GROHTH OF JUVENILE ABALONES (HALIOTIS RUFESCENS), MUSSELS (~IYTILUS CALIFORNIANUS), AND SPOT PRAHNS (PANDALUS PLATYCEROS), IN AN EXPERIPIENTAL POLYCULTURE

A thesis submitted to the faculty of San Francisco State University in partial fulfillment of the requirements for the degree

Master of Science in Marine Science

by

John Hilliam Hunt

San Francisco, California

June, 1987 Copyright by John William Hunt 1987 Growth of Juvenile Abalones (Haliotis rufescens), Mussels (Mytilus californianus), and Spot Prawns (Pandalus platyceras) in an Experimental Polyculture

John H. Hunt San Francisco State University 1987

Polyculture techniques were applied to the hatchery production of

juvenile abalones Haliotis rufescens, mussels Mytilus californianus,

and spot prawns Pandalus platyceras to investigate the effects of

coexistence and food quantity on shellfish growth and fouling control.

Growth of abalones and spot prawns was significantly greater in

monoculture treatments than in polycultures, and growth increased

significantly with increasing amount of food supplied. Mussels gre1<

relatively slowly in all treatments, and were not significantly

affected by any experimental manipulations. Abalones were found to be

capable of ingesting and assimilating shrimp meat that was intended as

food for prawns in the polyculture system. Overlap in utilization of

this food source is suggested as one reason for decreased growth of

abalones and prawns in the polyculture. Fouling was greatest in

mussel monoculture containers, significantly less in prawn containers,

and least in abalone and polyculture containers, which were not

significantly different from each other.

I certify that the Abstract is a correct representation of the content of this thesis.

· (Thesis Advisor) (Date) / / ACKNO\vLEDGEHENTS

I would like to thank Drs. H:tchael S. Foster, Gregor H. Cailliet,

George A. Knauer, and James IV. Nybakken of the Hoss Landing Harine

Laboratories, and Dr. Ralph J. Larson of San Francisco State

University for their assistance in designing the experiments and in editing the manuscript. Special thanks to Earl E. Ebert, director of the Harine Culture Laboratory at Granite Canyon, for his continuing support and advise, and for allowing me to do this work at that fine facility. Thanks also to James Houk, Arthur Hazeltine, Gino Segna, and Cathy Thaler of the Harine Culture Lab for their help and many helpful suggestions during the course of the project. I am grateful to

Sheila Baldridge, who, as usual, went out of her way to find references to the appropriate literature; and to Joe Aliotti for trapping and delivering healthy ovigerous spot prawns for broodstock.

Of course, I owe much more than thanks to Teresa Clayton, who not only helped with the collecting, sampling, and data analysis, but also enthusiastically supported the project for far longer than either of us had bargained for, and then married me anyway.

This work was funded in large part by a grant from the David and

Lucille Packard Foundation, and their support is gratefully acknowledged.

v TABLE OF CONTENTS

List of Tables . • ...... • ... . • . • • . • • . . • • • • . • • . • • . • • . • • vii

List of Figures . . • • . • • ...... • . • ...... • . . . . • ...... viii

List of Appendices ...... • . . • . . . . • . . • . • ...... • • . • • . . . . ix

Introduction • . . • ...... • . • . • ...... • . . • • . • • • • . . . . • • . . • • . . 1 Red Ahal one • • • • • ...... • . • . • . • . • • . • • • . • ...... • 3 California t1ussel . . . . . • . . • . . • . • • . . . . • • . • . . • . . • • . . • . • • • . 3 Spot Prawn • • ...... • • • . • . . . • • • . . . • • • • . . • • • • . • . . • . . . • • • . . 5

Materials and Methods . • . • • ...... • . . . • • . • . . . . • ...... • . . • • 7 Experimental design ...... • . . . • . • . . . • ...... • . • . . • • 7 Data analysis ...... • • . • • ...... 8 Test containers . . . • ...... • ...... • . • . • ...... • . . . . . • 9 Culture of test organisms . • • . • • . . . . • • . . . • . . . . • . • . . . • . . . 11 Test densities . . • . . . . • ...... 12 Mortality and replacement ...... 13 Food, feeding, and ration size ...... •...... •..• 14 Cleaning schedule • • . • . . • • ...... • ...... • ...... • • 14 Accuracy .. and precision of gr01;th measurements ...... 15 Competition experiment ...... •...•..•.••..•.•.•..•.•.. 15 Measurement of container fouling ...•.•••.•...... 16

Results • • • . . • • . . • • . . • ...... • . • • • . • . • ...... • . • ...... 18 Growth of test shellfish ...... ••.••....•.••.•....• 18 Comparisons between unfed treatments •...... 19 Total production from all species ...•••.•...... 20 Interactive effect . • . • ...... • • . . . • • ...... 20 Mortality . . • . . • ...... • • . . • . • . . . . . • ...... 21 Competition experiment . . . . • . . . . • • • ...... 21 Fouling of test containers ...... •.... 22

Discussion 44

References 53

Appendix • • • . . • . • • ...... • . . • . • . . . • ...... • . . . • . . . 63

vi LIST OF TABLES

Table Page

1. Analysis of variance: Abalone growth ...... 24 2. Analysis of variance: Mussel growth ...... 25 3. Analysis of variance: Prawn growth ...... 26 4. Analysis of variance: Total production ...... 27

vii LIST OF FIGURES

Figure Page

1. Experimental design •...... ••... 28

2. Test container ...... • . . • • . • . . . . . • . • • . • ...... 29

3. Shellfish wet weight vs. dry weight .....•..••..• 30

4. Phytoplankton growth curve ...... 33

5. Abalone gr01;th . . . . • ...... • . . . . • . . . • . . • 34

6. Hussel growth ...... • ...... • • . . . 36

7. Prmm gr01

8. Tot;al production . . • • . . • • . . . • ...... • . • . . . • 40

9. Growth of abalones fed meat •.••.•.....•...... ••• 41

10. Fouling of test containers ...•.•..•.....•.•••.•• 43

viii LIST OF APPENDICES

Appendix Page

l. Preparation of diets for test shellfish ...•.•••. 63

Benthic diatoms ...... • . . . . . • . . . . • • . . • • • . 63

Cultured phytoplankton ...... ••..••...... 65

Neat for prawns • . . • . . • . • ...... • ...... • 66

Nutrition provided by unfiltered seawater •.• 66

iX INTRODUCTION

Polyculture techniques have been used to increase production of fish in freshwater ponds for centuries (Yashouv, 1966; Bardach, et al., 1972; Cruz and Laudencia, 1980; Dimitrov, 1984). More recently, polyculture has been proposed as a means of increasing production in a number of other aquaculture systems, using such organisms as: penaeid shrimp with milkfish (Eldani and Primavera, 1981); penaeid shrimp with pompano (Tatum and Trimble, 1979); penaeid shrimp with tilapia, milkfish or rabbitfish (Gundermann and Popper, 1977); oysters with penaeid shrimp (Maguire~ al., 1981); oysters in fish ponds (Hughes-

Games, 1977); oysters, flounder, and lobster (Mitchell, 1975); and pandalid shrimp with salmon (Rensel and Prentice, 1979). Polyculture systems have also been explored as a means of converting treated waste water into usable animal biomass through controlled marine food chains

(Ryther, 1972, 1975; Tenore and Dunstan, 1973; Tenore et al. 1973,

1974; Tenore, 1976; Mann and Ryther, 1977).

The primary potential advantage of polyculture is increased production, the increase in cultured biomass in a given space and time. Greater production is achieved through the greater utilization of available food and space within the culture system by species having varied feeding and habitat requirements. The classic example, developed over centuries in China, involves the stocking of up to six different carp species in the same pond. Grass carp feed at the surface on macrovegetation, silver carp feed on midwater phytoplankton, bighead feed on zooplankton, mud carp and common carp feed on benthic animals and detritus, and black carp feed on molluscs

(Bardach et al., 1972). This diversity of feeding patterns all01;s

1 greater utilization of available resources, and can have the added advantage of turning organisms that might otherwise overgrow and foul the system into food for harvestable species (Avault, 1965; Rensel and

Prentice, 1979). Detritus produced by increased incorporation of the available food energy is retained in the system where it can be utilized directly or indirectly by the appropriate cultured species to increase the harvested biomass (Tenore, 1974).

A further potential advantage, for which there is evidence in terrestrial agriculture, is an increased resistance to disease in polycultured organisms, resulting from the host-specific nature of most pests and pathogens (Ehrlich et al., 1977; Levandowsky, 1977).

At the present time little is known about this phenomenon in aquatic systems.

