times to metamorphosis also change, but the size of veligers at metamorphosis tends to remain the same for a given species. Preliminary data indicate that the larvae of H. hippopus metamorphose at a length of 185-I 95 pm. The effect of an exogenous food supply appears to be to increase veliger growth rates and hence perhaps, shorten the time to metamorphosis. Although our data intervals are not close enough to show, this might be the reason we observed increased survival at metamorphosis of fed veligers when compared to unfed controls. It is apparent from our experiments and those of Gwyther and Munro (1981) that larvae of different spawns can be of markedly different size. A possible reason for this may be that eggs of different spawns (especially “induced” spawns) contain very different proportions of stored nutrients which may be reflected in egg size. We hypothesize that those eggs with larger nutrient stores would grow faster, survive longer, and give a larger probability of attaining metamorphosis than eggs with less stored nutrients. Our data, showing that many more starved control veligers from the group of larger veligers survived through metamorphosis (Table III) than the group of smaller veligers (Fig. 21, and that growth of unfed veligers stopped halfway through the veliger stage, are consistent with this hypothesis. The failure of some “spontaneously” spawned batches of tridacnid larvae to yield reasonable quantities of viable spat (G. Heslinga, personal communication, 1982) may be explained by this hypothesis. Adult tridacnid clams maintain the symbiotic dinoflagellate S. microatlriaticrrm in hemal spaces of their hypertrophied siphon tissues as well as the digestive gland and stomach, where they are thought to provide significant contribution to their host’s nutrition (Yonge 1936, 1980; Muscatine, 1967; Trench et al., 1981). However, the symbionts are not inherited by the offspring and must be acquired from the environment by the larval or juvenile clams. No eggs from any species have ever been seen with symbiotic algae (Stephenson, 1934; LaBarbera, 1975; Jameson, 1976; Fitt and Trench, 1981), and no confirmed observations of trochophores or veligers with ,symbiotic algae in their tissues have been reported (cf. Gwyther and Munro, 1981). The larval forms of the tridacnid clams are apparently unable to establish a syrnbiosis with S. tnicroadriaricrrtn until after metamorphosis. We have shown here for T. gigas and H. hippopus and previously for T.
194
squurnosa (Fitt and Trench, 1981) that veligers are capable of ingesting all strains of S. microadriaticum offered, especially the motile forms which are more likely to be encountered by the veligers. These algae are taken into the stomach where they apparently contribute to larval nutrition as made evident by the fact that veligers of T. squamosa “fed” different strains of S. microadriaticum had higher survival rates than controls (Fitt and Trench, 1981). It is not known if the veligers digest live and/or dead S. microadriaticum, or receive photosynthetic products released by live S. microadriaticum while they are in the stomach. Intact algae are often seen passing out of the intestine of veligers fed S. microadriaticum and in many cases veligers having S. rnicroadriuticum in their stomachs are seen at a later time with no algae in their stomachs. Only after metamorphosis are S. microadriaticum seen in tubules extending from the posterior portion of the digestive gland anteriorly into the developing siphon tissues. At this point in the development of the clam, a symbiosis has been established (cf. Fitt and Trench, 1981). There is apparently a mechanism of recognizing and establishing a symbiosis with only this species of algae, because the symbiotic dinoflagellate Amphidinium klebsii isolated from Palauan flatworms, and numerous free-living phytoplankton species which may be taken into the gut did not establish a symbiotic relationship in postlarval clams. The establishment of symbiosis with S. microadriuticum is not essential for metamorphosis in tridacnids (Fig. 3). This is in contrast to the situation demonstrated by some coelenterates such as the jellyfishes Mustigias papua (Sugiura, 1964) and Cassiopeia xumachana (Trench et al., 1981). ACKNOWLEDGMENTS
We would like to thank the entire staff Demonstration Center, Republic of Belau, visits. We especially appreciate experimental and E. Oiterong and help in obtaining Ngiramengioir, and 0. Orak. We also thank This study was supported by the Agency for DPE-5542-G-SS-1085 to R.K. Trench).
of the Micronesian Mariculture for their cooperation during our help from S. Derbai, D. Fisher, animals from J. Heslinga, M. M. Jess for graphical assistance. International Development (grant
REFERENCES Beckvar,
N., 1981. Cultivation, spawning, and growth of the giant clams Tridactta gigus, T. derasn, and T. squamosa in Palau, Caroline Islands Aquaculture, 24:21-30 Bonham, K., 1965. Growth rate of giant clam Tridanca gigas at Bikini Atoll as revealed by
radioautography. Science, 149:300-303. Bryan, P.G. and McConnell, D.B., 1975. Status of giant clam stocks (Tridacnidael on Helen Reef, Palau, Western Caroline Islands, April 1975. Mar. Fish. Ref., 38:15-l& Comfort, A., 1957. The duration of life in mollusks. Proc. Malacologica! Sot. Land., 32:219241. Epifanio, C.E., 1976. Culture of bivalve mollusks in recirculating systems: nutritional requirements. In: KS. Price, W.N. Shaw and K.S. Danberg (Editors), Proc. First Int. Conf. Aquaculture Nutrition. U. Delaware Press. Fitt, W.K. and Trench, R.K., 1981. Spawning, development, and acquisition of zooxanthellae by Triducna squamosa (Mollusca, Bivalvial. Biol. Bull., 161:213-235.
