Advances in the Remote setting of oyster larvae.

Jones, G. & B. Jones, 1988,
BC Min. of Agriculture and Fisheries, 808 Douglas St. Victoria BC Canada V8W 2Z7
PREFACE
The purpose of this publication is to update and clarify the information contained in the Marine Resources report titled METHODS FOR SETTING HATCHERY PRODUCED OYSTER LARVAE, by Jones and Jones (1983). The emphasis of this report is on the tank setting of artificial cultch since significant progress has recently occurred in that methodology. The focus is on problems that larvae setters have recently experienced. We hope the information presented in this text will assist in achieving greater setting success and save the lives of countless unborn oysters.

BACKGROUND HISTORY

The development of modern shellfish hatchery methods has evolved over the last hundred years. The first report of successful artificial oyster spawn occurred in 1879 when Brooks produced "free-swimming" oyster larvae by stripping eggs and sperm from ripe adult oysters. Other researchers attempted to rear larvae in their laboratories; however, it was not until 1920 that W.F. Wells successfully reared and set oyster larvae.

During the past 50 years a great deal of literature has been written on oyster hatcheries. Excellent reviews are contained in the following papers: Wells (1920), Loosanoff and Davis (1963), Matthiessen and Toner (1966), Walne (1974), Breese and Malouf (1975), Dupuy, Windsor, and Sutton (1977) and Jones and Jones (1983). The first commercial transfer of eyed oyster larvae was done by Bill Budge of Pacific Mariculture in the early 1970's. They were producing their larvae at Pigeon Point, California and moving them in buckets of seawater to their setting facility at Moss Landing, California. The method didn't come into widespread practice until 1978 when Lee Hanson built the Whiskey Creek Oyster Hatchery near Tillamook, Oregon, exclusively for the production of larvae for remote setting operations. The first commercial oyster hatchery in British Columbia was built in 1976 by Innovative Aquaculture Products (IAP) on Lasqueti Island. In 1982, IAP's production was large enough to supply four other growers with larvae and subsequently remote setting came into practice on the B.C. coast.

INTRODUCTION

The rapid growth of remote setting in the last few years has now almost replaced the unreliable and costly collection of natural spat on the west coast of North America. Currently, over 40 remote setting operations are in production within British Columbia, more than any other region of the world.

Oyster larvae settlement involves two phases: (1) attachment to substrate and (2) metamorphosis. Settlement is a general term to describe the transition from larva to juvenile. During transition larvae cease feeding, search for a suitable substrate and finally attach to their chosen location. At the time of attachment metamorphosis is initiated.

After the larva has attached to the substratum, the velum and foot are no longer needed. Their disappearance marks the transition from free-swimming to a sedentary mode of life. During the metamorphosis the larval organs disappear and there is an anatomical reorganization of the permanent organs. At this time the relative size of the organs and their orientation are changed. (Galtstoff, 1964) The recent practice of remote setting of larvae a considerable distance from hatcheries or natural setting areas has caused dramatic changes in the British Columbia oyster industry. Since many growers shuck their own oysters, large amounts of discarded shell are on hand for cultch. In the past this material had to be hauled to and from the natural setting locations or hatcheries. Now, instead of hauling cultch long distances, cultch can be artificially set with spat close to the grow-out site.

GENERAL HATCHERY & SETTING METHODS

Ready-to-set larvae are now available to remote setting operations from several hatcheries on the Pacific Coast. The method for producing setting larvae is as follows: Broodstock are conditioned in 15 to 20 C seawater for four to eight weeks. Spawning is then induced by suddenly raising or lowering the water temperature. Should temperature shock not elicit spawning, gonads from ripe oysters are added to the water, In brief, once the larvae have been placed in a tank of approximately 25 C After settlement, spat are held in the tanks two to seven days. The cultch is then removed from the setting tank and kept continuously immersed sub-tidally or covered on the beach until the spat are 5 to 30 millimeters in size. Thereafter, cultch can be spread on the beach. The basic procedure outlined above is frequently fine-tuned by hatcheries and setting operations to suit. Prior to shipment larvae are sieved out of the tank, wrapped in nylon cloth and moist paper towels to prevent dehydration and transported in an insulated container at 50 C. Two and a half million eyed-larvae are the approximate volume of a golf ball. The study by Carlson (1981) showed that holding eyed larvae in this way at 50 C for five to eight days before setting appears to have no effect on the viability of the larvae, However, this length of storage is not recommended by the authors since this stress may contribute to post-set mortality problems.

