' 'I \ \\{ 32 Technical Report Series 32 I Number 73-7

A MANUAL OF REARING

J

by David B. White and Robert R. Stickney

31 31

Georgia Marine Science Center University System of Georgia Skidaway Island, Georgia

81 A MANUAL OF FLATFISH REARING

by

David B. White

and

Robert R. Stickney

Skidaway Institute of Oceanography P. 0. Box 13687 Savannah, Georgia 31406

September 1973

The Technical Feport Serits of the Georgia Mari.ne Scie nce Center is issued by the Georgia Sea Grant Program and the Marine Extension Service of the University of Georgia on Skidaway Island (P. 0. Box 13687, Savannah, Georgia 31406). It was established to provide dissemination of technical infor­ mation and progress reports resulting from marine studies and investigations mainly by staff and faculty of the University System of Georgia. In addition, it is intended for the presentation of techniques and methods, reduced data and general information of interest to industry, local, regional, and state govern­ ments and the public. Information contained in these reports is in the public domain. If this prepublication copy is cited, it should be cited as an unpublished manuscript. Table of Contents

I. Table of Contents ...... 11

II. List of Tables ...... 111

II I. List of Figures . • ...... • • • 1v

IV. Introduction ...... 1

V. Culture Facilities at Skidawav Institute of Oceanography . . • ...... • . • 2

VI. Basic Culture Techniques ...... 4

Supplies of Eggs for Culture. . . . • • • • • . • . • . • • . . . • • • • • • 5

Larval Food and Disease Control 8

Collection and Care of Postlarvae and Juveniles 12

Nutrition of Postlarvae and Juveniles ...... 13

Environmental Conditions for Paralichthys Culture • ...... • . • • • 18

VI I. Conclusions and Recommendations 19

Choosing the Site for Culture ...... • . . . . • . . . . • . . . . . 19

Building the Physical Plant . • ...... • • . • • . . • • • 21

VI II. Bibliography ...... • ...... 25

IX. Tables ...... • ...... 28

X. Figures ...... 31

ii List of Tables

Table 1 Selected Environmental Parameters of some Laboratory Reared

Flatfish . . . 0 • • • • • • • • • • • • • • • • • • • • • • • • • • 28

Table 2 Essential Amino Acid Requirements of Plaice and Sole ..••.•.. 29

Table 3 Composition of Artificial Pelleted Diets of Cowey et al (1970)

and Stickney and White...... • • . • • . • • . . • . • • • . 30

iii List of Figures

Figure 1. Flatfish under culture at the Skidaway Institute of Oceanography:

Paralichthys dentatus (bottom) and Ancylopsetta

quadrocellata • ...... 31

Figure 2. Fiberglass swimming pool filters used for secondary

filtration ...... 32

Figure 3. View of water table with aquarium sized fiberglass tanks in

which postlarval fish are maintained...... • . . . . • . . . . . • . . • 33

Figure 4. Schematic of 1 meter diameter fiberglass tanks for rearing of

juvenile flormder ...... 34

Figure 5. The lower side of a Paralichthys dentatus demonstrating both

ambicoloration and extensive papillomas associated with an

outbreak of the virus Lymphocystis. • ...... • • . . • • • • • 35

Figure 6. Growth of Paralichthys dentatus during 1972 and 1973 demonstrating

increased growth caused by physical plant improvements between

those years...... 36

lv INTRODUCTION

Severe worldwide protein deficiencies that are evident today and promise to increase with the Earth's population have made man aware of one of his last apparent exploitable sources of food -- the sea. However, with our current knowledge of the world ocean as an ecosystem it has become increasingly clear that man is presently exploiting this system at close to its maximum sustainable yield (Ryther, 1969). This fact, coupled with the enormous expense and legal problems that arise when a country maintains a national fishing fleet which fishes the coastal waters of other nations has caused many countries to look into the potential for management of sections of the oceans in an attempt to increase the yield per unit area and thus to minimize the cost of harvesting food from the sea.

Culturing of aquatic ~ se dates back at least two thousand years; however, most of the knowledge gained by early culturists was not recorded and hence is lost to present generations. The late nineteenth century saw an awakening of interest in the rearing of aquatic organisms in North America.

At that time effort was mainly directa:i toward the augmentation of commercial fisheries by the release of large numbers of hatchery reared larval and postlarval forms into the oceans. Tremendous amounts of capital and energy were thrown into work in this area and consequently a considerable amount of knowledge was collected on spawning and rearing techniques, although attempts at supplementation of nat­ ural populations were eventually abandoned when they proved ineffective. The flatfish (Pleur onectiformes) as a group were well represented in this early research

1 and information on this group began to appear in scattered publications as early as the 1880's. In the 1920's financial hardships and unfavorable returns from their efforts caused the closing of many hatcheries. Within this period of time, however, flatfish became established as suitable animals for laboratory studies due to their ease of maintenance. For this r eason scattered information has been compiled until the present.

The object of this report is to gather the pertinent flatfish literature, to condense the most relevant information and to present generalized procedures and recommendations for the production of marketable flatfish from eggs. The techniques presented in this paper were taken from the literature and from work conducted at the Skidaway Institute of Oceanography on three of flatfish

(Paralichthys dentatus, P. lethostigma and Ancylopsetta quadrocellata) (Figure 1).

Cons ide ring that information on numerous species is presented in this paper, it is obvious that a detailed account cannot be given on each; however, further information is available in the bibliography. This paper is meant to be an introductory presentation to the layman who desires to work with one or more phases of the life cycle of the flatfish, and should serve as an introduction to the literature and culture techniques for interested scientists. An annotated bibliography bas been prepared by us to provide additional reference material (White and Stickney,

1973).

CULTURE FACILITIES AT SKIDAWAY INSTITUTE OF OCEANOGRAPHY

A flow through water system is utilized in our culture system. Water is taken from the Skidaway River, passed through a gravel filter, secondarily

2 filtered through sand swimming pool pressure filters (Figure 2), and UV steri­ lized water is then heated to desired temperature by a fuel oil heater equipped with a stainless steel heat exchanger. Prior to the 1973 experiments the secondary filter and UV systems had not been installed and bacterial disease problems wer e encountered. These problems have largely been avoided since the system was modified. A detailed description of the water system and culture building is presented in a technical report by White, ~ al. (1973).