195
Gwyther, J. and Munro. J.L.. 1981. Spawning induction and rearing of larvae of tridacnid clams (Rivalvia: Tridacnidael. Aquaculture, 24:197-217. Hirschberger, W., 1980. Tridacnid clam stocks on Helen Reef, Palau. Western Caroline Islands.
Mar. Fish. Rev., 42:8-15. Hester, F.J. and Jones, E.C.. 1974. A survey of giant clams, Tridacnidae, Western Pacific Aroll. Mar. Fish. Ref., 3617-22.
on Helen
Reef, a
Jameson, SC., 1976. Early life history of the giant clams Tririuctrn c’,‘oc’ea Lamarck, ~~;~~ff~,~z~ ~~~~~.~/~~?ff (Rodingl and ~i~/~~p~{s ~~j~~~~~~s (L~nn~eus). Pacific Science, 30:2 19-233. Kawaguti, S., 1968. Electron microscopy on zooxantheliae in the mantle and gill of the heart shell. Biol. J. Okayama Univ.> 14:1-I2. LaBarbera, M.. 1975. Larval and post larval development of the giant clams T?i&c!ra ~rr~\-inrn and Triducrra sylrar?zosa (Bivalvia: Tridacnidae). Malacologia, 15:69-79. Loosanoff. V.L. and Davis, H.C., 1963. Kearing of bivalve mollusks, In: F.S. Russel (Editor). Advances in Marine Biology, Vol. 1, p, I-136. Maclean, J.K., 1975. The Potential of Aquaculture in Australia. Australian Govt. Publ. Servtce. Canberra. McLaughlin. J.J.A. and Zahl, P.A., 1957. Endozoic algae. In: S.M. Henry (Editor), Symbiosis, Vol. 1, pp. 257-297. Academic Press, N.Y. McMichael, D.F., 1974. Growth rate, population size, and mantle coloration in the small giant clam ~TMxm ~2~~;~~~(Rodingl, at One Tree Island, Capricorn Group. Queensland. Proc. Second Int. Coral Reef Symp., Brisbane, I :241-254. Muscatine, L., 1967. Glycerol excretion by symbiotic algae from corals and Trif~at,i?uand its control by the host. Science, 156:516-519. Pearson, R.G., 1977. Impact of foreign vessels poaching giant clams. Austrdhan Fisheries. 7% 23. Stephenson, A. 1934. The breeding of reef animals. Part II. Invertebrates other than corals. Sci. Repts. Great Barrier Reef Exped., 3:247-272. Sugiura, Y., 1964. On the life history of rhizostome medusae. II. Indispensibility of zooxanthellae for strobilation in A4asti~;:inspop,ua. Embriologica, 8:223.233. Trench. R.K.. Colley, N.J., and Fitt, W.K., 1981. Recognition phenomena in symbioses between marine invertebrates and zooxanthellae: uptake. sequestration, and persistence. Ber. Deutsch Bot. Ges. Bd., 94529-545. Trench. R.K., Wethey, D.S., Porter, J.W., 1981. Some observations on the symbiosis with tooxanthellae among the tr~dacnidae ~Mollusca, Bavalvial. Biol. Bull., 161:180-198. Wada, S.K., 1954. Spawning in the tridacnid clams. Jap. J. Zooi.. 11:273-285. Walne, P.R., 1963. Observations on the food value of seven species of algae to the larvae of Ostreu e&k. J. Mar. Bioi. Ass. U.K., 43:767-784. Weingarten, R. and Tuxson, R., 1977. The giant clam. Mar. Aqua&, 8:32-40. Yamaguchi, M., 1977. Conservation and cultivation of giant clams in the tropical Pacific. Biol. Conserv., 11:13-20. Yonge, CM., 1936. Mode of life, feeding, digestion and symbiosis with zooxanthellae in the Tridacnidae. Sci. Repts. Gr. Barrier Reef Exped., 1:283-321. Yonge, CM., 1980. Functional morphology and evolution in the tridacnidae (Mollusca: Bivalvia: Cardiaceal Records Australian Museum, 33:735-777.
Aquaculture, (1984) 181~-195 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
LARVAL BIOLOGY
OF TRfDACNID
CLAMS
WiI_LIAM K. FITT, CHARLES R. FISHER, and ROBERT K. TRENCH Department of Biological Sciences and Marine Science Institute, University of California, Santa Barbara, CA 93106 (U.S.A.)
ABSTRACT Fitt. W.K., Fisher, Aqua&we.
CR.,
and
Trench,
R.K.,
1984.
Larval
biology
of
tridacnid
clams.
39: I8 l-195.
