Passive greenhouse heating, recirculation, and nutrient addition for nursery phase Tridacna gigas: growth boost during winter months

29

Ayuacul~ure. 108 (1992) 29-50 Elsevier Science Publishers B.V., Amsterdam AQUA40018

Passive greenhouse heating, recirculation, and nutrient addition for nursery phase Tridacna

gigus: growth boost during winter months

ABSTRACT Braley, R.D.. Sutton, D.. Mingoa, S.S.M. and Southgate, PC., 1992. Passive greenhouse heating, recirculation, and nutrient addition for nursery phase Trrdacna pigm: growh boost during winter months. Aquaculrure. IO& 29-50. The impetus for this study was winter-related mortality ofjuvenile Tridacno gigosalong Australia’s Great Barrier Reef. Heating nursery tank water by passive solar heating in a greenhouse and the addition of dissolved inorganic nitrogen (DIN) was assessed for elTeti on the growth and survival of cultured juvenile clams. Two age classes of X gigas were used, with means of .2 cm and 17.0 cm shell length. Treatments consisted of nutrient-spikes of 20 pM and 2 0 CM ammonium chloride daily or on alternate days. p!nus z :$kc of 2.3 p%piwrphale once per week vs. controls without nutrient additioa. Three rearing systems were used: ( 1) recircukating water enclosed in a greenhouse; (2) flow-through water enclosed in a greenhouse; (3) flow-through water with ambient conditions. In the older clams growth :II weight was best in system 2, while growth in shell length (SL) was best in system :, and DIN treatments significantly increased growth compared with controls. In the younger growth in SL was best in system I. DIN treatments produced significantly~atergrowth than controls. but there was no difference beiwcen 20&f and 40-W treatments. Survival was IOWa for larger clams but for smaller clams mean survival was highest overall in system I, while ZO+M DIN treatments within systems produced the best overall survival. The highest I~vels ofDIN in the nursery tanks were found in the 4D-/&I DIN treatments, particularly in system The wet tissue weight/shell length ratio for 40-a DIN treatments was highest in wswm 1 and decreased in systems 2 and 3, while controls were similar. Dry shell weight/sheil length &as highest in thr 4O+MDIN treatment over the control in sys~m only. The zooxanthellae index (no. of algal cells/g clam) was significantly higher in the 40-m DIN treatment than in lhe control in all three systents. Biochemical analysis of whole animals showed higher carbohydrate content in System 2 and in treatments receiving 20 pM DIN. Tissue pmtein content did not differ significantly between systems but increased with increasing cutrient mncentration. Lipid content was highest in system I and decreased with increasing nutrient mncen-

I

clams,

I.

1

Correspondence IO: Dr. Rick Braley, Giant Clam University, Townsville, Qld. 4811, Australia.

0044-8486/92/$05.00

0 1992 Elsevier

Science

Project,

Publishers

Zoology

B.V.

Department,

All rights reserved.

James

Cook

30

R D. BRALEY ET AL

tration. Tissue water content of clams at the 2044 DIN level was lower than clams in other treatments, indicating superior condition. The combination of passive solar healing, recirculated wafer. and nutrient addition for the giant clam land nursery phase opens possibilities for culture of this tropical bivalve in subtropical zones or in the tropics distanl from the ocean.

INTRODUCTION

There is considerable potential for increasing growth rates and survival of juvenile giant clams (Tridacnidae) during the land-based culture phase of culture. Recent advances have been made in both extensive and intensive mass culture techniques (Heslinga et al., 1984, Crawford et al., 1986; Braley et a!., !P88; Rraley et al.. l990), and in studies into the effects of nutrients supplied for the clam’s symbiotic zooxanthellae on the growth of the host (MMDC Bul!c;in, l9S8; Soiis ei ai., i 988; Heslinga ei ai., l9iiii j. Test groups of Tridacnn derasa (Roding, ! 798), wh i ch were treated with water fertilised with 100 w ammonium nitrate as daily spikes, grew 15% faster on average than controls after 2 months (MMDC Bulletin, 1988). In later studies, the addition of dissolved inorganic nitrogen (DIN) resulted in a near doubling ofgrowth rate in youngjuveniles ( < 1 cm), and a 20% increase in shell length (SL) in older juveniles over controls (Heslinga et al., 1990). In the Philippines, the results of growth in SL of juvenile Hippopus hippopus (Linnaeus, 1758) (2.9-4.6 cm shell length) showed a mean increase of 110% per month in three DIN treatments compared with controls (Solis et al., 1988). In both experiments, the fouling from filamentous algae increased because of DIN addition; the resulting algal overgrowth stressed the small juvenile clams. The positive effects on growth of juvenile clams with DIN addition suggest that seawater flow rates in nursery tanks would have to be much higher and circulation more efficient to compensate for the relatively low levels of nutrients in surface seawater. Typical nutrient concentrations in tropical reef regions are 0.1-0.3 fl nitrate, 0.2-0.5 w ammonia and co.3 m phosphate (Crossland, 1983 ). High densities of clam juveniles in nursery tanks would quickly reduce the levels of these nutrients in seawaier to undetectably low levels (W. Fitt, pers. commun., 1991). The use of inorganic nitrogen and phosphorus to increase nutrient levels is a minor cost compared with the alternative need for increased pumping of seawater. In land-based nurseries at higher latitudes m the tropics, where the minimum seawater surface temperature in winter drops :o 2 1 “C or below, there is a detrimental effect on survival ofjuveniie Triducmgigas (Linnaeus, 1758). Respiration rate decreases below 20°C and above 32°C (Mingoa, unpubl. data, 1990). Previous studies have shown thlat the growth rates of T. gigas grown in an ocean-based nursery system have a direct reiationship to seawater temperature (Nash, 1988; Lucas et al., 1989). The term ‘winter mortality’

has been used at the Orpheus Island Research Station, North Queensland, Australia, to describe significant mortality of juvenile T. gigas during several successive winters in the land-based nursery system (Braley and Lucas, unpubl. obs., 1990). To describe growth and survival of T. gigas over winter months (July-midOctober 1989) in a manipulated environment, a g:lseniruuse was erected over the nursery tanks to provide passive heating, and a recirculating biotiltration system was developed to retain solar-heated water. Also, our limited knowledge of the clam-alga! symbiosis indicated the need for a preliminary investigation into the effect of nutrient additions c 2 the quantity and quality of the clams’ zooxanthellae and into the biochemical changes that may occur in the clams.