Postlarval flounder are reared after capture in 50 l fiberglass culture tanks which are placed in water tables for temperature control (Figure 3). The water is static within these tanks where the fish remain until they reach between

0. 1 to 1. 0 g. The water tables can be accurately maintained at a desired temperature by adjusting a series of valves which mix ambient and heated salt water and allow it to flow around the culture tanks. The large reservoir of heated water within the water tables also serves to reduce heat loss from the culture tanks during power failures or shutdowns of the water supply system for maintenance or repair. One meter diameter fiberglass tanks, each supplied with running water and equipped with a Venturi drain system are used for fish once they outgrow the aquarium sized tanks (Figure 4 ).

During the design phase a recirculating system was considered as a possibility but abandoned in favor of the flow-through system presently in use.

Several reasons were involved in that dec is ion.

1) A recirculating system requires a biological filter to remove waste products from the water, and while such filters have been designed and used in freshwater systems with varying degrees of sue cess, the technology of biological

3 filters in conjunction with marine systems is generally lacking. After some experience with biofilters we realized that inordinate amounts of time and effort could be spent in developing and maintaining these filters.

2) A biological filter was deemed impractical since running several experiments simultaneously would require an individual filter for each experiment and treat- ment. For systems where the mixing of effluents would not create problems a closed or semi-closed system might be more practical.

3) Experiments testing various environmental parameters such as salinity and temperature would be difficult in a closed system because of mixing of treatment waters in reservoirs and filters. In practice only one treatment could be run at a time. This is neither temporally or scientifically desirable.

4) Because of the clay turbidity, included organic matter and bacteria asso­ ciated with raw Skidaway River water ,an elaborate filter system would be required even in a closed system since makeup water must be available on demand.

BASIC CULTURE TECHNIQUES

The pleuronectiform fishes generally have small pelagic (floating) eggs which follow normal symmetrical tEieostdevelopment through the early larval stages

(Norman, 1934). Pearcy (1962) discussed other flatfish with demersal (sinking) eggs. At a late stage of development, metamorphosis, the characteristic flatfish features (lateral compression of the body and migration of one of the eyes from one side of the body to the other) are manifested. After metamorphosis, the flatfish acquire their benthic habit which will persist for the rest of their lives.

As would be expected, the stress put on the animals in changing from planktonic or pelagic to benthic habits and from the physical changes in body configuration are

4 gr eat. Young flatfish are heavily preyed upon by larger fishes, including adult flatfish; thus , in nature only a small fraction of the larval flatfish survive meta­ morphosis . This is made up for by the fact that females often lay over one hundred thousand eggs per spawning season, depending on species.

SUPPLIES OF FLATFISH EGGS FOR CULTURE

There are three means by which fertilized flatfish eggs may be obtained by culturists:

1. Collection by plankton nets

Flatfish generally spawn in the winter months. Depending upon species, the spawning ground may be in inshore estuaries or in deep offshore waters.

Shortly after fertilization, the pelagic eggs may be easily obtained by surface plankton net tows over the spawning area with nets of 1 mm mesh size or smaller.

The eggs are first sorted from eggs and larvae of other species, then placed in appropriate sized containers and supplied with gentle aeration until they can be returned to the laboratory. Unfortunately, there is little a vai.lable information on identification of the eggs of flatfish and one usually ends up with an assortment of species.

2. Natural spawning in confinement

A second and more sophisticated means of obtaining fertilized eggs is to trawl for sexually mature or maturing adults on the spawning ground and re­ turn them to spawning tanks on the shore. Sexing of flatfish is usually accom­ plished by holding the up to a strong light source and observing the internal anatomy. In many species the sex can readily be determined by the presence or lack of distended ovaries which extend posteriorly in the abdominal

5 cavity. Of course, as the flatfish approach sexual maturity the distinction becom es more obvious even to the extent that most running ripe females exhibit such s wollen ovaries that the outline of those organs may be easily distinguished externally on the fish. In any case, several animals of both sexes are usually placed in the same tank to insure fertilization.

Shelbourne (1968) has had considerable success with plaice (Pleuronectes platessa) using tanks 3.7 x 3.7 x 1.2 meters deep and placing 30 to 40 adults

(30 to 40 em in length) in each tank. These tanks are kept in an area of low illumination and have a slow flow of sea water. Plaice readily spawn in these tanks with no artificial inducement. Of course, when using a flowing sea water system, screens must be placed over all exit ports to prevent the loss of the pelagic eggs.

This means of obtaining flatfish eggs eliminates the tedious step of sorting vast amounts of mixed plankton which often results in fatal damage to eggs.

Conversations with W. W. Anderson of the Georgia Game and Fish

Commission reveal that gravid summer and southern flounder (Paralichthys denta 1lls and !? . lethostigma, respectively) are not taken in trawls made by the

Commission in the estuarine and nearshore waters of the state. It is possible that

Paralichthys do not spawn in Georgia waters, or that their spawning ground is located in deeper water, as found for_!?. denta1lls off New England (Smith and

Fahay, 1970). During the winter of 1972-73 two_!?. lethostigma females which were maintained in a 2 m diameter fiberglass outdoor tank at Skidaway Institute of

Oceanography were found to have developing ovaries. No males were available at the time, thus no attempt at spawning could be made. One of these fish

6 subsequently died during a severe cold spell, while the other resorbed her eggs

when the water began to warm in the spring. The fact that some development occurred is encouraging, and it is felt that spawning under controlled conditions can

be carried out once sufficient brood stock are collected. It was initially felt

that deep water might be required for spawning; however, the development of_E.

lethostigma in the 1 m deep tanks indicated that laboratory spawning might be

feasible.

3. Artificial spawning in confinement

A third approach to obtaining flatfish eggs is by artificial spawning. This technique has been followed for years by fisheries biologists working with various species of fish. Basically the technique is as follows: The ripe female is grasped with one hand over the head region, the other hand about midpoint on the animal.