Previous attempts at the mariculture of tridacnid bivalves have emphasized parameters such as growth rates of metamorphosed spat and adults. Many of these studies agree that larval mortality in these clams is high, but there have been few attempts made to define culture conditions that would enhance larval survival. We have attempted to address aspects of the larval biology of Hippopus hippopus and Tridacnu gigas with an aim toward improving larval survival. We emphasize the effects of larval density and exogenous food (dissolved and particulate) supply on survival and growth of tridacnid larvae. The brief trochophore stage showed high mortality, but optimal survival was found at densities between 0.2 and 5 larvae/ml. Survival and growth rate of veligers were found to be optimal at densities of 0.2-10 larvae/ml. Control (unfed) veligers demonstrated high growth rates during the first half of the veliger stage, after which growth rates declined to zero, and mortality increased markedly. In contrast, veligers provided with particulate food (Z~oc~~sjs g~l~n~, Tahitian strain) had significantly higher growth rates (p < ,051 and lower mortality than the controls. Phaeoductylumtricornatum is not an effective food for tridacnid larvae. Optimal growth and survival of veligers fed I. gulbana occurred with algal densities of 104-l@ cells/ml. Dissolved organic nutrients in the form of vitamins and yeast extract significantly enhanced veliger growth and survival. Although all adult tridacnids possess symbiotic algae which contribute significantly to their nutrition, these algae are not passed from one clam generation to another. Under experimental conditions, veiigers take all strains of ~yrnbi~jffi~rnmier~driutic~m provided into their stomachs, but symbiosis is not established until after me~morphosis. A larval history of adequate nutrition may well enhance veliger growth, shorten the developmental time to metamorphosis, and improve growth after the establishment of symbiosis. INTRODUCTION
Tridacnid bivalves are currently the subject of mariculture efforts in the Indo-Pacific, mostly from the point of view of offsetting potential extinction as well as for their use as food (Jameson, 1976; Yamaguchi, 1977; Beckvar, 1981; Gwyther and Mum-o, 1981; Fitt and Trench, 1981; Heslinga er al., this volume). The commercial potential of the larger species is relativefy high because clam meat, especially the large adductor muscle, is considered a 0044-%4~6/84~$~3.00
0 1984 Elsevier Science Publishers B.V.
182
delicacy by the Japanese, Chinese, and Taiwanese (Gwyther and Munro, 19811, and now there are restaurants on the continental United States serving Tridacna meat. Tridacnid clams also form a significant portion of the protein in the diet of some populations in Micronesia and Polynesia (Maclean, 1975; Weingarten and Tuxson, 1977). In some areas of the Indo-Pacific, the larger species of tridacnids have been rendered virtually extinct (Hester and Jones, 1974; Bryan and McConnell, 1976; Pearson, 1977; Hirshberger, 1980). Tridacnid clams are an attractive mariculture subject because the juveniles and adults contain the symbiotic dinoflagellate Sym~~odin~umm~croadr~uticum, which contributes significantiy to the nutrition of the clam (Muscatine, 1967; Trench et al., 1981). Until reaching a size where they can be transpl~ted onto the reef, juvenile and small adult clams can be maintained in large tanks in natural illumination where they grow without food additions (Beckvar, 1981; Heslinga et al., this volume). As adult tridacnids do not pass their symbionts directly to their young (Stephenson, 1934; LaBarbera, 1975; Jameson, 1976; Fitt and Trench, 1981) the larvae or juveniles must acquire their complement of symbionts from the environment. The veliger larvae of Triducna squamosa are able to ingest S. microadriuticum into their stomachs, but a permanent symbiosis with this alga is not established until after metamorphosis when symbionts in juveniles somehow pass through the digestive gland and into the developing hemal sinuses of the hypertrophied siphon tissues (Fitt and Trench, 1981). Because the symbiotic algae do not appear to be the major source of nutrition of the larval forms, as they are in adults, basic questions concerning the nutrition of the larval clams become important, especially with respect to techniques for raising clams for mariculture purposes. Possible nutritional sources in nature include natural assemblages of phytoplankton (as the veligers are planktotrophic), suspended particles, and dissolved organic material (see Stephens and Manahan, this volume). Several studies have recorded very high mortality of the larval stages of tridacnid clams (LaBarbera, 1975; Jameson, 1976; Beckvar, 1981; Fitt and Trench, 1981, Gwyther and Munro, 19811, and many investigators place the emphasis in their mariculture attempts on the growth rates and survival of the metamorphosed juveniles (see Heslinga et al., this volume). Hence, very little is known about the nutritional requirements of the larval stages, In this study we have attempted to define optimum conditions for the culture of tridacnid larvae. We find that optimal densities of larvae and appropriate nutrition enhance the survival and growth rates of tridacnid larvae and may also have profound influence on growth of the metamorphosed juveniles. MATERIALS AND METHODS
Collection and maintenance of clams Adult specimens of Hippopus hippopus were collected from their habitats on
backreefs in the Republic of Belau, Western Caroline Islands and transported in containers of seawater to the Micronesia Mariculture Demonstration Center (~MDC~ on the island of Malakal where they were maint~ned in
183