Larvae should be put into the setting tank of 20 to 270C seawater within 24 hours of leaving the hatchery. In setting experiments maximum larval set occurred at temperatures between 25 - 300 C, with a decrease in setting success at temperatures above 300C (Bruce Henderson,l981). The lethal temperature limit for oyster larvae appears to be close to 300C. We believe that to avoid unnecessary stress setting should be done at approximately 250C. Setting normally starts as soon as the larvae are put into the setting tank with cultch, which can be either oyster shell or plastic collectors. For best success, cultch should be aged for one year in the ocean and be thoroughly cleaned. Some growers add algae to their setting water, although the feeding of setting larvae hasn't yet been proven to be beneficial. From our experience when metamorphosed larvae are fed there is greater initial survival. After the setting process is complete, the cultch is placed on the beach in the inter-tidal zone (covered to protect from the effects of the sun), or hung subtidally.

Several remote setting operators have observed a trend toward increased setting success early in the season, adding to the advantages at this time of a long high-growth period ahead and consequently an early marketability. Some growers have also noted a trend toward higher spat survival for earlier sets.

The environmental problems faced by mariculturists are usually site specific. This is as true for grow-out operations as it is for hatcheries and setting operations.

Our suggestions are based on the general environmental conditions in British Columbia. Each grower operating a setting system will, of course. adapt and improve these to meet their own resources and needs. We would advise growers who are contemplating using the eyed-larvae technique for the first time to visit and observe successful setting operations. Then, after they have evaluated the strengths and weaknesses of these operations, they should set up a medium sized tank to try it for themselves. They should begin by running sets every other week during the summer (keeping accurate records of the amount and condition of larvae added to the tank, quantity of cultch and densities of set). Bthe end of the summer they will not only have a large amount of seed, but a great of practical low risk experience.

The first consideration for a grower is the tank design. Larger tanks are more efficient and economical with regards toloading, unloading and construction. Less material is required per liter (or cultch bag) with a large tank; however, since the stresses are much greater, larger tanks must be built with a great deal of structural support. Smaller tanks conversely are much simpler and cheaper to engineer . Most hatcheries use small size tanks 2.6 meter square and 1.3 meters deep. This tank size gives more control during the set. TANKS SHOULD BE

REDONDA SEA FARMS (RSF), located at Refuge Cove in Desolation Sound, constructed a two tank setting system in 1983 on a 8.3 X 16.6 meter ferro-cement barge. These tanks are both 2.6 meters square and 1.3 meters deep and can be readily loaded and unloaded at any tide directly on or off of the transporting barge. The tanks are fabricated from plywood with styrofoam insulation and coated with a layer of fiberglass. The propane immersion heaters employed bring the tank water up to 23C in just a few hours. The styrofoam insulation is particularly important in keeping heating costs down. RSF found that by using two or more tanks there was greater flexibility to experiment with various cultch types and methods while at the same time setting a control group. Recently RSF has built a new setting facility which is equipped with concrete tanks that are 2.3 x 3.3 x 1.2 meters deep. These tanks are capable of holding 1,458 French pipes (spat collectors) and 4.5 million larvae. That is a density of 3,000 larvae per pipe.

Harmony Sea Foods is located at Granville Bay, Hotham Sound. Their 2.6 x 5.3 meter tank is British Columbia's largest remote setting tank. The water is heated with passive solar heating and has worked quite well during good weather; however, problems have been encountered in cold weather. Electric immersion heaters have since been added as a backup system on overcast days.