Slight pressure is applied to the sides of the fish (i.e. top and bottom of flatfish) by both hands while at the same time moving the hands toward each other. Eggs will flow freely from a running ripe female. The eggs are usually extruded into a bucket with about 1 em of sea water at the same temperature from which the adult fish were taken. Milt from the male is deposited in the same manner over the eggs and the mixture is gently stirred and allowed to sit for 3-5 minutes. Excess milt

is then washed from the eggs by filling the bucket with fresh sea water. Fertilized pelagic eggs will float to the surface within a few hours and can be skimmed off by dip netting. The remaining water, milt and unfertilized eggs are then discarded and the fertilized eggs are washed with several changes of the proper temperature

7 sea water (see Orcutt, 1959; Hildebrand, 1930; Smith and Fahay, 1970; Mito, et al.,

1960; Gutherz, 1970 ; Houdi, ~ al. , 1970; Miller and Marak, 1962, for details on embryological development). This method is the primary method used in selective breeding.

Difficulty may arise when fish eggs are crowded, the consequences of which often cannot be distinguished until after hatching. Our work and work conducted by Shelbourne in Great Britain has indicated that a hierarchial structure is set up in flatfish populations early in their lives. Hence, the early hatchlings become the dominant fish in the tanks and may prevent smaller fish from obtaining sufficient food. Juvenile oscellated flounder, Ancylopsetta qu3()rocellata, and both Paralichthys dentatus and ~· lethostigrna with which we have worked showed this pattern when post­ larval individuals of different sizes were stocked together at low stocking densities.

The pattern often disappeared at high densities. Shelbourne (1968) suggested a stocking density of 1, 000 to 2, 000 eggs per tank (60 x 30 x 30 em filled to a depth of 20 em), but the optimum stocking number probably varies considerably and should be determined for each species.

LARVAL FOOD AND DISEASE CONTROL

To obtain the largest percentage survival of flatfish eggs and larvae environ­ mental conditions must be optimized. Ideal temperature and s alinity requirements for development vary according to species. The effects of parameters such as photoperiod, light intensity, stocking density and other s may also vary. Optimum environmental parameters will have to be discovered by additional work on each species since, in general, such work has not been carried out (see Table 1).

There are, however, two major concerns common to all larval fish development

8 that should be considered: food (including food presentation) and disease control.

Early flatfish culturists used phytoplankton as larval food, apparently believing that larval pleuronectiform fishes were herbivores. In all cases m ortality rates were near 100 percent. Rollefsen (1939) discovered that several marine larval fish are carnivorous and readily accept the easily cultured brine shrimp,

Artemia salina. Since that time nearly all flatfish rearing techniques have in­ cluded the use of brine shrimp as a food for larvae and postlarvae. Several other types of food, including chopped oysters, fish, shrimp and naturally occurring zooplankton have been used as food with some success. Since most of these foods are dead when served, two major drawbacks are present: 1) the fish must be trained to accept non-living food since they are sight feeders and normally accept only living food (de Groot , 1971), and 2) the dead and decaying food may promote bacterial and ammonia build-up in the tanks unless a running sea water system is utilized. Non-living foods may be excellent for juveniles after the flatfish have developed sufficient hunting prowess, but for larvae and postlarvae

A. salina is probably the best readily available food item.

The readily available brine shrimp A. salina is collected from two main sources: The Great Salt Lake, Utah; and San Francisco Bay, California. Nauplii, hatched from canned eggs, are the main food source for larval marine fishes in the lab, although freeze-dried adult A. salina are also available. Hatching is accomplished by vigorously aerating sea water to which the brine shrimp eggs have been added. The greatest percentage hatch occurs at 25 C and 25-30 ~ salinity. Hatching takes place in 24-48 hours depending upon environmental

9 conditions.

To facilitate feeding and minimize bacterial buildup problems in fish culture tanks, the hatched nauplii are usually separated from the unhatched eggs and egg fragments. This is easily done by taking advantage of~. salina's positive photo­ taxic response. In our culture system a light is placed over the open end of a half covered opaque container and the nauplii and egg mixture is added to the covered side. The nauplii readily swim to the lighted end and are concentrated below the light. An even more thorough separation is obtained by placing a solid partition between the lighted and dark ends of the tank. When a narrow slit is left parallel to the bottom of the tank near the bottom, the brine shrimp quickly swim through the slit to the lighted side of the tank. Most of the unhatched eggs and egg frag­ ments remain on the dark side.

To make brine shrimp more visible to larval Paralichthys we have found that an opaque rearing tank with bright overhead lights is advantageous. Shelbourne

(1968) recommends a black lining inside the rearing tanks and a light intensity of 500 m. c. (meter candles) at the water surface (as measured with a light meter sensitive to the vertebrate eye) for the culture of plaice. Light intensity can be reduced gradually as the eyes of the fish become better developed and as the animals become efficient hunters [see Blaxter (1968) for a discussion on light and vision in plaice].

Normal bacterial counts of about 1, 000/ml are average in the sea (Zobel!,

1946) but may increase several thousand-fold when eggs have been added to sea water under hatchery conditions. Attempts to control marine bacteria with ultra­ violet light have met with some success. Oppenheimer (1955) found that several

10 combinations of drugs controlled marine bacteria in the presence of marine fish eggs and that a combination of penicillin with streptomycin in concentrations of 50 parts per million (ppm) of each drug in the water was effective in reducing bacterial populations. yet not detrimental to the larval fish. Shelbourne (1963) found that a single dose of 50 IU of sodium penicillin G per ml and . 05 mg strep­ tomycin sulphate per ml of sea water in a static system was effective in controlling bacteria in the presence of plaice eggs. Shelbourne placed fertilized eggs in the static system until the eggs hatched and removed newly hatched larvae daily to another tank of running water to begin their development through the larval stages.

Not only did treatment produce a larger percentage hatch of plaice eggs, but those larvae which hatched in the treated tanks were significantly stronger than those in untreated tanks.

Due to contamination of the Skidaway River by coliform and other bacteria, disease problems plagued our research during 1972. The placement of secondary filters and UV sterilization in the water system prevented a recurrence during

1973. Among the diseases we experienced during 1972 was an outbreak of

Lymphocystis virus (FigureS~ While the fish were not killed directly by this disease, their feeding was impaired by growths around the mouth, even1ually leading to starvation , and growths on the fins cleared the way for bacterial infec­ tions. The danger of introducing disease by obtaining fish from the wild is great, another good reason why controlled spawning should be attempted.