12004iter concrete raceways under ambient conditions of temperature and insolation. The specimens of Tridacna gigas used in this study were the same large adults maintained by Heslinga et al. (this volume). Spa wnirlg oj’ clams For I-I. hippopus,
the spawning procedure began by stopping the water flow to the raceway and adjusting the water level to a height just above the top of the clams. When the temperature had risen about 2-3°C above ambient (2-3 h), fresh or frozen macerated gonad tissue was dispersed in the water as previously described by Fitt and Trench (1981). Spawning usually began within 5 min after the addition of gonads and followed the pattern described by Wada (1954) and Fitt and Trench (1981). About 50 ml of water containing sperm from one individual, collected early in the spawning period, was mixed in a 35-liter aquarium containing eggs later spawned by another individual. The fertilized eggs were sorted into containers of 0.45-,um filtered seawater (FSW). T. gigas spawned spontaneously. Trochophores and veligers were siphoned from raceways and sorted into containers of FSW. All experiments were conducted under laboratory conditions of 27 -t l”C, and a 14:10 h (light:dark) photoperiod. Photon flux density was 100 pE/m2/sec. ,C@cts oj‘density
on survival
and growth
of larvae
The effect of density on growth and survival of trochophores and veligers was tested for the larvae of H. h~~~o~~s. Six replicates of densities ranging from 0.2 to 30 aninlals/mi, and at least 2 replicates of 50 and 100 animaIs/ml were established in plastic petri dishes, Zsochrysis galbana (5 x 104/ml) was used as food for veligers. Survival between days 1 and 2 after fertilization was determined for trochophores with no food additions by counting the number of veligers in the cultures on day 2. Veliger survival was determined after 4 days at the experimental densities (on day 6) and after a water change and addition of I. ga~ba~a (5 x 104/ml} again on day 11. No aeration or agitation was provided in any of the experimental conditions. The size distribution of veligers on day 8 was determined. l$$x~s of‘ particulate and dissolved organic supplements
on larval
survival
and grow&
Various particulate and dissolved organic substances were added separately to culture media containing trochophores of H. h~~~o~z~sand T. gigas at densities of 3 animals/ml. Particulate material included 1. galbana (at concentrations of lo3 to lo6 cells/ml), and live yeast (5 x 104/ml). Dissolved organic substances provided were a vitamin mix (described by McLaughlin and Zahl, 1957, for the culture of S. microadriaticffm, but with putrescine omitted) and yeast extract (1 pglml). Combinations of vitamins, yeast extract, and I. ga~ba~a (the latter at 5 x 104/ml) were also employed. Controi larvae were maintained in FSW only. Survival of trochophores was assayed by the number of live and dead veligers observed in six replicate cultures after 36 h, each original1.y containing 30 trochophore larvae in 10 ml of medium.
184
The effect of food concentration on veliger growth and survival was investigated using only 1. gu~~~~~. Stationary phase I. gff~~~~?ffwere added to 2-day-old (day 4) cultures of H. ~~~~0~~sveligers (3 veligerslml) in FSW in IO-ml plastic petri dishes. Densities of algae tested were 103-lo6 ceils/ml. Controls were maintained in FSW. Survival and growth were monitored and new FSW and food added at approximately 3-day intervals. Survival was measured as number of animals alive out of the original 30 present on day 4, and expressed as the mean percentage of six replicates. Growth was calculated as the mean daily increment from a minimum of I8 measurements made on animals from the six replicate dishes. Survival and growth of veligers fed the three phytoplankton species 1. tricornatum and all subcordijtirmis, Phaeodactylum galbana, Plaiymonas combinations of these species was investigated using If. ~~~~0~~~larvae. Six replicate IO-ml plastic petri dishes, each containii~g 30 veligers in FSW and fed a total concentration of 5 x lo4 algae/ml were monitored for each condition. Controls were maintained in FSW. Water changes, new food additions, and measurements were made at 2- to 3-day intervals. The effect of vitamins and yeast extract (1 yglml) on growth and survival of veligers was tested in the same set of experiments. Acquisirion qf symbiofic algae Cultured S. microadriaticum, isolated from the anemones Aiptasia tagetes (strain A), Zoanthus sociatus (strain Z), Heteractis lucida (strain H), Cassiopeia xamachana (strain C), T. gigas (strain T), and H. hippopus (unidentified strain) were added to petri dishes containing pediveligers of H. h~ppopusor T. gigas. In addition motile cells of A~hjd~n~urn klebsii isolated from a flatworm tentatively identified as Amphiscolops lungerhansi, found in Flatworm Lake on the Island of Koror in Belau, were introduced to cultures of pediveligers of H. hippopus. Microscopic observation of the veligers and metamorphosed juveniles was done periodically. RESULTS
Spawning of adult clams and behavior of larvae During the period July 10 to September 15, 1982, H. hippopus spawned sperm on 10 different occasions when exposed to macerated gonads, but only spawned eggs twice. Both egg-spawning events occurred in the late afternoon (1500-1900 h), within a week after the new moon. These observations are consistent with the summer spawning for this species observed by Stephenson (19341, Jameson (19761, Yamaguchi (19771, Beckvar (1981), and Gwyther and Munro (1981). Tridacna gigus spontaneously spawned both eggs and sperm in the late afternoon about a week after a new moon on July 27-28 and August 26-27 (see Heslinga et al., this volume). These observations compare favorably with assays of gonad condition and observations of natural spawning of T. gigas on the Great Barrier Reef, suggesting a summer breeding season for this species (R.D. Braley, personal communication, 1982).