A polyculture approach can have obvious disadvantages if the species chosen are not compatible. Inhibitory interactions such as competition or predation will clearly have a negative effect on production. Furthermore, many modern intensive cultivation systems are designed to address the needs and habits of a single target species. To include additional species can complicate the process, requiring greater inputs of labor and materials which may not be justified by any increase in production. Whether the advantages of a polyculture approach outweigh the disadvantages will depend on the choice of species and culture systems. For most situations it is difficult to predict whether one species or many will provide the greatest production from the resources available.

The purpose of this study was to investigate the suitability of a polyculture system incorporating three marine invertebrates presently being used or considered for commercial mariculture on the west coast

2 of North America. These are the red abalone Haliotis rufescens, the mussel Mytilus californianus, and the spot prawn Pandalus platyceras.

They are an appealing combination for polyculture because of their different food and habitat requirements, and similar tolerances to physical and chemical conditions.·

RED ABALONE

Juvenile red abalones are being considered for polyculture because they are known to graze on the ubiquitous marine microalgae that readily colonize most marine culture systems (Mottet, 1978;

Ebert and Houk, 1984; Owen et al., 1984). The red abalone has been a candidate for commercial mariculture in the United States since the early 1960's (Ebert and Houk, 1984). It is the largest of the

California marine gastropods, reaching a maximum shell length of 30 em. It natur?lly inhabits rocky areas from the intertidal to a depth of at least 30m, feeding mainly on benthic algae (Cox, 1962).

Mariculture-related investigations of the red abalone have defined nutritional requirements (Leighton, 1967; Tenore, 1976), reproduction

(Boolootian ~ al., 1962; Morse, 1977), larval rearing (Leighton,

1974), settlement (Morse, 1984), and integrated production techniques

(Ebert and Houk, 1984). Two commercial operations currently produce red abalone in California.

CALIFORNIA MUSSEL

The mussel is an appropriate addition to polyculture because it can filter phytoplankton and other suspended material that would otherwise pass through the system unused. rlussels trap this material and deposit the unassimilated fraction into the system as digested

3 feces and undigested pseudofeces, each of which may contain

considerable quantities of algal biomass and detritus that can support

other organisms within the system (Foster-Smith, 1975; Haven and

Norales-Alamo, 1966; Hildreth, 1980; Kaspar et al., 1985; Tenore."'..!::.

al., 1973, 1974). It is unclear whether I:!_,_ californianus is capable of

assimilating suspended detritus. Zobel and Langdon (1937) suggest it

can use bacterial particles, but this ability is apparently lacking in

-M. edulis (Wright ---et al., 1982). Mussel culture is increasing throughout the world, but nowhere

is it practiced as intensively as in Europe, especially in the rias of

northern Spain where it is the most productive of aquacultural systems

(Aquirre, 1979; Bardach et al., 1972; Figueras, 1979). Mussels have

steadily gained acceptance in U.S. markets, and there has been an

increase in commercial cultivation on the west coast in recent years

(Chew, 1984; Beattie, 1980). Mvtilus californianus is being

considered as an alternative to the traditionally cultured I:!_,_ edulis

for a number of reasons. Although its growth rate is slightly less

than that of M edulis (Harger, 1970), both reach marketable size in

about a year (Johnson and Chew, 1982). M. californianus has been

successfully introduced to several western markets (Skidmore and Chew,

1985). I:!_,_ californianus has a higher meat yield per shell length than

I:!_,_ edulis, and the stronger shell is better suited to mechanized

cleaning (Skidmore and Chew, 1985). Most importantly, M.

californianus does not exhibit the high summer mortality

characteristic of M. edulis in some locations (Skidmore and Chew,

1985).

There is a wealth of information on the ecology and physiology of

t1. californianus (e.g., Zobel, 1937; Coe and Fox, 1944; Harger •

4 1970; Bayne, 1976; Bayne et al., 1976; Wright and Stephen, 1977).

There is also a gr01;ing literature on the mariculture potential of N. californianus (e.g., Morse, 1977; Winter, 1978; Gallager and Mann,

1981; Johnson, 1981; Johnson and Chew, 1982; Falmagne, 1984; Skidmore and Chew, 1985). The potential ~f the California mussel for polyculture has not been previously studied.

SPOT PRAWN

The spot prawn has been considered for commercial culture in temperate regions where water temperatures are too low for penaeid shrimp (Wickens, 1972). Its range includes most of the coastal North

Pacific from Korea and Japan north along the coast of Russia, and from

Alaska south to San Diego, California (Butler, 1964, 1970). It normally occurs on rocky substrates at depths from 4 to 487 m, and supports a small commercial trap fishery throughout much of its range

(Butler, 1970). Pandalid shrimp are opportunistic feeders (Berenbojm,

1981; Weinberg, 1981), and f.:_ platyceras scavenges for and eats a wide variety of foods (Wickens, 1972). It is a protandric hermaphrodite, maturing first as a male at one and a half years of age when it averages 140 mm in length and weighs 14.2 g. At three and a half years it becomes a female and ultimately reaches a mean length of

180 mm and a weight of 35.5 g, the largest of the Pacific pandalids

(Butler, 1964).

Aquaculture studies on the spot prawn have focused on laboratory rearing of larval stages (Price and Chew, 1968, 1969, 1970); nutrient assimilation, feeding habits, and compounded diets (Forster and

Gabbott, 1971; Forster, 1973); culture techniques, growth, and survival (Wickens, 1972); fecundity measurements, nutrition, growth,

5 and temperature and salinity tolerances (Kelly,~ al., 1977); and reproduction, growth, polyculture with salmon in net pens, and control of fouling organisms (Rensel and Prentice, 1976, 1977, 1979, 1980).

The spot prawn is considered suitable for polyculture for a number of reasons. It is adaptable to various substrates (Rensel and

Prentice, 1980), it grows more rapidly than other pandalids (Butler,

1964), it has demonstrated faster growth in polyculture with salmon

than in similar monocultures (Rensel and Prentice, 1979), and it consumes a variety of foods, including dead individuals of other cultured species (Rensel and Prentice, 1980). There are no indications from previous studies, however, that spot prawns are capable of benefiting directly from the detritus of other polycultured organisms (Rensel and Prentice, 1979).

In this study, these three organisms were grown together in

polycultures Bnd separately in monocultures to look at the effect of

their coexistence on their growth and on the growth of fouling

organisms. The amount of food provided was also manipulated to

determine how food availability interacted with interspecies

coexistence to affect growth.

6 ~1ATERIALS AND NETHODS

All work was done at the Marine Culture Laboratory (California

Department of Fish and Game), located at Granite Canyon, California,

20 km south of the Nonterey penninsula at longitude 121 o SS'Iv, latitude 36° 26'N. The laboratory is supplied with continuously flowing seawater that is pumped directly from the ocean to a large holding tank then delivered throughout the laboratory by gravity flow

(see Ebert et al. 1974). Source water is well mixed by upwelling throughout much of the year and is relatively rich in nutrients and dissolved oxygen. Water temperatures ranged from 12° to 15° C during the course of the experiment, and salinity ranged from 33 to 34 ppt.

EXPERINENTAL DESIGN

Three factors were manipulated. The first factor was interspecies coexistence: test animals were grown either in single species assemblages as monocultures or in combination with members of the other two species in polycultures. The second factor was the amount of food provided. Food quantity was manipulated to test the hypothesis that growth rates in polycultures vary from those in monocultures because of trophic dynamics and the greater availability and utilization of potential food sources. By manipulating food quantity and species composistion, it was possible to look for the interactive effect of the two. In addition, a larger polyculture treatment was included to assess the effect of animal density on growth within the polyculture since the polycultures contained more animals in the same amount of space as the monocultures.

All treatments were replicated three times. In all, there were five culture treatments (polyculture, larger polyculture, and a

7 monoculture for each of the three species), three food quantity

treatments for each of the five culture treatments, and three

replicates of each, for a total of 45 test containers (Figure 1).

The variables measured ~

(shell length and live ~;eight). and gro~

~;eight and percent cover). The experiment ~

DATA ANALYSIS

To determine shellfish gro~

mean ~;eight per individual in each replicate ~;as subtracted from the

final mean length and ~;eight. Each of the three replicates of each

treatment contained the same number of individuals (either 10 abalones,

20 mussels, 4 pra~;ns, or all of these in the polycultures). To obtain

mean length, the maximum shell length or carapace length of each

animal ~;as measured individually and the average length per replicate

~

in each replicate ~;ere blotted dry and ~;eighed collectively, and this

total ~

mean. ~lean gro~;th in length and weight per individual per replicate

was then used in statistical comparisons between treatments.

Dry weight and percent cover of fouling organisms were measured

for each test container at the end of the experiment, and these values

~

cover data were transformed to the arcsine of their square root before

analysis.