Prophylactic treatments of formalin (about 25 ppm) biweekly have been used to retard or prevent disease outbreaks. We do not recommend such treatment unless

11 indicated by poor water quality (in terms of bacteria) or suspected disease. Several

U.S. government publications are available which help the culturist identify and control disease problems and usually can be obtained from County Extension

Agents of the U. S. Department of Agriculture.

COLLECTION AND CARE OF POSTLARVAE JUVENILES

Since gravid Paralichthys sp. have not been obtained from Georgia coastal waters, it has been necessary to collect flounders for culture studies as postlarvae and juveniles. Initial attempts at collection of suitable stock by otter trawling during 1971 resulted in a low number of animals varying significantly in size at capture. During January and February, 1972, about 2000 postlarval Paralichthys sp. were collected from the vicinity of Beaufort, North Carolina in 1 meter diameter

1 mm mesh plankton nets. During the same months in 1973 postlarvae were collected both from North Carolina and from the Skidaway River in Georgia.

Nets are set from stationary objects on incoming tides after dark and checked for contents every 1/2 hour. Towing of nets usually results in poor success since the animals are large enough to a void the boat wake and are, thereby, missed by the net. Smaller mesh or diameter nets have also proved to be impractical because the increased size of the hydrostatic cone which exists in front of these nets also causes avoidance.

Upon return to the laboratory the postlarval flounders are placed under static water conditions in fiberglass 50 1 capacity tanks at the temperature and salinity of capture, or the temperature and salinity in which they were brought to the lab. They are maintained under those environmental conditions for about one

12 week after which the tempera1llre is gradually raised to 25 C (from about 5- 10 C

at cap1llre) in two degree increments per day for several days. The salinity is

maintained during this period at about 25 °/oo .

The fish are fed brine shrimp ~ salina) nauplii ad libi1llm twice daily

beginning immediately after their arrival at the laboratory. We have experienced

no difficulty in establishing the postlarvae on Artemia.

The fish are stocked in the fiberglass tanks at a density which allows them

all to settle on the bottom of the tanks with a total coverage of no more than

50 percent of the substrate area. Aeration is provided by a blower and air stones.

Ammonia tends to concentrate in the static system and the water must be changed

at least twice weekly . The ammonia level should never be allowed to exceed 0. 5 ppm,

although the fish can survive levels over 1 ppm for short periods.

NUTRITION OF POSTLARVAE AND JUVENILES

With the use of~ salina as food it is possible to produce metamorphosed

flatfish. The small amount of a·vailable literature on cultivation of flatfish past

this stage of development is recent because, as already noted, earlier culturists were interested only in producing larval flatfish to supplement natural populations.

Juvenile flatfish have been reared on several types of natural food: chopped fish or

shrimp, oysters and even living shrimp; however, these items are often difficult

to obtain or maintain and may vary widely in nutritional content. Artificial diets,

on the other hand, can be easily stored and have precise nutritional content.

Aquaculture and many types of research on flatfish must rely on suitable artificial

diets. Unfor1llnately, little nutritional work on marine or estuarine species has

13 been undertaken. Thus, initial dietary investigations must be carried out using basic nutritional principles as well as nutritional information obtained from work conducted on freshwater or anadromous fish.

Early in our research a means was found by which flounders caul d be converted from natural living foods to artificial diets. We found that only a small percentage of flounder would directly consume artificial fry food (pellets) after they had been feeding on~. salina. The majority of the postlarvae would, however feed on frozen chopped penaeid shrimp. After a two week period floating, laboratory prepared freeze-dried shrimp was substituted for the frozen shrimp. The fish then began feeding from the surface. When floating commercially available pellets were fed a few weeks later the majority of the flounder converted to the artificial diet. This procedure was relatively simple since several thousand animals at the postlarval stage can be held in a small area. A single freeze dried shrimp was sufficient to feed several hundred animals for one day. Floating pellets proved to be superior to sinking pellets since uneaten portions could easily be removed from the tank with dip nets. This prevented fouling of the water which often occurs with sinking pellets. Also, since the fish feed by sight and are attracted by motion, the floating pellets may have stimulated feeding behavior for a longer period than did sinking pellets which, once they reached the bottom, seemed more difficult for the fish to locate.

Aquaculture presents several unique problems which are not found in terrestrial culture systems - one of these is the method of presentation of food.

In terrestrial animal husbandry a major problem is presentation of the ration with

14 an odor or taste that leads the animal to consume the feed. In aquatic systems one has this problem also, but more importantly one must present the ration in a form that does not disperse rapidly in water (a condition which results in benefit only to microorganisms). Most often the ration is presented as a pellet, varying in s ize depending upon age and species being cultured. Feeds used for small scale laboratory studies usually are high protein rations containing a binder such as gelatin, agar or finely divided collagen. Water may be added to form paste which is extruded through a large syringe and either dried or frozen for storage. Larger scale operations require a pellet mill or mechanical extruder which have the advantage that both the length and diameter of the pellets may be easily controlled. In addition, much larger amounts of ration can be prepar ed at one time, insuring homogeneity of the diet throughout an experiment. Freezing of a ration after it has been dried prevents microbial decomposition of the pellets and subsequent poisoning of the fish as a result of peroxidation of lipids in the ration. Normally no more than two or three weeks ration is left at room temperature.

Rather extensive dietary research has been carried out on the freshwater salmonid fishes (trout and salmon) and on the channel catfish, Ictalurus punctatus.

Of the five general classes of nutrients: vitamins, lipids, carbohydrates , minerals and proteins, detailed work with flatfish nutrition has been conducted only on proteins.

However, a preliminary study by us showed that sucrose and glucose produced better growth than does corn starch in the oscellated flounder (A ncylopsetta quadrocellata). Kitamikado, et al. (1964) have published similar data on the rainbow trout. Their work demonstrated that the digestibility of certain proteins

15 decreased as the starch content of the diet increased. J . W. Andrews (personal communication), on the other hand, has shown that channel catfish, !: punctatus, are able to utilize starch at high levels. He feeds corn starch at up to 40% in purified diets. It might be speculated that trout and at least some flatfish that are normally carnivorous have not developed the enzyme system necessary for utilization of large amounts of starch whereas catfish and other fish which ingest considerable amounts of carbohydrates, due to their omnivorous or herbivorous feeding behavior, have developed a system to obtain benefit from this class of food.