METHODS

Greenhouse A reinforced translucent plastic material (Solargro) was used to cover a metal framework canopy over six 3000-I circular tibreglass tanks. Walls and zippered doors were also made of Solargro. Light transmittance was approximately 50% through the Solargro material. Three tanks were set up, each with its own recirculation system. In the recirculation system (system I ), seawater was pumped from a 3000-I tank to a height of 3.5 m where the water sprayed intn =.trickle biofilter. Each tnckte aiofiirer ioi;si;ls?ed of four plastic bins tilled with coral rubble (total bed volume 600 I; anproximate rubble sze O.S- I .O cm diameter). Coral rubble *waschosen for its buffering capacity, ready availability, ease of collection and thresdimensionai space suitable for growth of marine nitrifying and heterotrophic bacteria. The seawater collected in a tray under the trickle filter, from which it ran by gravity into a tip-tray feeding an algal-covered panel before returning to the 3000-I culture vessel through l@f particle filter bags. The bags prevented any fragments which detached from the algal panel from entering the nursery tanks. The use of algae to remove nutrients from seawater in recirculating systems, similar to that described above, was reported by Adey ( 1983). The other three 3000-l tanks under the greenhouse canopy (system 2) each had flow-through seawater with particle filtration as described for system 1. Three 4000-l capacity tibreglass tanks (tilled to 3000 1) were set up adjacent to the greenhouse but not under a greenhouse canopy (system 3). Shadecloth which transmitted 50% sunlight was used over the metal framework canopy to approximate the light levels reaching the clams inside the greenhouse. The seawater was flow-through, and tiiter bags were used as in systems 1 and 2.

32

RD. BRALEY ETAI..

Clam juveniles Two age classes of T. gigas were used: clams spawned in January 1986 (42 months old) and Cscember 1988 (7 months old), hereafter referred to as older and younger clams, respectively. The mean SL ( 2s.d.) of the clams at the start of the experiment in July 1989 was 17.0 2 0.3 cm and I .2 2 0.1 cm, respectively. The SL measured was the maximum dimension from posterioventral to posterio-dorsal surface. For this revised anatomical terminology for giant clams see Lucas et al. ( 199 I ). Twenty older clams and IO0 younger clams were placed into each of the three tanks in each system (nine tanks total). Older clams were spaced evenly on the tank bottom, and younger clams were placed on 2.0-cm-size road gravel inside a tray in a position of maximum sunlight. Clams were cleaned every 2 w--kc* Ffi*+-- GQ+.G -I--- .was siphoned from the __._, lvul.,le

tank bottom, and, at times, the younger clams had to be removed and scrubbed carefully to remove filamentous algae. Shell length was measured with calipers, and survival determined from monthly counts. Drained wet weights (WW, obtained by inverting the clam for I5 min) were also measured on these occasions for the older clams only, all of which were individually tagged. There were no significant differences between initial WW (one-way ANOVA, P> 0.05) and initial SL (one-way ANOVA, PsO.05) of older clams. The younger clams were too small tti tag but all surviving clams were measured to obtain a mean for each group. Initial measurements of younger clams showed a significant difference (t-test, Pc0.05) between the conim! zzd treatment clams in system 1 on!y, with the 40qMDIN treatment being 10.4% larger than the control and the 20-MDIN treatment being 5.6% smaller than the control. Final SL were corrected for any prior differences by dividing by the initial group mean. The smallest number of clams surviving in the treatments ( 14) was used to achieve a balanced design. This number of clams was selected at random from all other treatments, and the significance level for the test was qet a priori at the more conservative level of P=O.Ol. A transformation of the type X=log(X+ 1) was applied to the data to restore additivity (Tukey’s ! DOF test, P=O.4855). The test which was used for both SL in younger clams and for both WW and SL in older clams was a two-way orthogonal ANOVA with system and nutrient regimens considered as fixed factors. The difference in the DIN application+background ammonia compared with ctmrluia, betwera sysrtms, was small enougn tor the assun,ptlon ot equal applicatio.1 to rema ~1 valid. In order to examine the relative rela:ionship between growth in WW and SL in the older clams, the ratio WW/SL was determined at the start and end of the experiment. The variable used was the difference in the ratio from start to finish. Data analyses were performed “sing the microcomputer statistical package Statist& V 3.1. For all statistical tests, the probability of type I error was set at P~0.05.

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Nutrients

Ammonium chloride was added to the test tanks in the late afternoon ( 17.00 h). Two concentrations were tested: 20 fl and 40 m ammonium chloride. Background ammonia in flow-through systems was about 0.5 ,ti. The tank trestments were: System I: recirculation greenhouse; 3.8 turnovers seawater day- 1 control (0 ,uMbackground ammonia day- ’ ) 20 @4 ammonia every other day 40 J&I ammonia every other day System 2: flow-through greenhouse; 2.9 turnovers seawater day-’ control ( 1.5 @f background ammonia day- ’ ) 20 @f ( 13.8x background ammonia day- i ) 40@f(27.6xbackgroundammoniaday-I) System 3: flow-through ambient; 2.9 turnovers seawater day-r control ( 1.5 fl background ammonia day-’ ) 20~&U( 13.8xbackground ammonia day-‘) 4Ow (27.6xbackground ammonia day-‘) Phosphate (garden fertiliser (9% PO., by weight) ) was administered at ! 7.00 h once per vveekas a nutrient-spike (2.3 w final concentration) to the seawater with the DIN treatment clams but not to the controi ciams. Ammonia, nitrite, and nitrate levels in the tank water were determined at intervals ranging from 3 to 8 days for all tanks in system3 1 and 2, but not for system 3. Both systems 2 and 3 were flow-:hrougb seawater so only one system was used to compare with system 1. The Bow rate in systems 2 and 3 was approximately 6 I min-I. The flow rate in the recirculating system 1 was about 8 I min-‘. Analyses for nitrite and nitrate were carried out usins methods described by Parsons et al. ( 1984). Ammonia analyses were conducted using the method of Garside ( 1978), using an Orion 9512 ammonia electrode. Phosphate concentrations were not analysed.