185
Fertilized eggs of H. hippopus remained on the bottom of containers through the ciliated gastrula stage. Trochophore larvae became free-swimming 16-20 h after fertilization, and actively swam at the surface. They did not appear to orient to light. Veligers first appeared 24 h after fertilization, and within 48 h all larvae had reached the veliger stage of development. Two- to three-day-old veiigers actively swam throughout the medium, whereas 4- to 9-day-old veligers were observed most often at the surface, their velums sometimes flush with the surface film. This behavior was not a function of larval density (lack of oxygen), as larvae at the lowest densities demonstrated this behavior. Pediveligers developed 9-14 days after fertilization and alternately crawled on the bottom by extension and retraction of the foot, or swam just off the bottom. Metamorphosis to juveniles occurred 9-13 days after fertilization in the first batch (day 4 veliger size = 187.5 pm f 6.3 sd., n = 201, and 13-18 days after fertilization in the second batch of spawned eggs (day 2 veliger size = 170.5 pm & 4.3 s.d., n = 15). Influence of’ larval density on survival
and growth
Fig. 1 shows that trochophore survival was greater at low densities (0.2-5 larvae/ml) than at higher (lo-100 larvae/ml) densities. Similarly, veliger survival was greater at low densities. Measurement of the size distribution of 8-day-old veligers showed (Table I) that animals maintained at low densities were significantly (p < .05) larger than those grown at higher densities. Influence
qf’added
particulate
and dissolved
organic
nutrients
on swvival
and growth
of larvae Survival
of trochophore
larvae
The addition of particulate or dissolved organic nutrients to cultures of trochophores of either H. hippopus or T. gigas had no obvious effect on the enhancement of survival (Table II). All treatments, with the exception of high densities of I. galbana, resulted in survival values that were insignificantly different from untreated controls. Trochophores of T. gigas exposed to high densities of I. galbana demonstrated significantly lower survival than controls. Microscopic observations of the trochophores showed no evidence that the algae were being consumed. Survival
and ,rrowtll
qf’ tleliger larvae
To determine the influence of particulate food on survival and growth, veligers of H. hippopus were provided with I. galbana at increasing densities ranging from lo3 to lo6 cells/ml. Control veligers were maintained without food. Table III shows that the highest survival of veligers through metamorphosis (day 13 in this experiment) was found among those fed I. galbana at densities between lo3 and lo5 cells/ml. During the early veliger stage (days 4-9), growth was optimal at densities of 1. galbana of 104-10’ cells/ml. During the later veliger stages, optimum growth was observed at lo5 cells/ml. Veligers characteristically demonstrated higher growth rates during
Fig. 1. Effect of density on survival of trochophores (Cl), day 6 veligers (e), and day II veligers (0) of H. hippoptis. Bars represent 95% confidence intervals. The number of replicates is indicated next to each point. See text for details. TABLE I Effect of density of larvae on growth of veligers of ~~~0~~~~~iip~~pu~ Density (veligers/mt) 0.2 1 5 10 30 50 100
Size km> 191.9 z!z4.3 195.8 i 4.2 192.2 -c 3.9
(101 (IOf (10)
190.9 185.4 185.6 184.1
(14) (14) (20) (20)
* f + k
4.2 2.7 3.7 2.8
“Numbers are means f 95% confidence intervals of &day-old veligers
the early stages, growth becoming progressively metamorphosis. In another experiment using a different batch hippopus at densities of 3 animals/ml were provided subcordiformis, and Phaeodactylumtricornatumsingly,
slower with approaching of spawn, veligers of H. with I. galbana, Plalymonas in pairs, or all together.
187
TABLE II Effect of additions
of various dissolved
and particulate
“foods” on survival and development
of
into veligers for Tridacna gigas and Hippopus hippop&
trochophores
Survival
Addition 1. galbana
Hippopus hippoprrs
Triducna gigas
1.3 f
103/ml
104/ml
(VI)
3.3
9.3 Ik 5.2
11.7 + 3.0
9.0 f
7.0
5x104/ml
6.8 f
1.8
lO’/ml
6.3 f
2.4
17.3 +
9.8
106/ml
5.3 + 1.6b
16.6 f
8.1
Vitamin and yeast extract
8.3 f
11.0 f
3.5
Vitamin
5.8 k 3.3
11.7 +- 4.9
Yeast extract
5.8 -t 2.7
15.0 +
17.3 + 10.2
1.1
9.1
Live yeast
6.7 + 1.5
12.3 f
8.1
Vitamin and yeast extract + 1. gulbuna (5x104/ml)
6.5 f
2.2
6.7 k
6.3
Control
9.0 * 2.2
9.0 f
4.2
aValues are the mean percentage of trochophores (k 95% confidence intervals) reaching the veliger stage from six replicate samples, 2.5 days after fertilization. See text for details. bSignificantly (p < .05) different from controls.