A two-factor analysis of variance was used to detect differences

in growth between treatments. Each factor had three levels. The first

factor, species coexistence, had the levels of monoculture,

8 polyculture, and large polyculture. The second factor, food quantity, had three levels corresponding to the three ration amounts described in detail below. For each species and each type of variable (i.e. growth in length or weight, or degree of fouling) a three by three two-factor analysis of variance was performed. If a significant difference was detected between treatments, a Student-Newman-Keuls multiple comparison test was used to determine where the differences lay.

TEST CONTAINERS

All test containers were arranged in a stratified random pattern on the same water table. The table was divided into three sets of replicate groups, with treatments assorted randomly within each. Each container received continuously flowing unfiltered seawater from a separate spout in a system of PVC pipes that distributed water from a single sourd§. Pipes were cleaned every other day at each feeding to clear debris that could interfere with uniform water flm<. Flow rates to each container were measured once immediately after cleaning and once after two days to assess the variability in water flow among treatments. The mean initial flow rate was 257 ml/min ± 18%

(coefficient of variation), and after the two days between cleanings the mean rate was 243 ml/min ± 53%. Analysis of variance revealed no significant differences in water flow between treatments. Outflows from containers went into a common \Vater bath that maintained even temperatures for all treatments.

The water table was illuminated by sunlight from a south-facing window. Light was diffused by two layers of 1 mm mesh nylon window screen that covered the entire top of the table.

9 Test containers were designed to provide variety of habitat, maximum use of three dimensional space, and to facilitate feeding by the three species (Figure 2). Abalone roamed freely throughout the containers, and had access to the diatom-covered grazing plates (see discussion of diatoms below) and to the floors and walls of both the container itself and the inner container that housed the mussels

(although a partial barrier of dry surface existed at the edge of the inner container). All incoming water was forced to pass through the inner container that confined the mussels. This arrangement gave the mussels first access to any material suspended in the unfiltered seawater. Prawns roamed freely throughout the container but were seldom found within the inner container, the dry edge apparently limiting their access. Grazing plates partially divided the main container volume into four areas where prawns could remain out of visual conta~t with one another.

The containers were made from commercially available polyethylene freezer storage boxes. Standard containers measured 12 em x 12.5 em x

12.5 em, while the larger polyculture containers measured 11 em x 15 em x 22.5 em. The center of the tight-fitting lid was cut out and

replaced with 1 mm mesh nylon screen to all01; water to enter from the

spouts above while filtering out larger particles. A screened outflow

tube was constructed to draw water from near the bottom of the

container, facilitating through-flow and the removal of water

associated with debris on the bottom. Total water volume of the

standard container was 1875 ml (including the inner container volume 2 of 320 ml), and total wetted surface area was 1604 em (including

inner container and grazing plates). Total water volume of the larger

10 polyculture container was 3712 ml (including inner container volume of . 2 641 ml), and the total wet ted sur face area was 2279 em, ~lean turnover time for water flowing through the standard containers was 7.3 min, and 14.5 min for the large containers.

CULTURE OF TEST ORGANIS~lS

Juvenile red abalones, Haliotis rufescens, averaging 15 mm in length were used in the experiment. These were selected after testing

1;ith spot prmms to determine the size at which abalones were immune from spot prawn predation.

All juvenile abalones were products of the same induced spawning which occurred 239 days before the beginning of the experiment. Three males and three females were used for spawning, and all gametes were mixed collectively at fertilization. Prior to the polyculture experiment, the abalones were raised in large fiberglass tanks and fed concentrated,:' filtered (5 urn), naturally occurring diatoms [see

Ebert and Houle (1984) for a description of the juvenile abalone culture facilities].

Small mussels, Mytilus californianus, averaging 11 ~~ in length, were collected from rocky intertidal mussel beds near the laboratory, and were of unknown age and genetic background. They were collected one week prior to the start of the experiment, and were maintained during that time in flowing unfiltered seawater with daily additions of cultured phytoplankton, Isochrysis galhana.

Spot prawns, Pandalus platyceras, were raised at the Narine

Culture Laboratory. Five ovigerous females were caught in commercial traps at a depth of 100 m off the Big Sur coast, and were transported hack to the laboratory in aerated, compartmentalized buckets filled

11 with sem;ater. Parent stock were maintained in an SO 1 aquarium with flowing filtered seawater, and were fed fresh adult mussels. An outflow pipe allowed newly-hatched zoea to be flushed from the aquarium into a collecting tank from which they were transferred to larval culture tubes. Zoea were raised to post-larvae on a diet which progressed from brine shrimp, Artemia, nauplii, to Artemia adults, to diced meat of commercially available frozen squid and shrimp. Prawn post-larvae used for the project were full siblings collected from a hatching which occurred 97 days prior to the beginning of the experiment.

TEST DENSITIES

Abalone monocultures had 10 abalones in each test container, 2 allowing 187.5 ml of water volume and 160.4 cm of wetted surface area per abalone. Mussel monocultures had 20 mussels in each test 2 container, g}ving each mussel 93.7 ml of water volume and 80.2 em of surface area, although mussels were generally confined to the 25 cm 2 area of the inner box screen (see Figure 2). Prawn monocultures were stocked with 4 prawns in each test container, and each prawn had 468.8 2 ml of water volume and 401 em of wetted surface area. Polycultures were stocked with 10 abalones, 20 mussels, and 4 prawns, giving the polycultures the higher density of one animal per 55.2 ml of water volume and 47.2 em 2 of surface area. In the larger polyculture

treatments, each animal had 109 ml of water volume and 67 em 2 of wetted surface area.

Abalones, mussels, and prawns were sorted before initial placement into the test containers to minimize initial variability in mean size or weight among treatments. The initial mean length and weight, and

12 the coefficient of variation between treatments were, respectively:

abalones, 14.9 mm -+2.1%, .37g -+5.3% ; mussels, 11.2 mm -+1.8%, .21 g ±3.2% ; and prawns, 14.1 mm ±6.2% (carapace length, tip of the rostrum

to the posterior mid-dorsal edge of the carapace), .20 g ±15.4%.

NORTALITY AND REPLACEHENT

Nortality was recorded at monthly intervals over the 100 day

experiment. Any individuals found dead were removed and replaced by

new individuals of similar size. A dead abalone was replaced with a

new abalone of the same age and parentage which had an equal shell

length. Dead mussels were replaced with mussels of equal shell length

which had been collected at the same time and place as the original

mussels, and had been maintained in the interim in flowing unfiltered

seaHater with batch additions of cultured Isochrysis. Dead prawns

were usually eaten, as were other dead animals, but since they left no

easily measu~~ble shell it was usually impossible to determine with

certainty the carapace length of the missing prawn. A dead prawn was

replaced with another prawn from the same hatch which had a carapace

length equal to that expected for the missing prawn. Expected lengths

were determined by adding the dead prawn's last recorded length to the

mean growth in length during that period of all prawns in the other

two replicates of the same treatment.

The rationale behind replacement of dead individuals in the

experiment was that the results would be biased more by variation in

animal density than by possible variability in growth rate caused by

replacement.

Of 270 abalones, 3 died and were replaced (two of the deaths were

caused by handling during measurement). Seven of 540 mussels died, and

13 23 of 122 prawns died during the course of the experiment.

FOOD, FEEDING, AND RATION SIZE

One suitable food was chosen for each of the three shellfish species: cultured benthic diatoms for the abalones, cultured phytoplankton (Isochrysis galban&) for the mussels, and diced shrimp meat for the prawns. To maintain equality in the amount and type of food presented to both the polyculture and monoculture treatments, all test containers at a given ration level were given the same amount of all three food types. There were three ration levels. Level I containers received nothing but flowing, unfiltered seawater, which provided an unknown but probably small amount of food in suspension.

Level II received unfiltered seawater and known additions of benthic diatoms (0.25 g dry weight/week), phytoplankton (10,000 cells/ml, added 9 times per week), and shrimp meat (3% of prawn wet weight/day).

Level III rec'eived unfiltered seawater and 0.50 g (dry weight) of diatoms per week, 50,000 phytoplankton cells per ml 9 times per week, and shrimp meat weighing 10% of prawn wet weight per day. The preparation and delivery of these foods are described in detail in the

Appendix.

CLEANING SCHEDULE

Pipes, spouts, and container lid screens were cleaned at each feeding to facilitate even water flow. Containers were siphoned to remove loose detritus once every two weeks, which allowed detritus to collect and cover the container floors before it was removed. This detrital residence time was chosen to allow potential growth of small invertebrates and algae that could serve as possible trophic intermediaries in nutrient cycling within the containers. Container

14 walls and the inner mussel screen were not scrubbed for the duration of the experiment to allow growth of attached fouling organisms.