Physicalparameters Daily temperatures were recorded with maximum-minimum thermometers in all nine tanks, and in the air adjacent to tanks. When conditions became too warm by late September, the greenhouse sides were lifted permanently. Salinities were recorded from refractometer readings in all tanks every other day. Light readings (pE m-’ s-‘) were taken every other day, around mid-day, in all tanks at the depth of the clams using a Li-Cor LI-I 88 Photometer and underwater sensor. Seawater Bow rates were checked regularly. Condition indices and zooxanthellae

Five clams from the younger group were sampled after the fmal measurement in October I989 from each of the control and 40-H ammonium treatments in the three systems. All specimens were collected at mid-day. Shell

R.D. BRALEY ETAl..

34

lengths were measured with a vernier caliper. Clams were then removed from their shells to obtain wet tissue weight (WTW). Shells were oven-dried at 60°C and weighed. The fresh mantle was homogenised by hand using a Tetlon tube homogeniser to extract the zooxanthellae. Zooxanthellae samples were diluted in a small amount of 0.45.@I filtered seawater (FSW ), and separated from clam mantle tissue by passing through four layers of cheesecloth, yieiding a brownish algal suspension. This suspension was centrifuged and the supematant discarded. The pellet was then resuspended in FSW, and the sample volume measured. The number of zooxanthellae was counted using a haemocytometer under a high-power microscope. Six replicate counts were made per clam, and mean values were calculated.

Four condition indices were determined, based on zooxanthellae, clam shell length (SL), and tissue parameters: (1) zooxanthellae density (ZD) index or number of zooxanthellae per clam WTW; (2) mitotic index, determined by counting the number of doublet cells (dividing zooxanthellae) per 1000 cells (non-dividing zooxanthellae) (Wilkerson et al., 1983): (3) dry shell weight/SL (g mm-‘); and (4) clam WTW/SL (g mm-’ ). Condition indices of clams in the three controls and in the ammonium and greenhouse treatments were compared. Proximale biochemical analyses

Clams from the younger group were removed from their shells, allowed to drain and weighed subsequent to being oven-dried at 50°C and re-.veighed. Dried tissue was powdered and homogenised in 2 ml distilled water using an Ultra-Turrax T25 homogeniser. Total lipid was extracted by the method of Folch et al. ( 1957) and quantified by charring with sulphuric acid (Marsh and Weinstein, 1966) using tripalmitin as a standard. Protein was precipitated from the homogenate by addition of trichloroacetic acid (TCA), dissolved in 1 M sodium hydroxide solution, and assayed by the biuret method as described by Layne ( 1957) using bovine serum albumen as the calibration standard. Carbohydrate was assayed from a TCA-soluble portion of the homogenate by the method of Dubois et al. ( 1956) using DaJucose as the calibration standard. Ash was determined by heating a portion of dried homogenate at 500°C to a con.stan! weight. The energy contents of whole clams were calculated using the caloric equivalents of 4.3,8.42, and 4.1 kcal g-’ for prctein, lipid, and carbohydrate, respectively (Beukema and DeBruin, 1979). Three whole clams were assayed per treatment and mean values were calculated. RESULTS

Growth and survival Older clams. Table 1 shows the initial and final mean shell length and mean wet weights ( + s.d.) for older clams in all systems. Absolute growth in WW was significantly higher in system 2 (2 > I = 3), but absolute growth in SL was

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TABLE 1 Initial mean shell lengths (T,,SL. cm) and mean wet weights (TOWW: 8) and tinal mean shell lengths (T$L) and mean wet weights (TrWW) for older (SL and WW) and younger clams (SL only) in systems l-3 and nuttie.> treatments. Standard deviations are shown with each mean ( f s.d.). Initial N=20 for older clams and N= IO0 for younger clams. Final N=20 for clams bul see Table 3 for final N foryoungerclams. System/treatment (SYS/+) IS indicared in column

older

SYS/T

Older clams

I

Younger clams

TSL

T,SL

TOWW

r,ww

TSL

T&L

17.2fl.4 l7.Of I.9 l6.8f I.5 17.3?; 1.4 17.5?1.6 16.8f t.5 17.l+L.6 l6.6f 1.7 l?.Ot I.6

l&4+1 6 19.1 t 1.9 18.9t I.7 l7.8? 1.4 18.6~ 1.5 ltl.lk 1.7 17.6f I.5 17.4* 1.4 18.1*1.8

978+261 893f291 9112271 9842271 999+269 903 + 264 878f238 784?293 934zk336

lO76f275 ll78+359 ll69t313 1128k319 13435326 11962353 1059+275 1003f282 ll98f406

l.l3+0.26 I .07 + Q.27 1.25?0.29 l.lO+O.32 l.10C0.28 t.OSfO.25 1.2310.31 1.13fO.27 1.16kO.26

1.9OfO.36 3.02tO.62 3.58AO.67 3.49*0.31 2.18?0:39 2.54kO.37 1.57co.13 2.15+0.48 1.99+0.47

l/C I I20 l/40 2/C 2flO 2140 3/C 3/50 3140

TABLE 2 Analyses results of weway ANOVAs run on the older clams for wet weight (WW), shell length (SL), and the combined WW/SL ratio. The younger clams awere analysed by two-way ANOVA for SL (adjusted. transformed data). A past hoc Least Significant Difference (LSD) painvise comparison of means was done for each ANOVA result. Effects are shown for system, nutrients. and interaction. System is represented as I. 2, and 3; nutrients are reprxnted by C (control), 20 (20~t.M DIN), 40 (4OMDIN) Older clams