Our own company, INNOVATIVE AQUACULTURE PRODUCTS,(IAP) on Lasqueti Island, British Columbia, uses a two setting tank system; one for shell and the other for artificial cultch. The tank used for shell is 5.6 x 2.3 x 1.3 meters deep. It is constructed of aluminum and plywood and finished on the inside with fiberglass. The tank is filled with sand-filtered seawater and heated with a propane immersion heater. All metals should be avoided in seawater systems except cast iron and ungalvanized steel; even some stainless steel has been reported to cause toxicity problems for oyster larvae. Some types or grades of aluminum and stainless steel may be used, but toxicity tests should be made beforehand, It is prudent to use fiberglass lined tanks and plastic plumbing in all seawater systems. In no case should the tank be painted on the inside as some paints contain toxic chemicals that will leach into the water. The toxicity of new fiberglass tanks is a potential problem and may have caused poor sets for many growers during their first sets, If the air inside a covered fiberglass tank has any "fiberglassy" smell, it hasn't cured well enough. The easiest way to cure a tank is via exposure to two months of natural sunlight. To speed up the process a large commercial steam-cleaner can be rented and the job can be done in less than a day. The tank should be covered, the nozzle clamped to point into the center of the tank so that it doesn't blast a hole in the side or bottom and the steam should be allowed to run into the tank for approximately 6 hours. After less than an hour the sides of the tank should be very hot to the touch if the unit is operating correctly, Curing may also be accelerated by placing a high wattage light bulb suspended in the tank and covering the top with a tarp.

REQUIREMENTS FOR TANK OPERATION

FILTERING

Seawater used for setting should be filtered with a 50 or 100 micron filter bag or sand filter and maintained at 22 to 28 C while the larvae are swimming. A non-breakable thermometer is useful. The plastic crystal types that are designed to stick on the surface of aquariums are good, but must not be left in direct sunlight, After the set has occurred, the temperature can be dropped and raw or filtered seawater pumped into the tank to feed the larvae. If algae is being cultured, it is usually added at a concentration that the larvae can consume in less than a day. It has not yet been proven whether feeding with algae improves the setting density but some growers consider it to be a good idea and is not harmful if done properly.
HEATING
The methods of heating are dependent on the source of energy locally available. Electricity is the most automatic and trouble free, but the current required to operate the large electric heaters is not available in many locations. Twenty-five amp quartz sheathed electric immersion heaters are a simple and proven way to heat seawater electrically. Propane is the second most reliable energy source. A simple propane immersion heater can be constructed by putting a propane flame thrower torch inside a 3" or 4" ungalvanized steel pipe that has been formed or cut and welded into a somewhat U shape. The bottom part is placed on the above bottom of the tank with the ends extending the surface of the water. The pipe a should not rest directly on the bottom of fiberglass tanks.

The relative costs of various methods of heating should be considered when designing a setting system. Heating 12,000 liters of water from an ambient seawater temperature of 17 C to a larvae setting temperature of 25 C requires about 500,000 BTUs. A "100 pound" propane tank contains more than enough energy to complete a set.