TABLE III Effect of different densities of lsoch~wis ga//xwa on growth and survival of veligers of Hppopus I
Density
”
Growth
(pm/day)
days 4-9
(cells/ml)
/~q~popusl
Survival (‘XI)
days 9- 13
day 4
day 6
day 9
day 13
0 (control)
20
100
19.2 f 26.2
60. I k36.2
lX.lk23.6
I03
22
0
0.13kO.17
100
R2.3k 18.9
69.2-1- 17.0
565t336b
104
1x
0.60k0.13b
0.13+-0.17
100
83.0*
9.x
62.2t31.9
42.St
67 Rt 10.6b
0.22kO.14
0
IO5
1R
0.8X*O.l4b
0.50+-o 19b
100
73.0*
11.2
70.0t
106
18
0.76+0.14b
0.05+0.15
100
17.3k
5.1
42.5-+33.2
“Numbers represent means + 9% confidence intervals; data are from six replicates. See text for details. “Significantly (p < .05) different from controls.
Dissolved
extract.
organic
nutrients
In this experiment,
n. number
97
of clams measured
14.0b
38.X-+35 2 for growth
Surwval
provided in the form of vitamins and yeast metamorphosis was complete after 18 days.
were
expressed as pm/day
2.64-t0.29 (22)
Control
Srowth
3.42~0.20
Vitamin + yeast extract
-
(14) (121 (19) (24)
0.58kO.18 0.57t0.23 0.7720.16 1.13+0.16
(19) (28) (12)
0.4720.14 0.90+0.13 0
1.02+0.15 (18)
(201
(t-t)
1.03t0.19
Days 8-13
& 95% confidence intervals.
(45)
2.56-+0.20 (201
(25) (241 (23) (28)
z.g. + P.S. + P.t.
P.S. + P.t.
1.16kO.22 1.76kO.23 0.80+0.22 1.28+0.19
2.50&0.26 (26) 1.90+0.21 (28)
I. galbana I? subcordifarmis P. tricornatum I.g. + P.S. I.g. + P.t.
(n)
Days 3-8
Food
0
0.36+0.16
0
0 0.46kO.26 0 0 0.70+0.15 0.2OkO.27
(4)
(20)
(6) (16) (151 (15)
(18) (9)
Days 13-18 (t-t)
(n>
180.9
194.8
1.62 ~~~0.03(20) 0.69-eO.09 (4)
186.7
186.6 185.4 177.8 183.9 184.0 184.0
Mean Iength (pm, day 18)
1.08+-0.04 (11)
1.07+-0.07 (18) 0.991to.04 (91 0.49kO.05 (6) 0.89z!zO.O4(16) 0.90-r-0.05 (15) 0.9OrtO.06 (15)
Days 3-18
Effect of phytoplankton “food” species and dissolved nutrients on growth of veligers of Hippopus hippop&
TABLE IV
189
Regardless of treatment, veligers grew most rapidly during the early stages, growth rates declining through the middle veliger stage and the onset of metamorphosis (Table IV). Unfed control veligers ceased growing after the first 8 days, and only 5% of the veligers survived to metamorphosis. In contrast, Table IV shows that all of the veligers provided with either particulate or dissolved organic matter, with the exception of those provided with P. tri~,o~r~a~L~nl~ showed enhanced growth and survival. Maximum sizes of metamorphosed juveniles were found among those that had been provided with vitamins and yeast extract, and with 1. ~~lb~~ff. Veliger survival (Fig. Za,b) was approximately five times higher than controls when provided with vitamins and yeast extract or with I. ~Q/~Q~IQ,alone or in combination with either P. subcordiformis or P. fricorrzatrm~. Veliger survival was lower when veligers were provided with either P. sabcord(ftirmis or P. tricornatum alone. Light-microscopic observations of veligers provided with the three algal species showed that i. galbana and P. sabcord0vri.s were consumed, that is, the algal cells were observed in the digestive tract. In contrast, P. tt+cornanrmwere not consumed.
The process of acquisition of algal symbionts by larvae of T. yigas and Fi. hippoptis was the same as that previously described (Fitt and Trench. 1981) for T. squamosu. As in other tridacnids and another symbiotic bivalve Corcaium cardissa (Kawaguti, 19681, the symbiotic algae are not passed from one clam generation to another, and as a result, symbiotic algae were not observed in the eggs or any of the larval stages of T. gigas or H. hippopus when maintained in FSW. This is consistent with the observations of LaBarbera (19751, Jameson (19761, and Fitt and Trench (1981), but is in contrast to the report of Gwyther and Munro (1981). All strains of S. rn~croudrjar~c~rn provided were taken into the stomachs of the pediveiigers of H. hippop~s and T. gigas within 24 h of their introduction to the larval cultures (cf. Fitt and Trench, 1981). The algae remained in the stomachs of pediveligers and newly metamorphosed clams, and about 7 days after metamorphosis could be seen in rows extending from the region of the stomach and digestive gland toward the developing siphonal tissues (Fig. 3). This observation is identical to that seen in the juveniles of T. squamosa (Fitt and Trench, 1981). Although under laboratory conditions the juveniles of tridacnids may establish a symbiosis with all strains of S. microadriaticum so far tested, they do not accept another symbiotic dinoflagellate, Amphidini~m klebsii. Finally, the veligers of T. gigus and H. hippopus do metamorphose to juveniles in the absence of symbiotic algae and phytoplankton food (cf. Gwyther and Munro, 1981) as shown in Figs. 2 and 3 and Table III. DISCUSSION
In the past, most studies of the mariculture of tridacnid bivalves have emphasized aspects of growth and survival of metamorphosed juveniles
a
Age
(days)
b
Age
(days)
Fig. 2. Survival of veligers of H hippopus fed (a) vitamins and yeast extract CX), I. galbana, (0). P. subcord@rmis (Cl), P. tricornatum (AI and (b), I. gaibana plus P. tricornatum (O), I. galbana plus P. subcordlformis (X), P. subcordiformis plus P. tricornatum (U), and I. galbana, P. subcordiformis, plus P. tricornatum (A). Unfed controls (*I. Bars represent 95% confidence intervals. See text for details.