Younger dams

ww

SL

WW/SL

SL

System LSD

P=O.O31 2>1=3

P2=3

P<0.0000 2=3>1

P2=3

Nutrients LSD

P<0.0000 20=4O>C

PC

P< 0.0000 20=4O>C

P<0.ooo0 20=4O>c

Interaction

P=O.OOl I

P>O.OS

P=O.O036

PzO.05

higher in system 1 ( 1) 2 = 3) (Table 2). This is illustrated clearly in Fig. 1 (top plot, WW, centre plot, SL). The analyses revealed a highly significant difference in growth in both WW and SL due to nutrient regirxn, but there was no significant difference between 20-@4and 40-Mf DIN treatments (Table 2; Fig. I ). The analyses detected a signifkont interaction effect betwee% system and nutrient regimen for WW but not for SL (Table 2). This interaction effect with WW arose from the ZO+MDIN treatments of systems significantly

R D. BRALEY ET AL 350 300 250 200 150 loo 50 0

01.

I

WJ

I

?DpM

Applied nutrient

40pM

concentration

I

37

I)

Fig. 2. Plat showing the mean log (adjusted shell length+ of growth in younger clams against applied nutrient regimen (control 20-F and 40-p&l DIN treatments) for the three systems.

I and 2>40 @Z>control, while in system 3 the pattern was 40 fl> 20 @fz control (see Fig. I, top plot). The lack of an interaction effect with SL indicated the relative relationship between DIN treatments (40s 2O>C) remaimd consistent between systems (Fig. 1, centre plot). The analyses on the WW/SL ratio showed that clams in system I grew significantly (and proportionally) less in WW than clams from systems 2 and 3 (Table 2; Fig. 1, bottom plot). In the case of the clams in the 20-,&l and 40w DIN treatments in system I, this result was due to disproportionate growth of the shell (Fig. I ). In the control clams of system 1, the extreme low mean value was due to both moderate SL and poor growth in WW (Fig. I ). There was an overall pattern within systems of control clams having significantly lower ratio than 20-$4 or 40-&f DIN treatments (Table 2; Fig. I, bottom plot). The analyses identified a significant interaction effect between system and nutrient regimen (Table 2). This interaction arose due to the mean value of the ratio increasing with nutrient level in system 3, while in systems I and 2 the pattern was 20 > 40> C (Fig. 1, bottom plot). The survival of the rider clams was 100% throughout the experiment in all controls and treatments.

Fig. 1. Three plots showing growth of the older clams wok. 20-m and 40-m DIN treatments) in the three (WW) increase (g), the centre plot shows mean shell plot shows the mean change in WW/SL ratio. Standard

against applied nutrient concentration (consystems. The top plot shows mean wet weight length (SL) increase (cm), and the bottom error bars are shown.

38

RD. BRALEYET.&

Younger clams. Table 1 shows the initial and final mean SL ( +s.d.) for younger clams in all systems. The two-way ANOVA identified a highly significant effect of both system (1>2=3) and nutrient regimen (20=4O>C) (Table 2). Fig. 2 clearly illustrates this trend in growth. No significant interaction effect was found between system and nutrient regimen. A general increase in growth was seen from control to 20-@f to 40-fl DIN treatments, with the exception of the 40qMDIN treatment of system 3 (Fig. 2 ). Fig. 3 is a photograph of younger clams in system 1at the end of the experiment, clearly illustrating the growth and colour difference between nutrient treatments and control. The colour is an important visual indicator of DIN treatment. Survival of younger clams for all tanks in systems 1,2, and 3 is shown for each measurement period in Table 3. The most consistent survival was in the 20-w DIN treatments; control and 40-@f DIN treatments had both good and poor survival rates. Best survival was in system 1 (Kruskal-Wallis nonparametric one-way ANOVA, P=O.O45I ). The overall best survival was in system I in the 40.jMDIN treatment, and lowest survival was in system 3 in the 4OqMDIN treatment. The overall population fitness was described by a joint function of growth and mortality. This population fitness index (NSL) was calculated by multiplying the number of clams remaining in a treatment at a measurement date, by the size of all the clams within that treatment at

Fig. 3. Photograph of system I younger clams at the end of the experiment, comparing darkness of colour and size of treatment clams against controls.

Survival (in percentage. where N= 100 initially) trolsand treatments

L

COIlId

20 @U 4Ofl 2

Control 2ojlM

3

Control 20 fir

4ow

4O/uM

of younger T. gigas from July-Ostober I989 in con-

83 100 100

71 62 100

65 :“8

63 57 68

100 100 IO0

64 57 48

33 43 35

20 40 33

loo 100 LOO

86 69 70

53 51 24

40 49 14

Fig. 4. Change in population fitness index (NSL) of the younger clams plotted against system and nulrients. This index takes into account both growth in shell length and nwtalily.

that date. Only system I showed consistent increases in NSL over the period of the experiment, while systems 2 and 3 showed moderate to severe declines in NSL (Fig. 4). System 1 had a greater mean change in NSL than systems 2 or 3 (one-way ANOVA, kO.0335). No significant difference was found between nutrient regimens (kc.6 I87 ). Each of the system and nutrient combinations vs. change in NSL are shown in the histogram of Fig. 4, which illustrates these results well.

40

Nutrients Table 4 shows the NH,/NH4 (reported as ammonia) and NO, concentrations detected in each system throughout the experiment. With respect to NHJNH,, concentrations were similar in equivalent treatments in both sysTABLE4 Nuuient concentrations (PM) detected in systems 1 and 2”

0 3 5 IO 17 24 31 38 45 52 60 66 76 80

(0s) No” 3 5 10 17 24 31 38 45 z 66 73 80

1.0

I.2 I.8 1.0 5.a 4.2 4.0 1.0

1.8 1.6 4.5 3.0 6.5

0.1

0.0 0.0

0.0 0.6 0.0 0.3 0.2 0.4 02

0.2 0.2 0.2 0.2

4.5 2.5 3.0 1.0 3.3 6.1 2.2 4.0 2.0 1.7 3.5 4.0 3.5

6.1 16.0 16.0 1.0 4.0 3.8 2.2 0.9 8.8 1.5 1.6 1.6 1.6

0.0 0.0 0.0 1.2 0.0 0.3 0.3 0.4 0.3 0.0 0.0 2.6 2.8 3.0

0.0 1.8 8.8 22.0 0.0 0.6 2.5 5.5 0.3 0.2 0.2 0.0 0.1 0.2

“System L (Sys I )=recirculsring. System 2 (Sys2)=flcwthrough. “Measured as am”onta. rDays from nuwient additions. Day O=daypliorioanaddirion.