AERATION
Aeration can be provided by any compressor, air pump or blower that does not produce an oil mist. (For example, do not use piston type paint sprayers which use petroleum oil in the crank case.) efficient and inexpensive blower is the "smog pump" used in General Motors automobile exhaust systems. These blowers are vane type and can be belt driven by a 1/3 hp or 1/2 hp electric motor or gas engine that is installed so that the pump will not pick up the exhaust gases. Wrecking yards should sell these blowers for less than $30. Oyster larvae do not require aeration for extra oxygen, Aeration is provided to create water movement to evenly distribute larvae throughout the tank and to prevent uneven heating of water. The air pump must be positioned above the setting tank so that it will not fill with water when shut off. Tank water should be only aerated gently, not at a boil and only while adding larvae or algae or intermittently during setting. Excessive aeration can cause the larvae to separate out of the water into the foam. On one occasion we witnessed larvae blowing in the wind! Some setters have indicated that heavy aeration greatly increased the number of larvae setting in the inside of their french tubes. The best method to distribute air evenly is to lay pipes drilled with small holes on the tankbottom.
LOADING AND UNLOADING CULTCH
Some sort of mechanical method for loading and unloading the tank should be designed into the system. It is more efficient to be able to unload the tank by an overhead rail or davit directly onto the barge that will transport the cultch. After a tank is unloaded it should be cleaned with dilute (100 ppm) sodium hypochlorite bleach and rinsed out. A plastic bristle broom is a good tool for scrubbing the surfaces.
CULTCH MATERIALS & PREPARATION
Cultch (whatever is used for a setting substratum) must be clean and leached for one year or longer by pre-exposing it to seawater or by placing it high in the intertidal.zone. The actual mechanism that is taking place during leaching is unclear, but it seems to neutralize or draw out toxins contained in the material. During the conditioning process the cultch becomes covered with a thin film of bacteria or micro-fouling that seems to attract the larvae at time of settlement.
SET INDUCERS
Once the environmental conditions are satisfactory for larvae to set, competent larvae appear to respond to a chemical cue to settle and attach themselves. In nature this cue is reported to be a pigmented bacterium called LST, which adheres strongly to surfaces like oyster shell (Weiner and Colwe11,1982). In a number of controlled experiments using different bacterial coatings, it was found that more larvae settled on the LST bacteria coated surfaces than any other. Setting tank operators should be aware that Weiner and Colwell (1982) found ultraviolet killed strains of LST appeared to repel larvae, Simple chemical compounds trigger attachment and metamorphosis of many larval marine invertebrates. DOPA (L-3,4-dihydroxyphenylalanine), an amino acid, has been used to induce the setting of oysters (Cooper,1982). Cooper showed that the use of DOPA will cause larvae to set faster, but does not cause a higher percentage of larvae to set. These findings suggest that DOPA will not increase the percentage set onto oyster shells when the setting is allowed to occur over several days. The use of chemical cues appears to be most applicable to cultchless setting systems where there is no natural attractant.
VEXAR BAGS
When vexar bagged shells are used as the cultch material, shells must be clean and aged for at least two years before being used. The shell chips and powder should be washed off prior to loading into the tank. If cement coated cultch is used, cultch should be leached until the cement no longer affects the pH of the water. Lee Wiegardt of Wiegardt and Sons in Washington State found that when dirty shell was used average counts were 16 per shell, while on extra clean counts of 44 per shell were obtained.

At IAP when bagged shells are used, the bags of cultch are washed with water under pressure from a fire hose before being placed in the tank. The bags are typical of what is used in the B.C. industry (usually 4 feet long, when filled approximately weighing 30 pounds and containing 120 large shells). Three hundred cultch bags are placed in the tank using a 25 foot boom and winch. Approximately 4 to 6 million larvae are added to the aerated water. The water is continuously aerated for the first 8 hours tocirculate the larvae so as to distribute the larvae evenly around the cultch. The tanks are then covered to hold in the heat and exclude the daylight. Daylight can cause an uneven set.

FRENCH PIPE
Redonda Sea Farms have been conducting research on artificial cultch for several years, particularly French plastic pipe. These 2 meter pipes are 25mm diameter hollow tubes with longitudinal groves 2mm deep on the outer surface. Their experiments showed that no larvae set on unleached pipes while those leached intertidally at the 3 meter level for only a week caught between 5-10% of the larvae and new pipes similarly leached for a year caught 2~%. Only those tubes that were put into the setting tank immediately following the leaching process secured a successful set. It appears that air drying of the "attracting film" kills it and makes the cultch repulsive to larvae. It is therefore advisable to leach pipes for as long as possible, and then just prior to use, condition them in seawater filled setting tanks for three to five days. Several growers have built washers for cleaning used pipes. After pipes are washed, they must again be conditioned for 3 to 5 days to allow the larvae attracting bacteria to repopulate the surface. With the older pipes (ones that have been used before) it is possible to get sets of 2,000 to 3,000 larvae per pipe. On new pipe leached for two or three months 400 to 500 is maximum.
SHELL CHIPS
The Frank M. Flower and Sons Co. of Bayville New York on Long Island use setting tanks that are 1 meter by 4 meters by .5 meters. They have 8 tanks and set on 225,000 6mm clam shell chips. This aged shell is crushed by 2 gears and graded by rotary screens, then passed by a fan blower that removes dust and flakes. The shell chips are spread over the tank bottom and the larvae added to the tank. This is a simple and easy method of producing single seed.
CHINAMAN HATS
At IAP we use French made spat collectors called "Chinaman hats" to produce single seed oysters. These plastic spat collectors are dipped in a slurry of cement at least six months prior to setting. The mixture of the slurry is 1/3 Portland cement to 2/3 sand. Water is added till it has a consistency that will leave an 1/8 inch coating on a stick. Each column has twelve Chinaman hats in it and we use 100,000 to 200,000 larvae per column to produce 10,000 to 20,000 seed. After setting the collectors are placed on the beach or hung on longlines. The spat are broken off several months later when they have reached the 10 to 30 mm size. A traditional method for producing single seed oysters is setting on ground shell chips in downwelling chambers. The shell is first smashed with a hammer and sieved on micron calibrated screens to obtain uniform sized shell chips of around 300 microns. These chips are then evenly spread over a 200 micron screen that has seawater pumped down through it. When setting, oyster larvae are added to this chamber and the larvae set on the shell chips. After the setting has finished, the direction of flow is reversed so that the shell chips are "fluidized" (just enough flow to move them around but not to blow them out). This is called upwelling. After the spat have grown slightly they are screened through the same screen the shell chips previously went through and the chips with the spat are retained. The spat are grown in the upweller until they reach a planting size or are moved to nursery trays.