Cowey et al. (1970a) succeeded in defining the essential amino acid requirements of the plaice (Pleuronectes platessa) and sole (Solea solea) and found them to be qualitatively similar to those of the Pacific salmon (Table 2). In a follow­ up study, Cowey et ~· (1970b) determined that increased protein levels up to 70 %

(as casein) produced increased growth. They did not determine if levels higher than this would pr oduce more rapid growth rates. Work conducted by us at

Skidaway Institute of Oceanography with.!: quadrocellata showed similar results.

Semi-purified diets with up to 70 % fish meal (43. 5% protein) produced better growth as the percentage of fish meal increased. In addition to pelleted diets, groups of

A. quadrocellata were fed chopped frozen fish and shrimp ad libitum. Results over the same period showed a growth rate on these "natural foods" of nearly twice that of the highest protein pelleted diet. Whether this is attributable to a high protein level found in these foods or to some other factor is not yet clear.

Summer flounder, Paralichthys dentatus, when fed in our laboratory a

16 commercially available fish feed of 40% protein have shown growth rates exceeding those in nature for this species as reported by Smith (1969), Hildebrand and Schroeder

(1928) and Smith and Fahay (1970). Figure 6 illustrates the growth of thirty X: dentatus from one gr am to over 120 grams during 1972. Several power failures and water system malfunctions during this time caused widely varying temperatures a nd other less than optimum conditions to exist for a large portion of the time. Figure 6 represents an encouraging growth curve for this species. Recent changes in the water system (White et l!J. , 1973) have reduced some of the earlier problems and allow the maintenance of a more suitable environment in the cultur e tanks. Growth of a group of 30 rapidly growing P. lethostigma captured during January, 1973, is also presented in Figure 6 and shows the results of modification in the water system.

Table 3 presents the composition of diets producing the best growth rates for Cowey et al. (19 70b), and our work with A. quadrocellata. Cowey fed a moist pellet extruded from a large veterinarian syringe. The dry components of the diet we re mixed 1:1 with water to which a taste attractant (assorted amino acids) had been added. The diet was stored under refrigeration until used. In our studies dry pellets were fed at a rate of 5% of the total biomass within each tank daily.

Pellets had been extruded wet from a small laboratory extruder (Model X-5,

Wenger Manufacturing Company, Sabetha, Kansas, U.S. A. ) and dried overnight at

50 C. Fish meal and fish oil present in the diet appeared sufficiently attractive to induce rapid consumption. It should be noted that corn starch -is the primary carbohydrate in this diet. As stated earlier, substitution of glucose or sucrose would be expected to produce better growth. It should also be noted that other

17 va riations between the two diets are also found in the vitamin, mineral and fat content, yet both produced positive growth.

ENVIRONMENTAL CONDITIONS FOR PARALICHTHYS CULTURE

Deubler (1960), Deubler and White (1962) and Peters and Angelovic (1971) have demonstrated that postlarval_£. lethostigma and~· dentatus grow most rapidly at salinities as high as 30 °/oo. Stickney and White (1973) have demonstrated s imilar results for postlarval Paralichthys lethostigma. Their data further indicate that salinity requirements change rapidly with age and that within a few months after capture juvenile southern flounder grow most rapidly at salinities as low as 5 to 10 °/oo . This change in optimum salinity requirement with age probably relates to the normal migrational pattern of Paralichthys. Fish of this hatch in highly saline offshore waters and then move to low salinity estuarine waters to grow. They eventually move back offshore during the winter months where they breed as 2 or 3 year old adults.

Peters and Angelovic (1971) demonstrated that 30 C produces the most rapid growth in postlarval summer flounder. However, the exact temperature requirements of summer and southern flounder juveniles have not yet been defined, although maintenance in the range of 20-30 C has provided excellent growth and feed consumption in our studies.

We have found that late larval and early postlarval R· dentatus and R· lethostigma reared in our laboratory feed well with a surface light intensity of 300 to 500 foot candles (1 foot candle = 10.76 meter candles). This intensity is obtained by placing a four foot double bulb fluorescent light fixture approximately three feet above the water's surface.

18 CONCLUSIONS AND RECOMMENDATIONS

Choosing the Site for Culture

Studies conducted with Paralichthys dentatus and P . lethostigma at

Skidaway Institute of Oceanography have acquainted us with many of the problems involved with flatfish culture and we have been able to overcome a few of them.

The feasibility of rearing Paralichthys sp. on a commercial scale has not been ruled oot, but a good deal more information will be required before a profitable venture can be undertaken.

The selection of a proper site is probably the most important step toward establishing a facility for the culture of P . dentatus orE· lethostigma. Ideally, the location should include:

1. Low levels of dissolved or suspended solids. Water of low turbidity is highly

desirable in culture facilities because outbreaks of disease organisms are

often associated with particulate material in the water. The lack of particulates

is also desirable in that extensive maintenance cleaning of the culture facilities

is eliminated and for aesthetic reasons. Feeding is also enhanced in clear

water since the fish are better able to see food particles. ·~ Turbidity

must be kept low in culture facilities. The culturist has the choice of

costly filtering or finding naturally low turbidity water prior to initiation of

culture.

2. Low bacterial and viral levels. Bacterial disease outbreaks are costly and

often difficult to control while viral infestations are nearly impossible to

control. The stresses placed on animals under aquaculture conditions create

severe problems. In general, good water quality and proper nutrition provide

19 a degree of protection against disease. All fish carry disease organisms

and outbreaks are likely following any period of stress.

3. The site should be located away from any type of industrial or municipal

pollution.

4. The water supply should be 5 to 20 °/oo salinity. Higher salinity water could

be diluted to the appropriate salinity but increasing salinity through use of

additives is not recommended for reasons of economics. Flounder are

able to live through most natural oscillations in salinity but may go off food

during these periods.