ACCURACY AND PRECISION OF GROIHH NEASUREMENTS

Length measurements were made using calipers accurate to 0.1 mm.

The balance used to weigh test animals was accurate to 0.01 g.

Accuracy was determined by comparison with standards.

The precision of the length and weight measurement was determined by a series of 10 replicate measurements of one individual of each species. Animals were taken live from the water, blotted dry, measured, weighed, and then returned to the water for 0.5 min before repeating the procedure. The coefficients of variation for replicate measurements of length and weight, respectively, were: for abalone

0.2% and 3.1%, for mussel 0.0% and 1.6%, and for prmm 0.0% and 1.4%.

Variability in the moisture content of measured animals was investigated by determining the wet and dry weights of 10 individuals of each species chosen to vary in size across the range used in the experiment. Animals were rinsed with distilled water and dried on absorbent paper at 60° C for 72 hours. The coefficient of variation for the ratio of wet to dry weight among 10 sampled abalones was 3.4%, among mussels 11.6%, and among prawns 15.2% (Figure 3a, b, and c).

COMPETITION EXPERINENT

During the course of the experiment, it became evident that the growth of prawns and abalones was slower in polyculture treatments than in the monoculture treatments. One possible cause for this was inter-species competition for available food. Since all three types of food (diatoms, meat, and phytoplankton) were present equally in

15 monocultures and polycultures, the use by one species of another species' intended ration would work to the advantage of monocultured animals and would cause competitive interactions in the polyculture treatments.

To investigate the possibility of competition for food between prmms and abalones, two abalones were placed in each of six isolated containers with flowing filtered seawater. Three of the containers were given no food, the other three were fed equal quantities of diced shrimp meat, the food which was intended for the prawns in the poly culture experiment. Similarly, one prawn was put in each of six separate containers; three were starved, and three were given diatom covered plates, the food provided for the abalones in the polyculture test. After 33 days, growth and survival were measured for each replicate of each treatment and the results were compared using a t-test.

MEASUREMENTS OF CONTAINER FOULING

Material that clung to container walls and blocked the submerged inner screen was measured to determine the relative degree of fouling which developed in each container. Two measurements were taken for each container: dry weight of material attached to the inside walls of the test container, and percent cover of material on the submerged inner screen. No taxonomic resolution of the fouling material was attempted, but it appeared that the majority of fouling was caused by algal/bacterial films, small macroalgae, sponges, serpulid worms, and various other small invertebrates.

To determine the dry weight of fouling material at the end of the experiment, the shellfish were removed, the outside of each container

16 was scrubbed clean, loose debris was rinsed and drained from the

inside of the container, and the containers were left to dry for one

week in a sunny window. The dried containers were then weighed. After

weighing, the inside walls were scrubbed free of dried fouling

material, and the empty containers were dried and re-weighed. Dry

weight of fouling material was then obtained by subtraction. The

inner mussel container was measured separately, after being weighed as

part of the entire test container, to test for differences in the

control of fouling which might have been caused by differential access

to the various areas of the container by the shellfish present.

Percent cover of the submerged mussel screen by fouling organisms

was measured by simply holding a 4 x 4 square grid below the whole

------screen, counting any grid space which was more than half obscured by

fouling material, and dividing by 16, the total number of squares.

This measure was considered important because restriction of water

flow across the screen reduced water circulation and the supply of

suspended natural food to the mussels.

17 RESULTS

GROWTH OF TEST SHELLFISH

Manipulation of both factors, interspecies coexistence and food quantity, resulted in significant differences in the growth of both

prawns and abalones. Mussel grm;tfl was not significantly affected by changes in either factor.

A comparison between culture treatments showed very significant differences in abalone growth with manipulation of coexistence (ANOVA,

p <<.001; Figure 5). Mean growth of abalones in monocultures was significantly greater than mean growth of abalones in either

polycultures or large polycultures (ANOVA, p < .001). Mean growth in

length and live weight, respectively, for abalone monocultures was 4.6

mm and 0.48 g, for polycultures 3.1 mm and 0.28 g, and for large, lm; density polycultures 3.9 mm and 0.33 g (Table l, Figure 5).

Differences in abalone weight were significant between all

treatments (SNK, p <.OS). Differences in abalone length were

significant bet1;een monocultures and either of the polycultures, but

not between the two polyculture treatments.

Growth of the young abalones was also significantly affected by

the amount of food provided (ANOVA, p «.001). Abalone mean growth in

length and weight, respectively, \188: for food group I (unfiltered

seawater only), 2.2 mm and 0.15 g; for food group II, 3.7 mm and 0.33

g; and for food group III, 5.8 mm and 0.60 g (Figure 5, Table 1).

Differences between all three groups were significant (SNK, p < .05).

No significant differences in mussel growth were detected between

culture treatments (Figure 6, Table 2). Overall grm;th of mussels was

very poor, with total mean growth in length and live weight of only

18 0.6 mm and 0.04 g over the 100 day span of the experiment.

Mussels did not respond significantly to changes in the quantity of food added (ANOVA,/.25 > p > .10; Figure 6, Table 2).

Prawns in monocultures grew in both length and weight significantly more than did prawns in either standard or large polycultures CANOVA, p <.001). The t1;o polyculture treatments had no significant differences between them (SNK p ).05; Figure 7, Table 3).

Mean growth in length and live weight for prawns in monocultures was

10.0 mm and 0.72 g, in polycultures 6.8 mm and 0.41 g, and in large polycultures 6.8 mm and 0.44 g.

Prawn growth also differed significantly between food quantity treatments (p « .001). Prawn growth in length and weight, respectively, was: for food group I, 4.2 mm and 0.21 g; for food group

II, 7.8 mm and 0.47 g; and food group III, 11.6 mm and 0.88 g (Figure

7, Table 3). Differences between all three groups were significant

(SNK p < .05).

COMPARISONS BET\o/EEN UNFED TREATMENTS

Although there were significant differences in abalone growth between culture treatments when all food levels were considered together, as described above, there were no significant differences between abalone monoculture and polyculture treatments that received only unfiltered seawater for nourishment (ANOVA, p < .25; Figure 5).

In contrast, prawn growth was significantly greater in monocultures than in polycultures at the ration level receiving only unfiltered seawater CANOVA, p < .005; Figure 7). This was similar to the results obtained above by combining all food levels in the comparison of culture treatments.

19 Growth of mussels was not significantly different between culture treatments at any individual ration level, or for all levels combined

(Figure 6).

TOTAL PRODUCTION FROH ALL SPECIES

Although prawns and abalone "grew better in monocultures than in polycultures, the combined growth of all individuals of all species was greatest in the large polyculture. All polycultures had 10 abalones, 20 mussels, and 4 prawns, while monocultures had only the individuals from one of those species groups. The combined growth for all individuals of all three species in the large polycultures was significantly greater than that of any of the monocultures or the standard polyculture (ANOVA, p <<.001; SNK p <.05, Table 4, Figure 8).

However, combined growth in the standard sized polyculture was not significantly different from that of the abalone monoculture (SNK, p

>.OS), despii:'e the greater number of individuals in the polyculture.

Combined gro1

As could be expected from previous results on individual growth rates, the combined grm;th rate 1

INTERACTIVE EFFECT

Coexistence and food quantity produced a significant interactive effect on the total combined growth of individuals of all species in the test containers (ANOVA, p <<.001). An interactive effect bet1

(ANOVA, p < .025).

20 t·lORTALITY

·During the course of the experiment, 3 abalones, 7 mussels, and

23 prawns died. Neither co-existence nor food quantity had any significant effect on the mortality of any species CANOVA, p >.25).

COHPETITION EXPERINENT

There were no significant differences in growth or survival between prawns which were fed only diatoms and prawns which were starved. Growth was negative for all prawns in both circumstances. One of three prawns in diatom fed treatments died, while two of three starved prawns died. Apparently prawns were unable to successfully use diatom films as a food source, and there is no evidence to suggest interspecies overlap in the utilization of this food.

There wa.s, hm1ever, a significant difference in the grm;th of abalones given shrimp meat compared 1dth those which received nothing but filtered seawater (t test, p < .001 for abalone length and weight). Hean growth in length and weight for starved abalones was 0.2 mm and -0.05 g, respectively, while mean growth for abalones given shrimp meat was 2.3 mm and 0.32 g (Figure 9). This amounts to growth of 11% over initial length and 36% over initial weight in 32 days for abalones given nothing but diced shrimp meat. Observations made at each feeding showed that no meat from previous feedings remained in the containers, as was generally observed in abalone monocultures during the course of the polyculture experiment. No abalones died in either fed or starved treatments during this test.