SETTING TANK CONDITIONS

TEMPERATURE

Larvae of C. gigas have a broad tolerance range for temperature and salinity. Tests have shown, however, that larval set increases as temperature increases from 15 to 30C, then decreases beyond that (Henderson,1983; Carlson, 1981), During the summer of 1986 several companies set at 30 C and found very poor post set survival. It appears that while 30C may produce better initial set, the animals are being stressed if this temperature is maintained after setting. Therefore it is recommended that setting tank water be maintained at 20 to 25C,
SALINITY
Salinity also has been shown to effect setting. Maximum larval settlement was found at 30 parts per thousand by tests performed during experiments at Oregon State University (Henderson, 1983). However, Huranka (1981) demonstrated that the survival of C. gigas larvae in various salinities is directly influenced by the ambient salinity at which the adult broodstock were conditioned prior to spawning. It may prove important to adjust setting salinity to that of the broodstock. This would be done by pumping up deeper more saline water or by adding fresh water.
FEEDING
Metamorphosis is a critical stage for oyster larvae because this is the point at which mobility is lost and the internal organs are modified to adapt to a sedentary existence. This process of change in morphology reduces the ability to filter feed for 24 to 48 hours and as a direct response to the reduced feeding activity growth rates are also reduced. This may be one reason feeding has not been shown to be beneficial for setting larvae in all cases. Feeding setting larvae at elevated temperatures may have a negative effect on the percent set since most algae used for larval rearing are killed at temperatures above 25 C, and the resulting cellular decomposition can increase the bacterial load. The velum (the larval feeding organ) of the larvae then can become fouled with the dead algal debris and bacteria which cause disease. Many molluscan researchers (Walne,1965; Schulte,1975; Epifanio ~ Ewart,1977; Gerdes, 1983) have indicated that whenlarval food concentrations exceed a critical level, larval filtration rates decrease. It has been shown that there is an inverse relationship between pumping rate and particle density, so that the number of cells filtered during a given time is relatively constant. Epifanio and Ewart (1977) showed that oysters in algal concentrations greater than 50,000 cells/mi increased psudofeces (uneaten food) production substantially, It would therefore appear prudent to provide algae at densities below this limit to avoid bacterial build-up. Henderson (1983) and Carlson (1981) tested feeding different levels of algae to setting larvae. Both of these studies concluded that there was no statistical difference in the set density between the fed and starved larvae. However, there appeared to be a decline in percent set at concentrations above 50,000 cells/ mi.
ARTIFICIAL FOODS AND DRIED ALGAE
Several companies, including IAP, are conducting feeding trials with extended storage algae and microencapsulated artificial foods. Results to date are very encouraging; it is expected in the next year several larvae foods will be on the market that will have virtually unlimited shelflife.
RECOMMENDED ALGAL FEEDING RATES
A recent development in the feeding of bivalve larvae is the use of centrifuged algae subsequently made into a paste(Jones, Jones and Watson,1986). Algae paste is made by passing dense algae cultures through a continuous flow centrifuge called a separator or clarifier. This spins the algae cells out of the culture water and deposits them on the rotating bowls of the clarifier, These packed algae cells have the consistency of tooth paste and can be refrigerated for several weeks without any loss of nutritional value. Thalassiosira pseudonana clone 3H), a diatom, is an excellent food for large larvae and spat. One liter of 3H will spin down to 0.2 grams of paste. One gram of paste contains roughly 1X10, or 10,000,000,000 cells.