5. Water with a constant tempera1llre of approximately 25 C (77 F) should provide

r apid growth. Although no tempera1llre requirement experiments have been

carried out with juvenile flounder w ~. results in our lab have shown

that temperatures of 30 C (86 F) retard growth and mortality increases

rapidly at temperatures above this level. Both E. denta1lls and g. lethostigma

are able to withstand temperatures of less than 10 C (50 F); however, growth

is reduced at temperatures below 20 C (68 F). Optimum tempera1llres

(between 20 and 30 C) may not be available throughout the year at any

location; however, the site should be chosen in an area where extended

periods with water temperature within this range occur. Water may be obtained

by pumping surface waters or from salt water wells (if such wells are

feasible in the area selected). Certain types of salt water wells may provide

constant temperature throughout the year, but in most cases some variability

should be expected.

20 6. The site should be located near readily available stocks of brood fish, eggs or

postlarvae. One of the greatest problems encountered in work with Paralichthys

sp. in Georgia was the unavailability of large numbers of postlarvae and

juveniles for stocking. Plankton netting for laboratory animals is feasible,

but the number required for an aquaculture venture would be prohibitive

in many cases unless an area was found in which extremely high densities

of these fish were found. Maintenance of brood stock, or collection of ripe

adults from the field and subsequent artificial spawning provide the only real

hope for successful aquaculture of these species. The development of strains

of rapidly growing, hearty animals will ultimately depend upon artificial

spawning of selected brood animals and will require a number of years to

develop. At present the natural variability in growth rate of flounder captured

from nature is a problem. Our work has indicated that temperature is the

most important aspect of initiation of gonad development in adult animals .

We strongly recommend that future aquaculturists learn to maintain and

propagate brood fish now that the feasibility of rearing the offspring has

been demonstrated.

Building the Physical Plant

Certain characteristics are required in the construction and operation of a

proper physical plant for the cul1ure of flounder. In our attempts to evolve a

working physical plant we have found that the least costly facility requires a high

initial capital outlay. Many of our recommendations are described in detail in a

previous technical report (White ~al., 1973).

1. Construction of a well insulated laboratory. During embryonic and larval

21 development flounder must be reared indoors under precise control of

environmental conditions. Temperature control can only be considered if

a well insulated laboratory is provided. A block building over a cement

floor with good ceiling insulation provides adequate temperature control if

the proper water heating and or cooling equipment is installed. With proper

site selection, temperature modification can be avoided resulting in a great

economic saving. A network of permanent floor drains to carry water from

the building eliminates extensive plumbing. Epoxy paint over the walls

and ceilings facilitates cleaning and is well worth the extra expense. Plumbing

should all be of PVC (polyvinyl chloride) including all pipes, valves and

fittings. Metal valves (even brass) last for only a few months in the

corrosive atmosphere of salt water. If metal is required anywhere it should

be of high quality stainless steel.

2. Construction of outdoor culture facilities. Our research has been confined to

laboratory scale studies, and it is obvious that a viable aquaculture operation

would require much more physical space. Tank culture does not appear to

offer a realistic possibility for a commercial venture at this time, and most

aquaculture operations would presumably be confined to ponds or enclosed

lagoons, bays, cages, etc. We have not had an opportunity to work with

such outdoor facilities, but some of the limitations to be expected with

such installations are readily apparent. Water quality deterioration associated

with increased oxygen demand as a result of high density stocking, supple­

mental feeding and potential noxious algae blooms is a primary consideration.

If the system is open to a natural saltwater area of any type the presence of

22 predators and fouling organisms within the ponds or other enclosures can

be expected. In addition, fish and other organisms which enter the ponds

from the outside will consume food meant for the flounder. As we have

already mentioned, the constant threat of disease outbreak is always

present and would be enhanced in cages, lagoons and ponds receiving

tidal flushing. These realities are associated with any outdoor marine system,

and many of them apply to freshwater also. Since salt water wells are not

always a real alternative, some filtration of pond water may be required

to remove unwanted organisms.

3. Substrate requirements. Virtually all of the fish which we have reared beyond

a size of 5. 0 em have shown greater or lesser degrees of ambicoloration.

By the time the fish reach a year of age the extent of ambicoloration is often

great (Figure 5 ). This could have an effect on marketability of fish which

are normally sold with head and skin intact. The development of pigment

on the underside of flounder may be preventable if the fish are provided with

a substrate in which they can bury (Stickney and White, submitted for publica­

tion). Such a substrate should be made available soon after the fish are

captured and must consist of extremely fine material in which the small

flounder are able to bury. Fine sand effectively reduces the amount of light

reaching the lower surface of the flounder buried in it and thus prevents

the development of dark pigment on the blind side of the fish.

In conclusion, it appears feasible to rear some species of flatfish under controlled or semi-controlled laboratory conditions; however, basic knowledge on

23 specific environmental and nutritional parameters is not now available for most

flatfish species. Some of this information is beginning to appear in the literature

as interest in flatfish aquaculture increases and as scientists are discovering the

importance of these fish in the oceanic food web. When experience has led to the

development of adequate skills by aquaculturists good survival rates should be

achieved through the critical larval stages and be yond to market size. Once the

postlarval stage is reached it appears that the flatfish are characterized as being

extremely hearty and easily adaptable to laboratory conditions.

ACKNOWLEDGMENTS

The authors with to thank Messrs. Daniel Perlmutter and Rodney Zeigler

for assistance in obtaining and maintaining the flatfish used in research carried

out at Skidaway Institute of Oceanography. We also wish to thank Ms. Barbara

McNair and Nancy Fair for typing the manuscript.

This work was supported by the Sea Grant Office of the National Oceanic

and Atmospheric Administration under Grant Number 1-36009. Tbis paper

is meant to serve as a final report to Sea Grant as regards the flounder project of Sea Grant Number 1-36009, as well as serving as an outlet for

information derived from that project.

24 Bibliography

Barry, R.J., S.B. Saila and D.B. Horton. 1965. Growth studies of winter flounder. Pseudopleuronectes americanus (Walbaum) in Rhode Island. Trans. Am. Fish. Soc. 94:259-264.

Blaxter, J. H. S. 1968. Light intensity, vision and feeding in young plaice. J. Exp. Mar. Biol. Ecol. 2:293-307.