2.3 4.2 3.3 4.9 0.0 5.4 4.2 2.0 7.0 4.1 2.1 1.6 2.8 5.0

3.3 18.0 40.0 8.2 1.0 3.2 4.6 12.5 2.5 4.0 17.0 2.2 5.2 8.0

3.8 3.5 6.1 13.7 0.0 17.0 3.8 1.8 7.0 7.0

0.0

0.0 5.0 21.7 116.6 20.4 10.1 19.8 75.0 35.9 17.0 19.8 17.1 3.0 4.0

0.1

0.1

0.0 1.0 0.0 0.3 0.3 0.3 0.5

0.4 0.2 0.1 0.1 0.6

19.4

2.0 3.1 4.8

0.2

0.1 0.8 0.0 0.3 0.5 0.4 0.5 0.2 0.8 0.3 0.3 0.2

terns, and were usually less than 8 @4. Only infrequently were higher concentrations detected, this occurring predominantly in the 40-M DIN treatments. Peak concentrations were detected in system 1 (20+&f and 40@4 DIN treatments) around day 5. Nitrate concentrations in most treatments were general!y brlow 1.O,uA$ however, in the 2O-@f and 40-@f DIN treatments in system 1, there were initial peak concentrations at day 10. Subsequent measurements were generally in the order of 20Mnitrate (40-@DIN treatment) and less than 5w nitrate (20~MDIN treatment). Measured nitrite concentrations (data not presented) in all treatments were usually less than 0.5 @, and always less ihan 2 w.

Physical parameters Weekly mean tank-water temperatures are shown over the experimental period for the 40-N DIN-treated tanks for systems 1,2,and 3 (Fig. 5). System I tanks (greenhouse, recirculating) were 57°C warmer than system 2 or 3 tanks throughout July, August and September. After the greenhouse sides were lifted permanently, there was a decrease in October to temperatures closer to

those of other systems. There was a 1-2”C’hlgher temperature within system 2 (flow-through greenhouse) than with system 3 (ambient flow-through) throughout the experiment. The means of the s.d. of tank-water temperatures for systems I, 2, and 3 were 0.99,0.67, and 0.7”C, respectively. There was no overlap between system 1 and the other systems but there were some mean weekly temperatures for which the s.d. would slightly overlap between sys-

terns 2 and 3. During the day, air temperatures in the greenhouse enclosure regularly reached 740X, while night-time temperatures would often be < 19°C. Ambient night-air temperatures were often < 14°C in the first half of the experiment. The biological filter was not damaged by the high air temperatures in the greenhouse because there was constantly flowing cooler tank water passing through the filter. There is a similarity between the temperature plot of Fig. 5 and the plots of mean SL increase for older clams (Fig. 1, centre plot) and smaller clams (Fig. 2), though this is not the case for mean WW increase in older clams (Fig. 1, top plot ) . A decrease in irradiance intensity generally reflected cloudy or partly cloudy conditions and the general increase with time reflects the change of season toward summer (Fig. 6). The position of the systems 1 and 2 tanks generally

o-r0

1

2

3

1

5

6

I

8

9

10 II

12 1.3

WEEK Fig. 6. Mean weekly imdiance intensity for all trealmem (e.g. ES1 , AT3 ) shown in legend with treatment.

tanks of systems I, 2, and 3. Tank codes

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subjected them to the highest irradiance intensities while the system 3 tanks had slightly lower overall irradiance intensity in the first 11 weeks ofthe study (Fig. 6). Also, it is notable that in the first 7 weeks of the study the 20-w tank in system 1 and the control tank in system 2 had much lower irradiance intensities than the other two tanks in those two systems. This was due to the position of the tank receiving lower irradiance intensity (Fig. 6). Salinities were in the range of 32-36s. Clam-condition indices Higher shell condition indices were obtained from clams supplied with ammonium than control clams, except for system 3 which showed no difference in DSW/SL between control and ammonium treatments (Table 5). The

greatest difference between controlsand the treatment tank was seen in clams from system 1, followedby system 2. Using WTW/SL in a ratio for 40-~64 treatment/control the result was: system I 4854/o, system 2 2800/o, and system 3 177%. Using DSW/SL in was: system 1 168%, system The zooxanthellae indices indices (MI) (no. of doublet

a ratio for 40-m treatment/control the result 2 127%, and system 3 103%. (ZD) (no. of algal cells/g clam) and the mitotic cells/ 1000 cells) for these same tanks show that

although there is very little difference between controls and their respective treatment tanks for MI, there is a consistently higher ZD in treatment tanks over control in all systems (Table 5). Using ZD expressed as the ratio for 4% m treatment/control the result was: system 1 543%, system 2 280%, and system 3 356%. Proximate composition of tissues The proximate compositions of clam tissues are shown in Table 6. These data were analysed using two-way ANOVA, and significantly different means TABLE 5

Conditionindicesofcontrol (C) and 409MDIN lreatmcnlyoungerclams(N=5) at the endofthe (g/mm)=WTW/SL,

expenmenc.Shell weight index (g/mm)=DSW/SL: Tissue weight index Zooxanthellae density( X IO”) =ZD; Mimic index (x IO’) =MI System/ tremnell,*

DSW/SL

WTWISL

ZD

M1

I/C I I40

19.8 33.3

2.5 I II.70

7.17 38.46

9.0 7.5

2/c 2140

26.6 34.3

3.53 9.53

10.08 28.20

8.0 11.7

3/C 3140

24.4 24.7

3.06 5.52

10.62 40.20

13.9 10.7

RD. BRALEYETAL.