To maintain an adequate concentration of algae in a 6,000 liter setting tank with 3 million larvae at 25 C., we recommend the following feed program:

The algae paste is resuspended by washing it through any mesh size nitex bongo screen. Put about 3 inches of filtered seawater in a plastic bucket and with a plastic cake spatula take the appropriate amount of algae out of its container. Wash this through the bongo by lifting the screen in and out of the water in the bucket and by rolling the algae clumps around on the screen with the spatula or paint brush while it is immersed in the water. Be gentle when washing the algae through the screen because if algae are squeezed through, the cells will clump and crush. When a grower wishes to add more seawater or algae during the sets, he will probably have to lower the level of the water in the setting tank. By draining the water out through a 200 micron (or less) nylon filter he can retain the still swimming larvae and dump the water. Whether or not algae needs to be grown, instead of merely changing the water, depends on the individual farmer's site and on the time of year. We recommend that after the setting is finished(two to three days), a third of the water should be changed per day.

LARVAE DENSITY
During Redonda's 1984 season, their setting success was high, yielding a mean of 624 spat per pipe, a greater density set than is required for grow-out on pipes in suspended cluster culture. This average number of spat per pipe for the year represents a setting success of over 20% ofthe 3 million larvae added to each tank. The higher counts, approaching or exceeding 1000 spat per pipe, were useful for stripping off single seed oysters for grow-out in trays or on the beach. The number of larvae added to the tank depends on the density of set required. Since most growers seem to prefer 10 to 20 spat per shell, we recommend a larval density of 150 larvae per shell. A tank containing 300 cultch bags would take 5 million larvae. Underestimating the amount of larvae needed for a set is a common mistake made by beginning setters. Studies in Washington State (Woelke, 1959 and Jones, 1978) measured the relationship between initial spat per shell and numbers reaching harvest size. These studies showed that the relationship between the number of oysters reaching harvest and the percent survival, yield increased up to 23 spat per shell at planting. The optimum density on unbroken seed was determined to be between 20 and 25 spat per shell. These survival studies also found that initial density must be at least 6 spat per shell to be economically viable.

From the data Redonda collected on "French pipes" during the summer of 1985, the highest percent spat survival resulted from sets of 300-500 per tube. It appears that aiming for 300 to 500 spat per tube makes the most efficient use of larvae and tubes. This would be done by putting 3000 setting larvae per tube into the setting tank. However, Porpoise Bay Shellfish has found they need 5,000 larvae per pipe when pipe is set for the first time.

The F.M. Flower and Sons Co. of New York puts 1.5 million C.virclinica larvae in a 1500 liter tank with 225,000 6 mm clam shell chips on the bottom. This yields about 500,000 single spat at the 30mm size. The hatchery operators, Dave Relyea and Joe Zahtila have kept accurate records of the average monthly yields at the 30mm size for their production. The seasonality of setting success is very well illustrated by this data, however their operation is on the Atlantic Coast which has different oceanographic conditions than B.C, The authors believe that in B.C. surivial of early sets vs. late is even greater.