Breder, C.M. , Jr. 1923. Some embryonic and larval stages of the winter flounder. U.S. Bur. Fish. Bull. 38:311-316.

Brice, J. J. 1898. A manual of fish-culture, based on the methods of the United States Commission of Fish and Fisheries. Reports of U.S. Comm. Fish. 1897.

Cowey, C.B., J.W. Adron, A. Blair. 1970a. Studies on the nutrition of marine flatfish: The essential amino acid requirements of plaice and sole. J. Mar. Bioi. Assoc. U.K. 50:87-95.

Cowey, C .B., J. W. Adron, A. Blair and F. Pope. 1970b. The growth of 0-group plaice on artificial diets containing different levels of protein. Helgolander Wess. Meer. 20:602-609. de Groot, S. J . 1971. On the interrelationships between morphology of the alimentary tract, food and feeding behavior in (Pisces: Pleuronectiformes). Neth. J. Sea Res. 5:121-196.

Deubler, E.E. 1958. A comparative study of the postlarvae of three flounder (Paralichthys) in North Carolina. Copeia. 1158:112-116.

Deubler, E.E. 1960. Salinity as a factor in the control of growth and survival of postlarvae of the southern flounder, Paralichthys lethostigma. Bull. Mar. Sci. Gulf and Carib. 10:338-345.

Deubler, E.E. and J.C. White. 1962. Influence of salinity on growth of postlarvae of the summer flounder, Paralichthys dentatus. Copeia, 1962:468-469.

Gutherz, E.J. 1970. Characteristics of some larval bothid flatfish and development and distribution of larval spotfin, Cyclopsetta fimbriata (Bothidae). Fish. Bull. 68 (2):261-283.

Hildebrand, S.F. and W.C. Schroeder. 1928. Fishes of the Chesapeake Bay. Bull. U.S. Bureau Fisheries. 43:366pp.

25 Hildebrand, S. F. and L. E. Cable. 1930. Development and life history of fourteen teleostean fishes at Beaufort, N. C . Bull. U. S. Bureau of Fish. 46:383-488.

Houde, E.D., C.R. Futch and R. Detwyler. 1970. Development of the lined sole, A rchirus lineatus, described from laboratory-reared and Tampa Bay specimens. Fla. Dept. Nat'l. Res. Research Ser. No. 62:43pp.

Howell, B. R. 1972. Preliminary experiments on the rearing of larval lemon sole Microstomus kitt (Walbaum) on cultured foods. Aquaculture 1:38-44.

Kitamikado, M. , T. Morishita and S. Tachino. 1964. Digestability of dietary protein in rainbow trout II. Effect of starch and oil contents in diets and size of fish. Bull. Jap. Soc. Scient. Fish. 30:50-54.

Miller, David and R.R. Marak. 1962. Early larval stages of the fourspot flounder, Paralichthys oblongus. Copeia. 1962 (2) :454-455.

Mito, S. , M. Ukawa and M. Higucch. 1969 . On the egg development and rearing of the larvae of a flounder, Kareius bicoloratus (Baselewsky) with reference to its spawning in the culturing pond. Bull. Nanei Reg. Fish. Res. Lab. 1:87-102.

Norman, J.R. 1934. A systematic monography of the flatfishes (Heterosomata). Vol. 1: British Museum N.H.: 459 pp.

Oppenheime r, C. H. 1955. The effect of marine bacteria on the development and hatching of pelagic fish eggs and the control of such bacteria by antibiotics. Copeia. 1:43-49.

Orcutt, H. G. 1950. The life history of the starry flounder, Platichthys stellatus (Pallas). Calif. Dept. Fish. Game Fish Bull. 78:64pp.

Pearcy, W.G. 1962. Distribution and origin of demersal eggs within the order Pleuronectiformes. J. Cons. Perm. Inter. Explor. Mer. 27(3):232-235 .

Peters, D. S. and J. W. Angelovic. 1971. The effect of temperature, salinity and food availability on growth and energy utilization of juvenile summer flounder, Paralichthys dentatus. ·Proc. Third National Symposium on Radioecology, in press.

Poole, J. C . 1966. Growth and age of winter flounder in four bays of Long Island (Pseudopleuronectes americanus). N.Y. Fish and Game. J. 13:206-220.

Rae, B.B. 1965. The Lemon Sole. The Whitefrtars Press Ltd. London. 106pp.

26 Rollefsen, G. 1939. Artificial rearing of fry of sea water fish. Preliminary communication. Rapp. Cons. Explor. Mer. 109(3):133-134.

Rythe r, J. H. 1969. Photosynthesis and fish production in the sea. Science. 166:72-76.

Scott, W.C.M. 1929. A note on the effect of temperature and salinity on the hatching of eggs of the winter flounder (Pseudopleuronectes americanus, Walbaum). Contrib. Can. Biol. 4:137-141.

Shelbourne, J . E. 1953. The feeding habits of plaice postlarvae in the Southern Bight. J. Mar. Bioi. Ass . U.K. 32:149-161.

Shelbourne, J . E. 1963. A marine fish-rearing experiment using antibiotics. Nature (London) 198:74-75.

Shelbourne, J. E. 1968. In "Marine Aquaculture" Edited by W. J. McNeil, Oregon State University Press. Corvallis, Oregon:15-36.

Smith, R . W. 1969 . Analysis of the summer flounder (Paralichthys dentatus L.) population in the Delaware Bay. U. of Delaware M.S. Thesis. 71pp.

Smith, W.G. and W.P. Fahay. 1970. Description of eggs and larvae of the summer flounder Paralichthys dentatus. U.S. Fish and Wildlife Ser. Res. Rep. 75:21 pp.

Stickney, R.R. and D.B. White. 1973. Effects of salinity on the growth of Paralichthys lethostigma postlarvae reared under aquaculture conditions Proc. 27th Ann. Conf. S.E. Assoc. Game and Fish Comm.: in press.

White, D.B. and R.R. Stickney. 1973. Annotated bibliography of flatfish (Pleuronectiformes) research, Ga. Marine Science Center Technical Report Series: in press.

White, D.B., R.R. Stickney, D. Miller and Lee H. Knight. 1973. Sea water system for aquaculture of estuarine organisms at the Skidaway Institute of Oceanognp hy. Ga. Mar. Science Center Technical Report Series: in press.