21 FOULING OF TEST CONTAINERS

The dry weight of fouling material that accumulated in the test containers over the 100 days of the experiment varied significantly between culture treatments (ANOVA, p << .001; Figure 10), but was not affected by changes in food quan~ity (ANOVA, .25 > p >.10). ~lussel monocultures accumulated the greatest amount of fouling material, with a mean C± S.D.) dry fouling material weight of 3.64 ± 0.80 g for each mussel monoculture container (Figure 10). Prawn monoculture containers were also covered with a growth of fouling organisms, but the prawns' continuous feeding apparently prevented the growth of much filamentous algae, and there were also fewer invertebrates visible in the containers. The mean dry weight of fouling material in prawn monoculture containers was 1.95 + 0.71 g, which was significantly less than mussel monocultures, but significantly greater than polycultures or abalone mo,nocultures (p <.OS). Containers ~.05; see Figure 10). The mean dry ~

0.35 ± 0.16 g, and for large polycultures 0.87 ± 0.24 g.

Fouling material on the inner container ~

(see Figure 2).

The second measure used to determine the degree of fouling ~

22 (see Figure 2). This measure gave results similar to those above.

There was a significant difference in the percent cover between culture treatments (ANOVA, p <<.001), but no significant difference in fouling due to changes in the q~antity of food provided (p > .25).

~lussel monocultures had screens with a mean cover of 98. 7%. Mean cover of screens in prawn monocultures was 16.7%. Mean cover of screens in abalone monocultures was 9.8%; and both polyculture and large polyculture treatments had 0% cover. Mussel and prmm monocultures were significantly more fouled than polycultures, while abalone monocultures were not significantly different from either

polycultures or prawn monocultures. The alpha level for significant difference in all multiple comparisons is 0.05. TABLE 1a. Analysis of Variance: Abalone Growth in Length.

Results of Analysis of Variance testing of abalone length data. Each treatment is replicated 3 times. Culture treatments are monoculture, polyculture, and low density polyculture. Food groups I, II, and III are explained in the text. DF = degrees of freedom, SS = sum of squares, t•lS = mean square, F = the test statistic, P the probablity that observed differences are due to random chance, SD = significant difference, NSD = no ~ignificant difference.

Source of Variation DF ss NS F p Result

Among subgroups 8 70.29 8.79 culture 2 9. 29 4.64 17.76 <.001 SD food 2 59.16 29.58 113.13 <.001 SD interaction 4 1.84 0.46 1. 76 <.25 NSD \vithin groups 18 4. 7l 0.26 Total 26 75.00

TABLE lb. Apalysis of Variance: Abalone Growth in Weight.

Results of Analysis of Variance testing of abalone weight data. Each treatment is replicated 3 times. Culture treatments are monoculture, polyculture, and low density polyculture. Food groups I, II, and III are explained in the text. DF = degrees of freedom, SS = sum of squares, NS = mean square, F = the test statistic, P the probablity that observed differences are due to random chance, SD = significant difference, NSD no significant difference.

Source of Variation DF ss NS F p Result

Among subgroups 8 1.189 0.15 culture 2 0.20 0.10 27.27 <.001 SD food 2 0.93 0.47 129.42 <.001 SD interaction 4 0.06 0.02 L, .18 <.OS SD \hthin groups 18 0.06 0.004 Total 26 1.25

21. TABLE 2a. Analysis of Variance: Mussel Growth in Length.

Results of Analysis of Variance testing of mussel length data. Each treatment is replicated 3 times. Culture treatments are monoculture, polyculture, and low density polyculture. Food groups I, II, and III are explained in the text. DF = degrees of freedom, SS = sum of squares, MS = mean square, F = the test statistic, P = the probablity that observed differences are due to random chance, SD significant difference, NSD no significant difference.

Source of Variation DF ss MS F p Result

Among subgroups 8 1.68 0.21 culture 2 0.30 0.15 0.91 >. 25 NSD food 2 0.66 0.33 2.00 > .10 NSD interaction 4 0. 72 0.18 1.10 >.25 NSD \hthin groups 18 2.97 0.16 Total 26 4.65

TABLE 2b. Analysis of Variance: Hussel Growth in \veight.

Results of Analysis of Variance testing of mussel weight data. Each treatment is replicated 3 times. Culture treatments are monoculture, polyculture, and low density polyculture. Food groups I, II, and III are explained in the text. DF = degrees of freedom, SS = sum of squares, MS = mean square, F = the test statistic, P the probablity that observed differences are due to random chance, SD significant difference, NSD = no significant difference.

Source of Variation DF ss MS F p Result

Among subgroups 8 0.008 0.001 culture 2 0.002 0.001 2.30 >.10 NSD food 2 0.002 0.001 1.94 >.10 NSD interaction 4 0.004 0.0009 1.71 > .10 NSD \hthin groups 18 0.009 0.0005 Total 26 0.017

25 TABLE 3a. Analysis of Variance: Spot Prawn Growth in Length.

Results of Analysis of Variance testing of spot prmm length data. Each treatment is replicated 3 times. Culture treatments are monoculture, polyculture, and low density polyculture. Food groups I, II, and III are explained in the text. DF = degrees of freedom, SS = sum of squares, NS = mean squar.e, F = the test statistic, P the probablity that observed differences are due to random chance, SD = significant difference, NSD no significant difference.

Source of Variation DF ss MS F p Result

Among subgroups 8 318.74 39.84 culture 2 62.30 31.15 21.03 <.001 SD food 2 242.07 121.04 81.70 <.001 SD interaction 4 14.37 3.59 2.43 <. 25 NSD Within groups 18 26.67 1.48 Total 26 345.41

TABLE 3b. A~alysis of Variance: Spot Prmm Growth in Height.

Results of Analysis of Variance testing of spot prmm weight data. Each treatment is replicated 3 times. Culture treatments are monoculture, polyculture, and low density polyculture. Food groups I, II, and III are explained in the text. DF = degrees of freedom, SS = sum of squares, MS = mean square, F = the test statistic, P = the probablity that observed differences are due to random chance, SD = significant difference, NSD = no significant difference.

Source of Variation DF ss ~1S F p Result

Among subgroups 8 2.64 0.33 culture 2 0.52 0.26 34.02 <.001 SD food 2 2.09 1.05 137.98 <.001 SD interaction 4 0.03 0.008 1.04 >. 25 NSD \Vi thin groups 18 0.14 0.008 Total 26 2.78

26 TABLE 4. Analysis of Variance: Total Production

Results of Analysis of Variance testing of combined weight of all animals in each culture container. Each treatment is replicated 3 times. Culture treatments are monoculture, polyculture, and low density polyculture. Food groups I, II, and III are explained in the text. DF = degrees of freedom, SS = sum of squares, ~1S = mean square, F =the test statistic, P = the.probablity that observed differences are due to random chance, SD = significant difference, NSD = no significant difference.

Source of Variation DF ss MS F p Result

Among subgroups 14 337.41 24.10 culture 4 128.65 32.16 76.72 (.001 SD food 2 160.20 80.10 191.07 (.001 SD interaction 8 48.56 6.07 14.48 (.001 SD \vithin groups 30 12.58 0.42 Total 44 349.99

27 ABALO~;E NUSSEL PRfo.h'~ STM\DARD Lm: DE:;snr l·tot;OCLUUR E ~10/WCliLTURE ~·\OiWCCLTURE PULYCULTL!RE POLYCl'LTI'RE I I _j_ I I I I FOOD GROUP ;;? 1- ~ .... ~ r? ..... - 1- - - 1-- I I ~ I I I I FOOD GROUP ll t;!fl" 1- ~ 1- ~ - ~tlJ - 1- - ~ - I I I I :J --- I I I I J FOOD ~ Jt. GROUP Ill ~ .... - !\Ill - r- ~ - 1- r- ~ 1- - ~ r-- FIGURE 1.

Design of the polyculture experiment showing 15 treatment containers each replicated 3 times. Replicate abalone containers hold 10 abalones, replicate mussel containers hold 20 mussels, replicate prawn containers hold 4 prawns, and replicate polyculture containers hold 10 abalones, 20 mussels and 4 prawns. The treatments were arranged as a matrix of 5 levels of the culture factor and 3 levels of the food quantity factor, both of which were manipulated to determine their effects on the growth of test shellfish and on the growth of fouling organisms.

28 r

FIGURE 2. TEST CONTAINER.