SETTING LARVAE CONDITIONS
Larvae should always be inspected and evaluated as soon as received from the hatchery. The larvae ball should have a dark brown colour and be moist without any "fishy" smell. The behavior of larvae in a bucket can be used as an indicator of condition and is very important to observe, especially if a microscope is unavailable. When the larvae are suspended in a bucket of water, a large number of the larvae will form vertical chains and produce mucus with which they string together. Since the larvae are crowded in the bucket, don't be too concerned if most of the larvae are staying on the bottom. After the larvae are poured out of the bucket, you should see that some have actually set on the bottom. A compound microscope with a measuring reticule is needed for measuring the larvae to see that the size is 300 microns or larger and that the eye-spot is approximately 14 microns. The lens you need on the scope will be 10X eyepiece and 10X objective lens. You should see foot activity of the larvae. If you don't see them crawling with their foot, remove most of the water from the slide and look again. During the time (usually two days) required to set the eyed larvae, the 25 C water temperature must be maintained. After the set has occurred, the cultch should not be moved until the spat starts to spread its shell out over the cultch surface. This will insure that it is well enough attached and will not wash off. This usually takes three or four days. Light appears to have an effect on the distribution of larvae. It is good practice to cover the tank to reduce heat loss and make a more even set as larvae tend to set in darkened areas away from light. Redonda Sea Farms studied larval batch variability to see if it was a major controlling factor in determining density of set. They tried to identify larval characteristics that would be indicative of setting success. Neither the size nor the range of sizes of the larvae, observed and measured microscopically, were diagnostic of setting success; neither was the temperature of the larvae on arrival. However, the speed with which the larvae began to swim after being placed in a bucket of warm seawater appeared to be somewhat related to setting success. Larvae that were slow to begin swimming generally yielded a lower percent set. Some larval batches which were relatively quick to become active (14 minutes) failed to produce satisfactory sets. Porpoise Bay Shellfish also evaluates the quality of their larvae by their ability to make chains and swimming activity. After a half hour in the setting tank, mast of the larvae should be actively swimming. Porpoise Bay Shellfish changes half of the water in their tank every other day and reheats the water twice a day until the set is finished. They then slowly lower the temperature by doing a half water change daily. This is a good practice as it reduces bacterial and metabolite build up in the tank and lessens the chance of temperature shock while adding fresh natural food.
RECORDS
It is important to keep detailed records of setting results. Wiegardt and Sons said that only occasionally do their average spat counts go below 20 per shell. A typical set would have the following range of spat counts: 7.4% have 0-5 spat per shell, 79% have over 11 spat per shell, 53% have over 20 spat per shell, 13& have over 61 spat per

shell. Since freshly set spat are so extremely delicate, it should be kept in mind that every time a test cultch is removed from the set tank for counting, you are probably causing some mortality. For this reason a different piece of cultch should be counted When sampling test pipes count all of the spat on the first 3cms, the mid 3cms and the end 3cms of at least 3 pipes. During the set, a short piece of fresh conditioned pipe should be added every day to see how the rate of settlement is progressing. Do counts on these as well as counting the number of larvae swimming in a cup of water in order to determine when the larvae have finished setting. Change a third of the water every day after the larvae have set.

POST-SETTLEMENT HANDLING & SURVIVAL
Postset survival is the largest problem facing the remote setter. We believe that the main killer of spat is heat and drying during transfer from the set tank to the nursery area. We have found that freshly set spat can be killed in less than an hour at room temperature by drying. Some cultch types hold moisture better than others. Plastic has very little water absorption and maximum surface exposure to air, so the setter must be very careful when transferring these. The time of larval set appears to be a significant factor in nursery survival. Our data has shown there is a weak correlation between decreasing nursery survival and progress of the setting season, as there also is between initial setting success and progress of the season. A toxicity problem associated with phytoplankton blooms, particularly when blooms are dense, may contribute to the variability in setting and early spat survival that many remote setters are experiencing. Cardwell, et al. (1979) found water samples containing the dinoflagelate Ceratium fucus and Gymnodinum splendens from Puget Sound in Washington State to be highly toxic to oyster larvae. These organisms occasionally bloom in B.C. waters during the summer months, visible as "Red Tides". They have been observed simultaneously with massive larval mortalities in our own hatchery. Ken Wing of Porpoise Bay Shellfish feels there is less problem with postset mortality in earlier sets that have a longer portion of the season to grow. Freshly set spat are very delicate and severe mortalities can be caused by exposure to heat and sun when the cultch is removed from the setting tank. The transfer should be done in cool temperatures, (early mornings or rainy weather) and as quickly as possible.