19
Fig. 3. Juveniles of H. hippopus about 2 weeks after metamorphosis. Clam on the left contains S. microadriuricum (z), but the clam on the right, maintained throughout larval development in vitamins and yeast extract does not have zooxanthellae. Dark bodies (db), found in all juveniles, are not zooxanthellae. st, Statocyst. Bar = 50pm. See text for details.
(Yamaguchi, 1977; Beckvar, 1981; Heslinga et at., this volume). These and other studies CLaBarbera, 1975; Jameson, 1976; Fitt and Trench, 1981) have shown that at a given spawn, tridacnids release large numbers of eggs, but less than 1% survive larval development. Therefore, from the point of view of mariculture, it seems important to determine conditions of larval rearing that
would reduce the high larval mortality that appears to be characteristic of these organisms. Compared to the life span of adults (Comfort, 1957; Bonham, 1965; McMichael, 1974; Yamaguchi, 1977), tridacnids spend a brief but critical period as larvae (Fig. 4). Development from trochophore to veliger is usually complete within 24-48 h. There is no evidence that trochophores are capable of particulate feeding. The veliger stage lasts from 1 to 3 weeks depending on the species and culture conditions, and vetigers are demonstrable planktotrophs. Veliger growth rates are highest during the early stages, growth rates becoming progressively lower with the onset of metamorphosis (see Jameson, 1976; Gwyther and Munro, 1981). It is evident that the highest mortality occurs during the transitions from trochophore to veliger and from pediveliger to juveniles. The high mortality at these transitions may partly be due to changes in the mode of acquiring nutrition. As these clams do not establish a nutritional symbiosis with Sjmbiodinium microadriaficum until after metamorphosis, their larval stages require a nutritional input from exogenous sources, and in this respect, the larval biology would trot be expected to be very different from that of nonsymbiotic clams. Thus, until trida~nid larvae are capable of feeding, they must rely on stored nutrients provided with the eggs. If, under laboratory conditions, no exogenous nutrients are provided, the clams would have to rely on stored nutrients throughout their entire larval existence. The amounts and quality of stored nutrients in the eggs of tridacnid bivalves are unknown.
192
-Id
:..*.
5-8 doys
:: :
3-8
days
<5 days
I veliger
pediveliger
metamorphosis
.a.*’, -IOh ,8&h.
Fig. 4. Representative
timetable
of the developmental
stages of H. hippopm.
The major goals of this study were to determine conditions of larval culture that would reduce the high mortality usually encountered in the mariculture of tridacnid clams (see Heslinga et al., this volume). Our results show that we have achieved a four- to fivefold increase in survival in larvae reared at low densities and fed, when compared to controls. We had little success in reducing the high mortality levels among trochophores. Despite all attempts, trochophore mortality remained at least 80%. It is possible that bacterial contamination may be responsible for the high mortality. Future judicious use of antibiotics may help to improve this situation. The optimum conditions for the culture of veligers appear to be maintenance at low densities and provision of an appropriate source of nutrition. Veligers maintained at densities less than lo/ml showed optimal survival and growth. The high mortality (80-90%) of veligers at high densities may have resulted either because of the accumulation of waste products (e.g. NH,+) or a lower quantity of food per veliger being available. That the latter explanation is less tenable gains support from our observation that control unfed veligers at low densities demonstrated only 30-400/o mortality after 6 days (see Fig. 2a). Mean growth rates for the entire veliger period were greater for all fed veligers (except those fed P. tricornatutn) than for unfed controls. The highest growth rates were found in veligers fed vitamins and yeast extract. Low growth rates of veligers provided with P. tricornatum are consistent with our observation that these diatoms were never seen in the digestive tracts of veligers and were probably not being used as food. Similarly, the largest recently metamorphosed juveniles (Table IV) were among those that had been provided with an exogenous food supply (with the same exception as mentioned earlier). However, growth increments throughout the veliger stages were not constant. Our data are consistent with those of Jameson (1976) and Gwyther and Munro (1981) showing a gradual decrease in daily growth rates, such that under most conditions, clams undergoing metamorphosis grow very little if at all. Our data on the apparent depressed growth rates of veligers fed P. tricornatutn and P. cubcordiformis (either singly or in combination), as compared to controls, in the early veliger stages, are difficult to interpret. Toxic and growth-inhibiting substances released from sotne species of algae have been
193