INFLOW

COVER

1 1 MM MESH SCREEN

; OUTFLOW PIPE ~ / / ~ . - --l / ~ INNER CONTAINER WATER - HOLDING.MUSSELS N "' - 1- --- ~ [( INNER SCREEN POLYETHYLENE BOX ------.. ,..- • 1. --. 12 CM X 12.5 CM X 12.5 • • I I :. ~ t I I I . II DIATOM PLATES I I I , I .' ' ' 'I "I I ~-·!I.- •.. ··~ .. ------...... - ~ ...... ---- , . / Figure 3a. Regression of Abalone Wet versus Dry Weight 1.00 ,..------"7-----,

0.80

~ 1/) E E o.so Cl ~ .c -Cl ~ 0.40 ?: Y=0.0112+0.4293x R=1.00 c 0.20

0.00 +---....,...---.-----.-----,.---....----1 0 2 3

Live Wet Weight (grams)

30 Figure 3b. Regression of Mussel Wet versus Dry Weight

0.70 -.------r-----,

0.60

0.50

0.40

0.30

0.20 y = 0.0149 + 0.4255x R = 1.00

0.10

-0.00 ;------...------,------~------1 0 2

live Wet Weight (grams)

31 Figure 3c. Regression of Prawn Wet versus Dry Weight

0.50.------.,

0.40

~ 1/) E Cll... 0.30 Cl ~ ..c: -Cl 'Qj s: 0.20 >- c...

0.10 Y= -0.0149+0.2411x R=0.99

0.00 +--'"------.------,.-----.------l 0 2

Live Wet Weight (grams)

32 Figure 4. Phytoplankton Growth Curve

4 Ill Ill Ill Ill Ill Ill 3 Ill E .. Ill Q) c. .!!!. Ill Qj Ill [J (.) 2 0 Ill -Ill r:: 0 Harvest ::1!! Ill Ill

0~------~------,------~_j------~ 0 10 20

Culture Age (days)

Growth of phytoplankton, lsochrysis galhana, used as food in the polyculture experiment. Phytoplankton cultures were grown for seven days, then used in feeding every other day for five days. Points are concentrations measured on a hemocytometer at various times over the course of the experiment. The curve is a third order polynomial fit to the data.

33 Figure 5a. Abalone Growth in length

8~------,

6 e §... "' u"'e 4 .E .c -.."' ..J"'

2

0 M1 M2 M3 P1 P2 P3 L1 L2 L3

Culture and Food Treatments

Bars indicate mean growth(+ l S.D., N = 3) in length or weight of 10 abalone in each of three replicates of each treatment. M = monoculture, P = polyculture, L = low density polyculture. Bars labelled l, 2, and 3 correspond to food levels I, II, and III for each culture treatment (see Figure l). Underline connects culture treatments that are not significantly different (SNK, .05).

34 Figure 5b. Abalone Growth in Weight

0.6 ~rn E ...Cll Cl ~ Q) rn Cll 0.4 ...Q) (J .5 .r:; -Cl "Qi 3:: 0.2

M1 M2 M3 P1 P2 P3 L1 L2 L3

Culture and Food Treatments

Bars indicate mean growth (±. 1 S.D., N = 3) in length or weight of 10 abalone in each of three replicates of each treatment. M = monoculture, P = polyculture, L = low density polyculture. Bars labelled 1, 2, and 3 correspond to food levels I, II, and III for each culture treatment (see Figure 1). Underline connects culture treatments that are not significantly different (SNK, .05).

35 Figure Ga. Mussel Growth in Length

~ E E ~ 1.0 Q) til C!l ...Q) I.) .5 .c: Cl 0.5 -c: Q) -I

M1 M2 M3 P1 P2 P3 L1 L2 L3

Culture and Food Treatments

Bars indicate mean growth(+ 1 S.D., N = 3) in length or weight of the 20 mussels in each of three replicates of each treatment. N = monoculture, P = polyculture, L = low density polculture. Bars labelled 1, 2, and 3 correspond to food levels I, ll, and Ill for each culture treatment (see Figure 1). Underline connects all culture treatments, none of which are significantly different (SNK, .OS).

36 Figure 6b. Mussel Growth in Weight

~ til E 0.06 ...(II Cl ~ (J) til (II ...(J) 0.04 (,) .E ..c:.... Cl 'Qj 0.02 :s:

M1 M2 M3 P1 P2 P3 L1 L2 L3 Culture and Food Treatments

Bars indicate mean growth(+ l S.D., N ~ 3) in length or weight of the 20 mussels in each of three replicates of each treatment. M ~ monoculture, P ~ polyculture, L ~ low density polculture. Bars labelled l, 2, and 3 correspond to food levels I, II, and III for each culture treatment (see Figure 1). Underline connects all culture treatments, none of which are significantly different (SNK, .05).

37 Figure 7a. Spot Prawn Growth in length

~ E E ~ Q) 1/) co Q) 10 ...u c:: s::. Cl -c:: ~

M 1 M2 M3 P 1 P2 P3 L1 L2 L3

Culture and Food Treatments

Bars indicate mean growth C± 1 S.D., N = 3) in length or weight of the 4 prawns in each of three replicates of each treatment. M = monocu1ture, P = polyculture, L = low density polyculture. Bars labelled 1, 2, and 3 correspond to food levels I, II, and III for each culture treatment (see Figure 1). Underline connects culture treatments that are not significantly different (SNK, .05).

38 Figure 7b. Prawn Growth in Weight

1.0

~ 1/) E ...(ll 0.8 Cl ~ Q) 1/) (ll Q) 0.6 ...u .5 ..c: 0.4 -Cl ·a; :s: 0.2

M1 M2 M3 P1 P2 P3 11 12 13

Culture and Food Treatments

Bars indicate mean growth C.±. l S.D., N = 3) in length or weight of the 4 prawns in each of three replicates of each treatment. M = monoculture, P = polyculture, L = low density polyculture. Bars labelled l, 2, and 3 correspond to food levels I, II, and III for each culture treatment (see Figure l). Underline connects culture treatments that are not significantly different (SNK, .OS).

39 Figure 8. Total Production

8

~ 1/) E co ... 6 Cl -Q) 1/) co ...Q) 0 .5 4 .s::. -Cl 'iii 3: 2

M1 M2 M3 S1 S2 S3 A 1 A2 A3 P1 P2 P3 L1 L2 L3

Culture and Food Treatments

Bars indicate total growth(± 1 S.D., N = 3) in weight of all species combined in each of three replicates of each treatment. M = mussel monoculture (total growth of 20 mussels per replicate), S = spot prawn monoculture (total growth of 4 prawns per replicate), A = abalone monoculture (total growth of 10 abalones per replicate), P = polyculture (total growth of 20 mussels+ 4 prawns + 10 abalones per replicate), and L = low density polyculture (total growth of 20 mussels+ 4 prawns+ 10 abalones per replicate). Bars labelled 1, 2, and 3 correspond to food levels I, II, and III for each culture treatment (see Figure 1). Underline connects culture treatments that are not significantly different.

40 Figure 9a. Growth in length of Abalone Fed Meat

~ 2 E E ~ Q) catil ...Q) () .5 ..c: Cl -c Q) ..J

s s s M M M

Starvation Controls Abalone Fed Meat

Each bar represents one replicate of one of the two treatments. Starvation controls were given no food for 32 days, the other three abalone containers were given shrimp meat three times a week in the amounts provided to food group III in the polyculture experiment.

41 Figure 9b. Growth in Weight of Abalone Fed Meat

~en E ...('ll Cl ~

s s s M M M

Starvation Controls Abalone Fed Meat

Each bar represents one replicate of one of the two treatments. Starvation controls were given no food for 32 days, the other three abalone containers were given shrimp meat three times a week in the amounts provided to food group III in the polyculture experiment.

42 Figure 10. Fouling of Test Containers

5~------~

4

~en E 3 ...t'O 0'1 ~ .c:: -0'1 '(jj 2 3:: ...>- c

M .:M M S S S A A A P P P L L L

Culture and Food Treatments

Bars indicate the mean dry weight (± 1 S.D., N = 3) of fouling material growing on container walls. M = mussel monoculture, S = spot prawn monoculture, A = abalone monoculture, P = polyculture, and L = low density polyculture. The three bars for each culture group correspond to food levels I, II, and III, respectively (see Figure 1).

43 DISCUSSION

The results of this polyculture experiment show that the growth of both abalones and prawns was significantly greater in the monoculture treatments than in the polyculture. The growth of these animals was diminished enough by [heir coexistence that the total production of all species combined in the polyculture was no greater than that obtained in the abalone monoculture alone. Only the larger polyculture, with about twice the water volume of the other treatments, produced significantly more shellfish biomass (Figure 8).

A number of factors are involved in the ecological interactions that lead to greater production in successful polyculture systems.

These include direct interactions between the cultured organisms themselves, and indirect interactions mediated by the environment in which the organisms are cultivated. Direct interactions include predation, irthibition, and various forms of competition; a successful polyculture would be one in which these types of interactions are minimized. Indirect interactions depend on such environmental conditions as the diversity and availability of food and habitat, and on the dynamics of intermediate trophic systems. This is especially true of systems based on detrital energy or photosynthesis that regenerate nutrients in a form that can he utilized by the cultured organisms.