When we have transferred cultch bags to the beach directly after setting, we have observed that the shells on the exposed top surfaces lose all of their set. We have concluded that the heat of the sun had been too much for the fresh spat. It is better if the cultch is kept submerged or covered for two weeks to a month prior to being exposed to the wind and sun. However, if cultch is kept submerged too long, it will become fouled with mussels, barnacles, starfish, etc. that will smother the spat. If newly set cultch must be held intertidally, mortalities can be reduced by covering with burlap or by spraying with salt water. If the spat on French pipes is to be stripped off to produce single seeds, the correct timing of spat stripping is critical and depends on the density of the set. Adhesion is a function of density; lighter sets have stronger adhesion and must be larger before breaking off.

The Washington State Fisheries department has done several studies on natural seed survival, [Burge. 1984). They found that in the absence of silt, seed mortalities could be substantially reduced. It is suggested that cultch be held up out of the silt during the first winter by placing them on a natural oyster reef, gravel beach or on racks. It appears that during times of poor seawater quality, survival is better if the spat are kept in the intertidal zone rather than hanging subtidally. We have observed this with our artificially set seed and also observed it in natural sets in Pendrell Sound. More research needs to be done on this. However, it appears that the beach seed is less stressed by the summer toxicity problems. Of course, they don't grow as fast on the beach, so each grower needs to evaluate his site and seawater conditions to determine what postset procedure should be used for each set.

CONCLUSION

It appears that the remote setting method is helping to revitalize the British Columbia oyster industry. However, setting problems that occurred during the 1986 season showed that straying too far from the commonly practiced methods can still result in seed shortages. During the 1987 season larvae setters experienced less problems and had few set failures. Larvae users are apparently learning from their past mistakes and now the mortality problems that occur are usually post set problems.

The most common setting problems are still: (1) toxicity of the tank surface, (2) cultch condition (too dirty or not conditioned) and (3) water temperature (too hot or too cold). There will always be unexplainable set variations; however, if detailed records are maintained most of the causes will become apparent.

Several years ago the future of oyster farming in B.C. seemed dependent on a "reliable source of seed". Now that there is an adequate supply of larvae (5 hatcheries are supplying B.C.) and the methods and procedures for its use are established and results consistent, the new problem appears to be water quality degradation. It is pointless to produce seed if the oysters are going to be killed directly by toxic water, transferred to nursery area with too cold water or too little food, or indirectly by diseases caused by the stress of toxic chemicals.

Oyster growers should become concerned about what chemicals are ending up in the water that they grow their animals in and what effects they might expect. Some of the sources of these pollution problems are industrial, (mills and factories) forestry, (defolients sprayed to discourage competing vegetation) pleasure yachts, (toxic antifoulents and bilge cleaners~ fish farms, (toxic antifoulents and from themselves.

APPENDIX A

To help standardize the data being kept by remote setting operations we have made the following setting check list. This should be photocopied and the data recorded for each set. This information will be invaluable for determining why some sets are better than others. A more detailed data collection form is available from the Ministry of Agriculture and Fisheries in Victoria. Most of the questions on the form are self explanatory, but a few need explanations: "Number of larvae swimming in 1 cup" is taken by pouring a cup of setting tank water onto a filter screen so the larvae can be easily counted. This number gives an indication of what percent of the larvae are still looking for a place to set. The approximate numbers that should be found are:
"Number on test cultch" is found by counting the newly set spat on an oyster shell that was hung in the tank the day before. Each day a new shell is added and the 24 hour old.

RECIPE FOR OYSTER SEED

Fill cultch tank with 2 year (or older) bagged shell, Add seawater through 100 micron filter bag. Preheat setting tank to 25C. Mix water well with heavy aeration and add eyed larvae by first putting it in a bucket of setting water, stirring well, then pouring the swimming larvae in scattered areas of the tank. Repeat until all of the larvae is in the setting tank, Maintain 25 C. temperature and intermitant aeration for 48 hours. After 24 hours add 10 gm algae paste. Then 24 hours later, during the cool part of the day, drain 1/2 of the water out of the tank, through the bongo filter, put any larvae retained back into the tank and refill and bring back up to 25 C and add 20 gm paste. Add 30 gm of paste after another 24 hours. Drain all of the water after another 24 hours (during the cool part of the day) and refill with unfiltered sea water. Repeat daily for 2 more days; then unload tank and store set cultch intertidally (covered to protect from drying at low tide) at the 3 to 5 foot level.

REFERENCES

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