44 TABLE 6 Proximate composition of whole clams (K mg-’ were used (N=3 per treatment1 System

Treatment

I

Control 2OpM 4ouM

2

COlWlJl 2Oti 4OW

3

C”ntrol

201ucI 40pM

Protein

Lipid

286.1 x33.3 343.0 t41.2 344.2 ?r 130.6

98.5 f8.1 71.4 ? 19.0 49.5 f16.4

215.0 It 33.5 285.9 %25.5 311.0 t 12.2 284.7 k45.7 336.4 + !3.9 378.9 +43.7

dry weight+

s.d. ). The yoww

Carbohydrate

WUPS of T. &as

Ash W)

Meall energy conlent (kcatS-‘)

Tissue waler soment

47.7 + 3.2 63.2 +7.7 28.6 +9.7

39.2 f 7.3 36.5 2 5.2 33.6 * 3.8

3.0 I

90.0 f2.2 74. I to.7 82.3 21.2

56.5 flO.5 61.0 f18.9 45.6 f 2.0

75.4 f2.6 94.2 f 10.3 49.2 ? 5.7

40.5 f3.1 36.5 t 4.9 39.9 i 1.7

2.77

62.3 * 10.7 65.9 f9.8 59. I f13.3

6.2 fO.5 74.0 f4.5 52.1 + 14.2

42.0 f4.8 33.6 &I.7 39.3 + 2.7

2.75

301 3.04

2.96 2.76

3.12 2.83

(96)

78.9 i 2.2 76.4 f 8.3 81.3 + 5.0 60.1 + 1.0 85.7 f 0.0 85.7 kO.7

TABLE 7

Protein

Lipid

Carbohydrate

3

324.4” 270.7’ 333.40

73.1” 54.4b 6X4=,”

46.5” 72.9b 44.1=

0 ?O@f 40 [LM

261.9” 32 I .gb.’ 344.7’

72.4’ 66 I”d x:46

43.1’ 77.1a 43.3’

system

1

7

o h,“.“Meons in columns sharing P common

superscript

are no1 significantly

ditTwcnt

(P> 0.05 1.

in each system and at each nutrient regimen were determined using the Tukey test (Zar, 1984) (Table 7). The mean protein content of clam tissue did not differ significantly (Pz O.G5) between systems but was significantly greater in 40-a DIN treatments than in controls. Clams from system 1 had the highest lipid content. Mean lipid content decreased with increasing nutrient level: the highest mean lipid content of 72.4 pg mg- ’ dry weight was found in clams receiving no nutrient addition. This value differed significantly (PiO.05) from that of clams in treatments receiving nutrients at the 40-@4 DIN level. Carbohydrate content was significantly higher (Pt0.05) in clams from system 2 and in clams held at the 2O-@I DIN level than those in other treat-

ments. In general, clams in greenhouse recirculating system I had higher energy contents than those in other systems. The ash content of clam tissues did not show any consistent trend between treatments. DISCUWON

Greenhouse and nutrient effects Significant water temperature elevation and the highest survival ofyounger clams occurred in the greenhouse recirculation system compared with the other

systems. The 5-7°C temperature increase in the water of the greenhouse recirculation system was the factor most likely to affect survival ofthese younger clams. System was significant as a main effect in all the analyses for older and younger clams and was significant in two of three interaction terms for older clams. Although we can not ascribe total effects of system to temperature, this was clearly the main difference between systems. The ‘winter mortality’ observed at Orpheus Island over several winters appeared to be directly related to low winter temperatures. It is not known whether protozoan parasites, such as Perkinszcs (Goggin and Lester, I987), are the cause of some winter mortality when low temperature adversely affects homeostasis of 7’. gigas juveniles. This area requires further study. Both size classes of clams in system 1, 2, and 3 showed highly significant

growth (SL) in the nutrient treatments compared with controls. In older and younger T. gigas, temperature elevation combined with enhanced nutrient levels produced clear advantages for growth in SL, but in older clams no benefit was realised for growth in WW. The SL growth is in agreement with the direct relationship between metabolic activity and temperature shown in poikilothermic animals (Newell and Branch, 1980). System 1 clams produced larger shcl!s but were no heavier than tkeir counterparts in systems 2 and 3, which suggests that the shell density was lower in system 1 clams. Calcification in giant clams may occur at a more rapid rate with increase in temperature, and this is enhanced even more with the presence of nutrients (N and P). Nutrient supplementation (5 w N, 5 @4 N+2 @4 P) significantly enhanced shell-extension rates in T. gigas but significantly reduced shell weights

46

R.D. BRALEY EThL.

at equivalent size (Belda et al., submitted) similarly. Phosphate enhancemznt (C, 2 #4, 4 a, 8 w), notably at 4 PM, resulted in a more porous skeleton, and narrower, abnormal growth in the coral Acropora sp. (Rasmussen, 1990). Although the younger clams were not monitored for WW increase, the clam condition index showed the highest WTW/SL in the greenhouse recirculation system I. There was a larger increase compared to systems 2 and 3 not only in SL but also in tissue content, as shown in the clam-condition indices. This indicates that the younger clams are more responsive than older clams to the synergistic effects of temperature and DIN addition. The lower WW increase seen in system 1 older clams may be related to trace compounds in seawater acting as limiting factors to WW increase. Standard media for the culture of unicellular algae (such as fi) contain about I dozen compounds, of which N and P comprise over 80% by weight. It is conceivable that the zooxanthellae in the clams of system 1 would not produce the quantity or quality of photosynthetic products usable by the clam for tissue growth, due to essential trace elements being in short supply. The flow-through greenhouse system 2 provided a slight advantage in elevated temperature and a