In this study, only direct effects of coexistence were investigated to explain the reduction in growth observed in the polyculture treatments. Predation was not significant, if it occurred at all. Few abalones or mussels died in the experiment. A number of prawns died, but it is unlikely that prawn mortality was due to

44 predation by ' either of the molluscs. Some types of physical interference or chemical inhibition of feeding activity may have occurred, but these were not explored.

The possibility of overlap in the exploitation of food was investigated as a possible source of competition between the prawns and abalones. This competition experiment demonstrated that although prawns seemed unable to feed on plates covered with benthic diatoms, abalones seemed fully capable of eating and assimilating the diced shrimp meat that was provided as food for prawns (Figure 9). \;hile starved abalones lost weight, abalones fed only shrimp meat grew at the very high rates of 73 microns/day in shell length and 10 mg/day in live weight. Mottet (1978) mentions incidental consumption of small hydrozoans, copepods, foraminifera, and bryozoans by abalones, and

VanBlaricom and Stewart (1986) observed black abalones consuming

pelagic red crabs. The present study, however, offers the first

reported evid'ence of growth by abalones fed only meat. The abalone's ability to consume food which was intended for the prawns in this culture system indicates the possibility of direct competition for

food between the two species, and may explain the decrease in

production observed in the polyculture. Since all monocultures (and

polycultures) in food levels II and III were given meat (and diatoms

and phytoplankton) regardless of the inhabitant species, monocultured

abalones and prawns each received a full ration of meat.

Polycultured organisms had to share among species, and probably

received proportionately less.

In the unfed treatments, naturally occuring diatoms and other

fouling organisms were available to abalones in all three culture

treatments, and there were no significant differences in abalone

45 growth (Figure 5). Grazing of naturally occuring organisms by abalones probably affected the amount of food available to prawns in the unfed treatments, since prawn growth was significantly higher in unfed monocultures than in unfed polycultures (Figure 7).

There were no indications thit nutrient recycling through detrital food chains offset losses in food availability caused by competition. Although detritus was allowed to build up in the test containers, substantial communities of detrital feeding organisms were unable to develop in the containers with prawns or abalone. In previous work on polyculture systems, polychaete worms were intentionally introduced into the culture system to feed on available detritus and provide food for cultured organisms (Tenore et al.,

1974). These detritivores were cultured in the same containers with the herbivores that produced the detritus, but the cultured carnivores that were to feed on the detritivores were grown separately. In the present study, all organisms were grown together, and continuous foraging by prawns and abalones apparently prevented the production of a large standing crop at the detrital level. In the containers with only mussels, the lack of active disturbance allowed the growth of a variety of organisms including amphipods, copepods, opisthobranch molluscs, various polychaetes and rich carpets of microalgae. A serial polyculture system like that described by Ryther (1972, 1975) and Tenore et al. (1973, 1974, 1976) may be a more efficient way to utilize detrital food chains, allowing them to develop before subjecting them to predation.

Adventitious organisms which colonize the culture containers can be viewed in two ways: if they can serve as food for cultured

46 organisms, they are beneficial trophic intermediaries; if not, they are generally considered as fouling organisms to be eradicated. Spot prawns introduced into salmon net pens in Puget Sound were able to clean nets by consuming fouling organisms which the salmon could not, indicating a distinct advantage for that type of polyculture (Rensel and Prentice, 1979). In the present study, polycultures and abalone monocultures were equally free of fouling organisms, prawn monocultures had significantly more fouling, and mussel monocultures had the most. Prawns and abalones were, therefore, able to consume the organisms which colonized the containers, but in this system, polycultures had no advantage over abalone monocultures in the control of fouling. In a system with only mussels, however, it might prove beneficial to add one of the more active foragers to help control fouling.

In interp_reting the results of this experiment, it is important to keep in mind the age of the organisms and the scale of the experiment. All organisms were juveniles, of an age intermediate between hatchery production and field grow out. Abalones of the size used here were at the weaning stage described by Ebert and Houle

(1984), the age at ~

(Korringa, 1976). Prawns were post-larvae of a size slightly smaller than that introduced into net pens for gro~< out by Rensel and Prentice

(1976, 1979). I proposed that polyculture at this stage might fit into the operations of a multipurpose hatchery, prior to transferring the animals to growout facilities. This polyculture arrangement did not increase production, but the same animals might possibly interact

47 differently when older, or when contained in a larger grow-out type of culture system.

Although the test containers were small and the proximity of the test animals was great, the test densities are comparable to those reported in other culture systems: Abalone were kept at a density of one per 160 cm2 of submerged surface area (including walls, plates, and habitats), or one per 15.6 cm 2 of bottom area. Large polycultures had about 50% more total area and about twice as much bottom area.

Japanese culture system densities range from one abalone per 4 cm2 to one per 89 cm 2 of bottom (Mottet, 1978). Owen et al. (1984) used one per 70 cm 2 as the highest density for similar sized abalone.

Mussels were contained primarily on a 25 cm2 screen in the test containers, giving a density of one mussel per 1.25 em 2 . From calculations based on data by Korringa (1976), densities of one mussel 2 .. · per 0.65 em ·are common for mussels of similar size seeded onto intertidal flats.

Prawns in monoculture containers were held at one prawn per 401 em 2 of submerged surface area, or one per 39 em 2 of bottom area.

Slightly larger prawns grown by Rensel and Prentice (1979) were kept in aquaria at one per 90 cm2,.and in net pens at densities ranging . 2 2 from one per 325 em to one per 1075 em of submerged surface area.

The difference in density between the large and regular sized

polycultures did not significantly affect the growth rates of any of

the test species individually, but it did have a slight but

significant effect on the total growth of all species combined (Table

4, Figure 8). This may indicate that test densities were not unreason-

ably high, but probably approached some optimum range as there was an

48 observable, slight decline in gro,;th as density increased. This test

,;as not designed to establish optimum densities, but comparison of the t~;o densities tested indicates that cro,;ding ~;as probably not a factor affecting the reduced growth observed in the polyculture treatments relative to the monocultures.

The growth of test abalones compares favorably with other published growth rates for similar sized red abalones. Abalones at the highest ration level in monocultures grew a mean rate of 67 microns/day and 7.8 mg/day. Abalones in polycultures at the highest ration level grew at the slower rate of 49 microns/day and 5.2 mg/day.

Abalones of similar size grown at the Marine Culture Labaratory during the same season averaged 62 microns/day and 7.3 mg/ day. Ebert and

Houle (1984) give a mean abalone growth rate of 67 microns/day for slightly larger abalones. Owen et a1. (1984), growing red abalones in

Chile on diatoms and other microalgae reported rates of 44 microns/day. Leighton (1974) reports an annual mean of 61 microns/day, with a monthly maximum rate of 91 microns/day during

October. Red abalones fed Ulva as part of the polyculture project at

Woods Hole grew 47 microns/day (Tenore, 1976). Abalones fed only diced shrimp meat in the present study grew 73 microns/day and 10 mg/day over a one month period in November, a faster rate than any reported except for the monthly maximum rate given by Leighton (1974).

Mussels grew very slowly compared to published values for similar sized M. californianus. The maximum growth rate, from the monoculture

III treatments, was 11 microns/day and 0.7 mg/day. Growth rates previously reported for mussels of similar size range as high as 206 microns/day (Coe and Fox, 1942; this value taken from their graph of a natural population at Scripps pier). Other reported values are 65

49 microns/day (Skidmore and Chew, 1985), 54 to 67 microns/day (Harger,

1970), and 69 microns/day (Tenore, 1973; forM. edulis in polyculture). The reasons for such poor growth in the present study are probably nutritional. A number of filter feeding bivalves have been cultured at the Marine Culture Laboratory, and none have shmm significant growth on unfiltered seawater alone (Earl Ebert, personal communication). The same appears to be true of~ californianus, despite the existence of thriving intertidal populations near the lab's seawater intake pipe. The algal concentrations presented to the fed mussels are within ranges used by other researchers (Schulte,

1975; Kiorboe et al. 1980, 1981; lhight ~ al., 1982), and discontinuous feeding has been shown to be effective with hatchery

reared oyster spat (Langton and McKay, 1976). However, the residence

time of added algae in the test containers (one hour) was less than

that used by>bther investigators. This shorter feeding period was used

because of concern for the health of spot prawns, which have been

shown to be adversely affected by static conditions of high algal

concentration (Rensel and Prentice, 1976).