Zobell, C. E. 1964. Marine Microbiology. Chronica Botanica Co. Waltham. Mass.: 240 pp.

27 TABI.l·. I

SJ:.Lf:CT£0 EN1.1110NMENTAL PAilAMf:TEils OF SOME L,\ BOHATOH\ Jli.AilLil FU rF1~11

1.(;(; IH. I LUlP~tf.:-11 LAlli AL OJ:. I ELOPMENT Jt \.EN 111.1ll:\ 1'101'\I J.N; time lo tlmt· rr()m ytk. abs. food [cd umc to sin· ul ,C), or uptlll\Uin pC' ak ll.mp. S;tliOJI\ rt·rt. lo Satlnlty :~ fl. hatch upon ylk. metamor- S;ilinll\ tlnw to ,ldUh 1'1.!~"' nf p.i\\'OinJ,!; spa wru n~ -,ji.IWIIIIll! !'ish l P. P. r. h:ttl'hln~ Temp. P.P.T. (rln,l's) nbsorp. phosls Temp. P. P.1. Sl':\ . nLit. ·~ml idlJi l ll'nl l ~ ~l' ISflll dqllh(lllt Art.cmla \l hirU"\ nauplu lint· LW:o; and small 10-11 -,-1,- 1\) .!i =2 :1 1""'2 J "'da •s zooolonk. davs .1:1 n-Jul' Sh:lilow 0> ;\IH' I'Oslnmus \;ti'H.'li h.iu 2-3 35 - 10 100,000 loc:tlh ft- :1) 11-1:1 :141 Hda ·s fi-10° 30 months 6-10° 30 :! years em 700,00(1 fi.S 1\t:t r- .\u • loll -1 1111 a r.t IC' 1 \ 'S flt.·nt;I W S growth 100 .000 f1-7l Artemia b~s t I•• :J:! li hours J0-1 5° 25-35 naup li i a1 40 :10 - 40 300 000 !'\0\ - Jk.·l' :.!-, - 111 Artemia Pl:ltit:hth\s nau plil .. td l:iw ~ and 0-35 11, 000- 0 l'l ).! ..-. ,,.., hours 12. 5° .. - 5 otht:rs tn nature 2-3 years 40 em 000 10-1:} l>t.:t·-.Ltn -, ..

l ~kUI'Orl l 'l"lt:~ pi.tlt.'S~:t Artemla 100-150 I~ I - Ill 1 11-l :r-, ~1 davs 7-~0 35 4 - 5 Dat.r.plii 49davs 11-12° 35 5 vea.rs oou 1•~ •.: udup ll'U- soo, uou ~ tnH'nt•;tnu~ 1.)- }M lll-151 :1-;J :~:? •., d;J\"!; 2- :l 32.3 12- H 60dil\"S 32 32- 3 3- 4) cars 1 billion 1-1 h ·h-.\ l.tl

11• -JIIIlltk', t.'t :11. oq~"· ' ffil- PC'Iers und A!lebvlc (1971) (11) - Bern , et ol. (19(ifi) I~~ -!lti\\P}J t 1 ·t':2t (il- Smith nnd Fahav ( 1970) (12) - Bredor (19nl .:,, - H :II,' t 1 ~~~;.-,I ( 8)- Orrutt (1950) (1 3) - Drice (11{9M) , ,, - Dl'uhl t· r 11!1.-, ... , (9)- Shclbourne (1953) (14)- Poole (1966) ',;, lh ldt•hr;mcl a nd Lahll' 1 J!l:lllt (I 0)- She! bourne (1968) (15) - Scott (1929) TABLE 2 ESSENTIAL AMINO ACID REQUIREMENTS OF PLAICE AND SOLE1

Regutrements in Requirements in Amino Acid Plaice Sole

Alanine

Arginine + +

Asp artie Acid

Cysteine

Glutamic Acid

Glycine

Histidine + +

Isoleucine + +

Leucine + +

Lysine + +

Methionine + +

Pheny!alanine + +

Proline

Serine

Threonine + +

Tryptophan N N

Tyrosine * *

Valine + +

1. Mter Cowey, Adron and Blair (1970) . + Indicates a dietary source required - Indicates a dietary source not required from ordinarily available material. * Probably from hydroxylation of Phenylaline. N Not determined.

29 TABLE 3

COMPOSITION OF ARTIFICIAL PELLETED DIETS OF Cowey et al., (1970) * and Stickney and White

COWEY et al. * Skidawa;y Ins t. Studies COMPONENT % of dry diet % of dry diet

Protein 70 (as casein) 43.5 (70% of diet as fishmeal)

Dextrin 7.3

Vitamins 4 . 5 1.0

Minerals 4.0 2.5

Corn oil 4.0

Cod liver oil 3.0

Fish oil 1.0

Alpha cellulose 2.0 10.5

Corn starch 10.0

Binder 4. 0 (as guar gum) 5. 0 (as agar)

Food attractants .2

* Cowey, Adron, Blair and Pope (1970)

30 Figure 1. Flatfish under culture at the Skidaway Institute of Oceanography: Paralichthys dentatus (bottom) and Ancylopsetta quadrocellata.

31 Figure 2. Fiberglass swimming pool filters used for secondary filtration. Figure 3. View of water table with aquarium sized fiberglass tanks in which postlarval fish are maintained. ..-A

B

On e meter culture tank. A. 6 inch .outer ~tand pipe. B. ll inch inne r s tand pipe. C. Floor of tank with slope t awa rd the te~te r. D. ltinchthreadedP.V.C. flangewithleginserted. E . l r tnch e lbow with flexible hose connection for drain .

Figure 4. Schematic of 1 meter diameter fiberglass tanks for rearing of juvenile flounder.

34 j I

Figure 5. The lower side of a Paralichthys dentatus demonstrating both ambicoloration and extensive papillomas associated with an outbreak of the virus Lymphocystis. 140

120

100

80

~ -0 u., ...,: 0) ~ 60

40

1972 • • 1973 A A 20

4 8 12 16 20 24 28 32 WEEKS

Figure 6. Growth of Paralichthys dentatus during 1972 and 1973 demonstrating increased growth caused by physical plant inprovements between those years.