replenishment of trace compounds compared with system 1. The results in WW increase supported this for the older clams. Calcium was not limiting in system 1,however, and superior growth in SL resulted. Zero mortality of the older clams during the experiment suggests that stress was not extreme in any tank: however, the combined effects of cold water and a large DIN dosage, as in system 3 40-#A4DIN treatment, may have been the cause of high mortality in the younger clams. Here, the lowered metabolic rate of the clams in cooler water and the algal overgrowth resulting from high nutrient levels, will have greatly influenced survival. In system 2 the levels of DIN detected remained low and similar to incoming seawater (i.e. system 2 control). On only three occasions were levels of NH3/NH4 elevated, possibly reflecting variations in clam uptake or flushing rate. In system 1 the initial high peak of NH3/NH4 detected at 5 days in both nutrient treatment tanks was followed several days later by peaks of NO,, indicating nitritication in the biological filters. Declines i:1 NHJNH, and NO3 after these peaks, indicates nitrification in the biological filter and/or uptake by the clams. The maintenance of elevated levels of NO, in system I 40-/1M DIN treatment was presumably due to overloading the biological fdter and the capacity of the clams to remove excess NO, as completely as in the 20-jlM DIN treatment. This excess nitrate which continued to recirculate through the tank may have contributed to the superior growth of clams in this system compared with the other systems. Heslinga et al. (1990) ctilised the NO, form of nitrogen as standard protocol at the MMDC in Palau. Most marine animals are stressed by high levels of nitrogen (Kinsey and Davies, 1979) but giant clams have been shown to utilise high levels @‘DIN wluch enhances activity of the symbiotic zooxanthellae (Wilkerson and Trench, 1986; Heslinga et al., 1990). For example, growth rates nearly doubled in juvenile T.

M*N*oEMENTOFN”R.sERY

PHSE

TR,DACN,l G,G.,S

47

lOO@fammonium nitrate, a concentration which represents a 500-fold increase in background nitrogen levels (Heslinga et al., 1990).

derasa with the addition of

Physicalparameters, condition indices. and biochemistry Lit$t irradiance levels were the !owest for system 1 20-m DIN-treated tank and for the system 2 control tank. Light levels probably affected growth in these tanks, possibly having more effect on the older clams since it has been shown that there is a requirement for higher irradiance intensities in older juvenile giant clams compared with very young clams (Fisher et al., 1985). The system 2 controi tank had poorer growth compared with system 3 control: both were flow-through seawater tanks. Here, only light levels and a l2°C temperature difference were varying factors. The higher temperature in system 2 control should have been a positive growth factor compared with system 3 control but the Ii& level was low enough in system 2 control to act as a negative growth factor. The clam condition indices show the highest WTW/SL where temperature and nutrients were highest (system 1). The MI did not prove useful in this experiment, but clearly ZD is related primarily to nutrient level rather than a combination of nutrients and temperature. The water content of bivalve tissues may serve as a useful indicator of condition (Lucas and Beninger, 1985); greater condition being shown by a lower water content. Using this as an index, small clams at the 20-w DIN treatments showed better condition than those at 40-@fDIN treatmentsandcontrols. These clams also had a significanily higher carbohydrate content than those at 40-m DIN or controls. Zooxanthellae produce carbohydrates as a major photosynthetic product (Muscatine, 1967; Grifftths and Streamer, 1988); a significant portion of these may subsequently be incorporated into host tissues (Griffiths and Streamer, 1988). The carbohydrate content of clam tissues was signilicantly greater (P< 0.05) in system 2 and at the 20-w DIN than in any other treatments. This indicates that conditions in these treatments were more favourable for photosynthetic activity of zooxanthellae than in other treatments. This was not reflected in growth in SL of the younger clams, but growth in WW of older clams showed a trend of higher growth in 20-w DIN in the warmer systems 1 and 2. The mean energy content of animals in the greenhouse/recirculating system was higher than in the greenhouse/flow-through system, indicating superior condition of clams held at higher temperature during winter months. Aquaculture application

The success of the recirculation/greenhouse system in maintaining higher water temperatures and the enhanced survival and growth of juvenile clams has major implications for tridacnid aquaculture. Using such a system, landbased nursery culture of seed obtained from tropical hatcheries may be pos-

sible in temperate climates. The use of partial recirculation would reduce costs in a coastal aquaculture operation; however, it would also be possible to use this system as a totally closed system in landlocked areas. Further studies are needed to determine growth-limiting compounds beyond N and P. The use of inexpensive garden-quality fertilisers in the commercial culture of giant clams will be an important saving in the production of seed. Rapid growth of young juveniles, especially when an ideal temperature is maintained, will result in

shorter residence time in land-based nursery tanks and earlier movement to the ocean-based nursery phase of culture. ACKNOWLEDGEMENTS

We sincerely thank all the people who assisted us in keeping the experiment running on a daily basis, especially Mr. Stephen Lindsay, Mrs. Katrina Miller, A/Prof. Johns S. Lucas, Mr. Russell Garrick, Mr. Jeremy Barker, and Mr. Geoff Charles. We thank A/Prof. John S. Lucas, Dr. Bill Fitt and Ms. Carmen Belda for comments on draft copies of the manuscript; Mr. Andrew Lewis and Mr. Peter Lee assisted with statistical analyses and with tinal production of some figures. We thank the Australian Centre for International Agricultural Research (ACIAR) for funding the Giant Clam Project (Project nos. 8332 and 8733).