suggested as being responsible for similar phenomena in cultures of veligers of other bivalves (see Loosanoff and Davis, 1963). Apparently, P. tricornatutn may occur in four morphologically distinct forms (Walne, 19631, and some forms have been found to be inadequate food for other bivalves as well (Epifanio, 1975). The data we have presented show that the veligers of H. hippopus survived and grew best when provided with vitamins and yeast extract. This would imply that the veligers may be able to utilize dissolved organic nutrients (see Stephens and Manahan, this volume), although our cultures were not axenic. Because we employed both of these substances in combination, it would be important to determine which one produced the observed effects. Studies of other bivalve larvae (Loosanoff and Davis, 1963) have shown that when parameters such as feeding regimes or temperature are manipulated, growth rates and developmental times to metamorphosis also change, but the size of veligers at metamorphosis tends to remain the same for a given species. Preliminary data indicate that the larvae of H. hippopus metamorphose at a length of 185-I 95 pm. The effect of an exogenous food supply appears to be to increase veliger growth rates and hence perhaps, shorten the time to metamorphosis. Although our data intervals are not close enough to show, this might be the reason we observed increased survival at metamorphosis of fed veligers when compared to unfed controls. It is apparent from our experiments and those of Gwyther and Munro (1981) that larvae of different spawns can be of markedly different size. A possible reason for this may be that eggs of different spawns (especially “induced” spawns) contain very different proportions of stored nutrients which may be reflected in egg size. We hypothesize that those eggs with larger nutrient stores would grow faster, survive longer, and give a larger probability of attaining metamorphosis than eggs with less stored nutrients. Our data, showing that many more starved control veligers from the group of larger veligers survived through metamorphosis (Table III) than the group of smaller veligers (Fig. 21, and that growth of unfed veligers stopped halfway through the veliger stage, are consistent with this hypothesis. The failure of some “spontaneously” spawned batches of tridacnid larvae to yield reasonable quantities of viable spat (G. Heslinga, personal communication, 1982) may be explained by this hypothesis. Adult tridacnid clams maintain the symbiotic dinoflagellate S. microatlriaticrrm in hemal spaces of their hypertrophied siphon tissues as well as the digestive gland and stomach, where they are thought to provide significant contribution to their host’s nutrition (Yonge 1936, 1980; Muscatine, 1967; Trench et al., 1981). However, the symbionts are not inherited by the offspring and must be acquired from the environment by the larval or juvenile clams. No eggs from any species have ever been seen with symbiotic algae (Stephenson, 1934; LaBarbera, 1975; Jameson, 1976; Fitt and Trench, 1981), and no confirmed observations of trochophores or veligers with ,symbiotic algae in their tissues have been reported (cf. Gwyther and Munro, 1981). The larval forms of the tridacnid clams are apparently unable to establish a syrnbiosis with S. tnicroadriaricrrtn until after metamorphosis. We have shown here for T. gigas and H. hippopus and previously for T.
194
squurnosa (Fitt and Trench, 1981) that veligers are capable of ingesting all strains of S. microadriaticum offered, especially the motile forms which are more likely to be encountered by the veligers. These algae are taken into the stomach where they apparently contribute to larval nutrition as made evident by the fact that veligers of T. squamosa “fed” different strains of S. microadriaticum had higher survival rates than controls (Fitt and Trench, 1981). It is not known if the veligers digest live and/or dead S. microadriaticum, or receive photosynthetic products released by live S. microadriaticum while they are in the stomach. Intact algae are often seen passing out of the intestine of veligers fed S. microadriaticum and in many cases veligers having S. rnicroadriuticum in their stomachs are seen at a later time with no algae in their stomachs. Only after metamorphosis are S. microadriaticum seen in tubules extending from the posterior portion of the digestive gland anteriorly into the developing siphon tissues. At this point in the development of the clam, a symbiosis has been established (cf. Fitt and Trench, 1981). There is apparently a mechanism of recognizing and establishing a symbiosis with only this species of algae, because the symbiotic dinoflagellate Amphidinium klebsii isolated from Palauan flatworms, and numerous free-living phytoplankton species which may be taken into the gut did not establish a symbiotic relationship in postlarval clams. The establishment of symbiosis with S. microadriuticum is not essential for metamorphosis in tridacnids (Fig. 3). This is in contrast to the situation demonstrated by some coelenterates such as the jellyfishes Mustigias papua (Sugiura, 1964) and Cassiopeia xumachana (Trench et al., 1981). ACKNOWLEDGMENTS
We would like to thank the entire staff Demonstration Center, Republic of Belau, visits. We especially appreciate experimental and E. Oiterong and help in obtaining Ngiramengioir, and 0. Orak. We also thank This study was supported by the Agency for DPE-5542-G-SS-1085 to R.K. Trench).
of the Micronesian Mariculture for their cooperation during our help from S. Derbai, D. Fisher, animals from J. Heslinga, M. M. Jess for graphical assistance. International Development (grant
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