Prawn growth was substantial, but less than that shown in

previous investigations. Mean growth in carapace length for the best

fed pra~;ns in the present experiment was 136 microns/day for

monocultures, 118 microns/day for polycultures, and 118 microns/day

for large polycultures. The mean growth in weight for the above three

treatments was 11 mg/day, 8 mg/day, and 9 mg/day, respectively. These

values compare to rates of 21 mg/day (Rensel and Prentice, 1980), 31

mg/day (Wickens, 1972), and 35 mg/day (Butler, 1964; from histogram

studies of natural populations). Slower growth in the present

50 experiment may again be indicative of a lower level of nutrition.

Prawns were not fed to excess, and probably could have ingested more food, since no surplus meat from any of the treatments containing prawns (or abalones) was ever left over at subsequent feedings.

The presence of a significant interactive effect on total production is evidence of the interdependence of food quantity and coexistence in the polyculture system. Optimal utilization of available food and regeneraton of energy into edible forms by trophic processes are two of the basic premises for improved production in polyculture systems (Bardach et al., 1972; Ryther, 1975; Dimitrov,

1984). Both of these concepts imply the interdependence of food and species composistion. This interdependence was evident in the present study even though coexistence did not enhance production. In this case, it appears that overlap, rather than diversification, in the utilization of food sources was responsible for the differences in production observed between polyculture and monoculture treatments.

The results presented here re-emphasize the importance of doing

preliminary experiments before implementing new aquaculture systems on a commercial scale. There is little in the literature to suggest the

possibility of competition for food between prawn and abalone, but in

the system described here, carnivorous behavior by juvenile abalones

resulted in competition that reduced production in the polyculture

treatments at all ration levels. Care must be taken in extrapolating

from the results of small scale experiments when designing commercial

scale aquaculture operations (Huguenin and Webber, 1981), but it is

probably a greater risk to assume the nature of a given biological

interaction without prior experimentation.

51 The success of a polyculture operation is most dependent on the choice of species used (Levandowsky, 1977; Gundermann and Popper,

1977), but also on the type of culture system. There are obvious ecological differences between a traditional, earthen, freshwater fish pond, where the polyculture concept originated, and the synthetic containers used in modern hatcheries. The most important distinction is the presence in ponds of a variety of pre-existing trophic levels capable of a wide range of energy transformations. These are difficult to replicate in an artificial setting, and in most cases will probably not be worth the effort in terms of increased production. There are a number of other ways to increase the use of three dimensional space in culture vessels. Furthermore, since the food present in hatchery systems is usually only that which is intentionally supplied [with the exception of microorganisms (Langdon and Siegfried~ 1984)], there is little reason to provide a variety of species to utilize it. Given the increased complexity of feeding and cleaning multiple species cultures in a hatchery setting, the benefit to production would have to be substantial to warrant a polycultural approach in this type of situation. In the case of the present study, it is not.

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62 APPENDIX

PREPARATION OF DIETS FOR TEST SHELLFISH

Three food levels were used in the experiment. Food level I was unfiltered seawater only; level II included additions of diatoms, phytoplankton, and shrimp meat; and level III included more of these three foods, which are described in detail below.

BENTHIC DIATOHS

Diatoms were grown on styrene plates of uniform size. Plates were notched half way up along the vertical axis so that a pair of plates could be fit together to form a free-standing cross, the ends of which fit into the corners of the test containers (see Figure 2).

Each side of each plate had a surface area of 100 em 2 , so that a pair 2 of plates had a total diatom-covered area of 400 em .

At regular intervals dictated by the feeding schedule of each ration group, plates were removed from the test containers and replaced with plates covered with a new growth of diatoms. Old plates were wiped clean of old growth (which was especially prevalent on plates removed from mussel monocultures) to minimize substrate variabilty as these plates were re-innoculated to provide diatoms for subsequent feedings. The diatom films washed from old plates were collected in a diatom culture tank and combined in a dense slurry with diatoms washed from the walls of the tank. The slurry was drained

from the tank and filtered through a 5 urn bag-filter to obtain new innoculum (see diatom slurry system as described by Ebert and Houk,

1984). The cleaned plates were then arranged randomly on three racks

in the diatom culture tank. Racks consisted of long, low, vertical

styrene strips which were vertically notched so that 30 plates fit on

63 each rack cross-wise, each plate parallel to the next, 2 em apart. The tank was then filled with seawater to cover the plates, and the filtered diatom innoculum was added. Plates remained in the static innoculum solution for 24 hours, after which flowing unfiltered seawater was delivered evenly thrciughout the tank by a pipe with multiple holes. Plates 1dth one week's growth of diatoms were re­ introduced into the test containers to be grazed by the shellfish.

The mean dry weight of diatoms on a pair of plates after one weeks growth was 0.25 g. Eight pairs of diatom plates were sampled at random time periods over the course of the experiment to determine the variability of the diatom cover over time, which was 10.1%

(coefficient of variation). Ten more pairs of plates were collected at the end of the experiment after one week's growth to measure variability in diatom cover among replicate plates at the same time, which was 21.0%.

Sampled plates were dried in sunlight for 48 hours and weighed.

They were then washed to remove dried diatoms, dried, and weighed again so that the dry weight of diatoms could be obtained by subtraction.

Ten plates were preserved for microscopic analysis. This revealed that the material covering the plates consisted of organic matter with at least a 50% cover of chain-forming and solitary benthic diatoms. The remainder was a composite of bacteria, detritus, other algae, and various small or larval invertebrates.

The schedules for diatom addition were as follows: food group I - no added diatoms; food group II -- plates replaced once a week (0.25 g diatom dry weight per week); food group III -- plates replaced twice

64 per week (0.50 g diatom dry weight per week).

CULTURED PHYTOPLANKTON

The single-celled, planktonic alga Isochrysis galbana was

cultured in 20 liter Pyrex carboys. Carboys were acid washed, rinsed . with distilled water, filled with an f2 medium of nutrients in 1 urn

filtered UV sterilized seawater (Guillard, 1975), and innoculated with

300 ml of axenic stock culture. Cultures were incubated at 20° C +1°

C, with 24 hour illumination.

Ten carboys were sampled to obtain measurements of phytoplankton

density and variability over time (Figure 4). To test sampling

precision, one carboy was sampled repeatedly 10 times, and the

coefficient of variation for sampling error was 11.0%.

After one week of incubation, at cell concentrations of 2.85 x 10

cells/ml ± 20.3%, volumes were withdrawn from the carboys every other

day to be used in feeding. A new week-old carboy was used each week.

Animals in ration group I received no added phytoplankton; those in

ration group II received 6 ml of phytoplankton 3 times per day, three

days per week, resulting in test container concentrations of 10,000

cells/ml at each addition; and ration group III received 30 ml of

phytoplankton 3 times per day, 3 days per week, resulting in

concentrations of 50,000 cells/ml at each addition. Large polyculture

containers received the same amount of phytoplankton as did the

regular-size containers, resulting in proportionally lower cell

densities within the larger containers.

Hater flowing through all containers was turned off for one hour

during each feeding, and flowing seawater was provided to the bath to

maintain even, ambient Lemperaturesa

65 ~!EAT FOR PRA\fflS

Commercially available frozen whole shrimp were thawed in 12° C seawater. The head and cuticle were removed, and the tail meat diced into small pieces of about .05 g. Animals in ration group I received no added meat; those in group II were fed meat equaling 3% of mean prawn wet weight per day ; and those in group III received a meat ration of 10% of mean prawn wet weight per day. Food was delivered to the test containers in equal servings three times per week. Feeding amounts were adjusted at each prawn weighing (about every 4 weeks) to maintain the ratio of food to prawn weight. Samples of the meat were dried 48 hours at 60° C to determine variability in moisture content.

The mean moisture content of 10 different sized samples of meat was

83%, and the coefficient of variation in moisture content between samples was 3.4%.

Although' diced shrimp meat is not an economical food source for

large scale mariculture operations, similar foods have been used by other researchers (e.g. Wickens, 1972), and they may simulate less expensive protein sources obtainable from seafood processing operations.

NUTRITION PROVIDED BY UNFILTERED SEAWATER

No measurements were made of the amount or type of nutritional

items that entered dissolved or suspended in the unfiltered seawater.

Certain qualitative observations were made which indicate that a

substantial amount and diversity of material was delivered to the

containers through the seawater system. Large particles were excluded

by the 1 mm mesh screening of the container lids, which had to be

cleaned three times a week to prevent blockage. Inorganic nutrient

66 levels were high enough to sustain a rich growth of benthic algae in ungrazed test containers. Phytoplankton concentrations and composition were unknown. Planktonic invertebrates and invertebrate larvae were present, and grew especially in the ungrazed mussel monoculture treatments, which often containe& a diverse fauna including copepods, nereid, serpulid, and other polychaete worms, amphipods to 1 em in length, and, at the end of the experiment, 4 em long opisthobranch molluscs with egg strings. Other material found in the containers included unidentified organic detritus and mineral debris.

67