REFERENCES Adey, W.H., 1983. The microcosm’ a new tool for reef research. Coral Reefs, I: 193-201. Belda, CA , Cuff. C. and Yellowle~s, 0. Perturbations io c;l;ifica;ior. by giarji clams al elevated nutrient levels: implications for coral reef poilution. (Submitted.) Beukema. J.J. and D?Bmin. W.. 1979. Calcrilic values of the soft pans of the lellinid bivalve ~fncoina balrhicu(L.) as deit-rmmed by two methods. J. Exp. Mar. Biol. Ecol.. 37: 19-30. Bmley. R.D., 1990. Manual for the Culturing of Giant Ciams. James Cook Umversity of North Queensland Publication, Townsville, Qld., 180 pp. Braley. R.D., Crawford, C.M.. Lucas, J.S., Lindsay, S.L.. Nash, W.J. and Westmore, S.P., 1988. Comparison of different hatchery and nursery culture methods for the giant clam Tridacna gigas.In: J.W. Copland and J.S. Lucas (Editors). Giant Clams in Asia and the Pacific.AC!AR

Monograph No. 9. Australian Centre for International Agricultural Research, Canberra. A.C.T., pp. 1 IO-I 14. Crawford, CM., Nash, W.J. and Lucas, J.S., 1986. Spawning induction, and larval and juvemle rearing of the giant clam. Tridacnagigas. Aquaculture, 58: 28 Crossland. C.J., 1983. Dissolved nutrients in coral reef waters. in: D.J. Barnes (Editor), Perspectives on Coral Reefs. Australian Instilute of Marine Science. Contrib. No. 200 (277 pp. ). pp. 56-68. Dubois. M., Gillies. K.A., Han ‘ton, J.K.. Rebers, P.A. and Smith, E. 1956. Colorimetricmethod for the determination of sugars and r&ted substances. Anal. Chem., 28: 350-356. F~sber. CR., Fitt, W.K. and Trench, R.K., 1985. Photosynthesis and respiration in Tridocrm gigus as a function of irradiance and size. Biol. Bull.. 169: 230-245.

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MANAGEMENT

OF NURSERY PHASE %Q,D,lc*!.l G,G,IS

49

F&h, J., Lees, M. and Sloane-Stanley, G.H., 1957. A simple method for the isolation and puriticaiton of total lipids from animal tissue. J. Biol. Chem.. 226: 497-509. Garside, C., 1978. Determination of submicromolar concentrations of ammonia in natural waters by a standard addition method using a gas-sensing electrode. Limnol. Oceanogr., 23 (5): 1073-1076. Goggin, CL. and Lester, R.J.G., 1987. Occurrence of Perktwo species (Protozoa, Apicomplexa) in bivalves from the Great Barrier Reef. Dis. Aqua. Org., 3: 113-l 17. Griffith% D.J. and Streamer, M., 1988. Contribution ofrooxanthellae to their giant clam host. In: J.W. Copland and J.S. Lucas (Editors), Giant Clams in Asia and the Pacific. ACIAR Monograph No. 9. Australian Centre for International Agric&nral Resw-ch, Canberra, A.C.T., pp. 151-154. Heslinga, G.A., Perron, F.E. and Orak, O., 1984. Mass culture ofgiant clams (F. Tridacnidae) in Palau. Aquaculture, 39: 197-215. Heslinga, G.A., Watson, T.C. and Isamu, T., 1990. Giant clam farming. Pacific Fisheries Development Foundation (NMFWNOAA), Honolulu, HI, 179 pp. Kinsey, D.W. and Davies, P.J.. 1979. Effects ofelevated nitrogen and phosphorus on coral reef growth. Limonol. Oceanogr., 24: 935-940. Layne, E., 1957. Spectrophotometric and turbidimetric methods for measuring protein. In: S.P. Colowick and N.O. Kaplan (Editors), Methods in Enzymology, Vol. 3. Academic Press, New York, NY, pp. 447-466. Lucas, A. and Beninger, P.G., 1985. The use of physiological condition indices in marine bivalve aquaculture. Aquaculture, 44: 187-200. Lucas, J.W., Nash, W.J.. Crawford, CM. and Braley, R.D., 1989. Environmentalinfluenceson growth and survival during the ocean-nursery rearing of giant clams, Tridacna gigas (L.). Aquaculture, 80: 45-6;. Lucas, J.W., Ledua, E. and Braley, R.D., 1991. Tridacnn tewrm Lucas. Ledua and Braley: a recently-described species of giant clam (Bivaivia. Ttidacnidae) from Fiji and Tonga. Nautilus, 105 (3): 92-103. MMDC Bulletin, March 1988. !ab test result: fertilizer speeds clam growth. MMDC Bull., 3 (l):2. Marsh, J.B. and Weinstein, D.B., 1966. Simple charring method for determination of lipids. J. Lipid Res., 7: 574-576. Muscatine. L., 1967. Glycerol excretion by symbiotic algae from corals and T.-idacnaand its control by the host. Science, 156: 516-519. Nash, W.J., 1988. Growih and monality ofjuvenile giant clams ( Tridncm gigas) in relation to tidal emersion an a reef flat. In: J.W. Copland and J.S. Lucas (Editors), Giant Clams in Asia and the pacitic. ACXR Monograph No. 9. Australian, Centre for International Agricultural Research,Canberra,A.C.T.,pp. 183-190. Newell, R.C. and Branch, G.N., 1980. The influence of temperature on the maintenance of metabolic energy balance in marine invertebrates. Adv. Mar. Biol., 17: 329-396. Parsons, T.R, Maita, Y. andLalli, CM., 1984.A Manual ofchemicaland Biologic&Methods for Seawater Analysis. Pergamon Press, Oxford, 173 pp. Rasmussen, C., 1990. Enhanced nutrient levels in the marine environment and their effects on coral reefs. In: Proceedings of the 4th Pacific Congress on Marine Science and Technology, Vol. II, Tokyo, Japan, 16-20 July 1990, pp. 13-20. Solis, E.P., Onate, J.A. and Naguri. M.R.A., 1988. Growth of laboratory-reared giant clams under natural and laboratory conditions. In: J.W. Copland and IS. Lucas (Editors), Giant Clams in Asia and the Pacific. ACIAR Monograph No. 9. Australian Centre for International Agricultural Research, Canberra, A.C.T., pp. 201-206. Wilkerson, F.P. and Trench, R.K., 1986. Uptake of dissolved inorganic nitrogen by the symbiotic clam Mdacna gigas and the coral Acropora sp. Mar. Biol. (Bed.), 93 (2): 237-246.

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Wilkerson, F.P., Muscatine, L. and Muller-Parker, G., 1983. Temporal patterns of cell division in natural populations of endosymbiotic algae. Limnol. Oceangr., 28: 1009%1014. Zar, J.H.. 1984. E&statistical Analysis. Prentice-Hall International, Inc., London, 7 18 pp.