LIFE CYCLE

Life cycle of Strombus gigas, a marine gastropod. (Illustration by Bonnie Bower-Dennis)

ANATOMY

Anatomy of an adult female queen conch. Male is similar except has verge (see previous page). (Image from Caicos Conch Farm)

Anatomy of a 4-day-old queen conch larva (veliger).

NAGUABO HATCHERY AND NURSERY

The Naguabo Fishing Association building provides space for the fisher-operated Queen Conch Hatchery and Nursery.

This facility is also a showcase for the wider community to learn about queen conch biology, fisheries, aquaculture, and restoration.

This 18′ × 18′ (324 ft²) facility is designed to grow 2,000 juvenile conch per year to 3.2″ (8 cm) in shell length to restock seagrass meadows or to stock coastal pens for production of sustainable seafood.

FIELDKIT

To collect egg mass sections, it is necessary to bring a field kit on the boat containing the following:

Note: A permit is required to collect egg masses from the wild

FIELD COLLECTION

INCUBATION SYSTEM

Now that the egg mass sections have been transported to the hatchery, they must be transferred to the incubation tank, which holds up to eight incubation cylinders. Egg masses will stay in this system until the day the embryos are ready to hatch. Temperature plays an important role in the development of the embryos. The temperature range to incubate the egg masses is 26- 30 ^oC, with 28 ^oC being ideal. At this temperature the eggs will hatch in three to four days after collection.

An incubation tank with eight incubation cylinders.

Illustration of an upwelling system. The bottom of each cylinder has a screen mesh (60 - 70 µm) which allows water to upwell through it and drain into a pipe manifold. This provides even water flow over the developing egg mass strands. The incubation tank is on a recirculating system, meaning the water is being used over and over again.

INCUBATION OF EGG MASSES

Continued

EGG DEVELOPMENT

CHAPTERO VERVIEW

LARVAL TANK SYSTEM

Water in the larval tanks is considered static, meaning the tank is filled once with filtered, UV-sterilized seawater, and does not leave the tank until a manual water change is done (p. 27). Optimal culturing temperature inside a larval tank is 28 ^oC (82 ^oF) and a salinity of 36. Veligers, however, can be grown at temperatures 24 - 32 °C (75 - 90 ^oF) and salinities 26 - 40.

Larval tanks have a conical bottom with a one-inch diameter drain at the lowest point. The conical bottom should be a gentle slope angled at 30 to 45 degrees. If the slope is too steep the conch larvae will continually touch the sides causing them to retract their lobes or damage their shells. Larval tanks come in many sizes, but they operate more or less the same way. Here a 1,000-L tank is set up for a water change.

Each tank is set up with a one- to two-inch diameter PVC standpipe that stands two to three inches above the water level. The standpipe, prevents veligers from getting trapped in the drain hole.The top of the standpipe should be even with the tank rim, or slightly below it, so that a lid can be placed on top. The lid prevents bugs and dust from falling into the tank.

To keep the veligers in suspension, a PVC airlift (6″ dia × 6″ high; 15 cm × 15 cm) with two airlines (no airstones) attached to either side is used. The airlift hangs on a thin polypropylene line from a notch in the top of the standpipe and hovers right above the bottom of the tank (0.5 cm).

Illustration of airlift and airlines. Air flows in a circular motion inside of the tank. This keeps the veligers from sinking to the bottom.

DENSITY IN THE LARVAL TANKS

Each larval tank is 68 liters or 18 gallons. It is very important that the conch larvae are stocked initially at a relatively low density (∼ 200 / L; 760 / gal) equaling approximately 13,600 veligers per larval tank. Throughout the larval cycle, the density in the tanks is reduced as the veligers get older and larger. The ending density should be 10 - 20 veligers per liter meaning 680 - 1,360 veligers per larval tank.

LARVAL DENSITY TABLE

This table is based on a 21-day larval cycle, but veligers can grow faster or slower depending on the environment (ex: feeding, density, temperature). Monitoring shell length and development will assist in determining the correct sieve mesh size to use for a water change. Growth rate is usually 35 - 50 µm per day. This table assumes a 40 µm average daily growth rate or 80 µm for a two day period.

SUPPLIES:

MICROSCOPE OBSERVATIONS

There are three columns in the Larval Rearing Data Sheet that require using a dissecting microscope, also known as a stereo microscope. The ‘number of lobes' and ‘food in gut' can be observed after the veligers have been placed on a depression slide. The ‘shell length’ is determined with the use of a small ruler built into one of the microscope eyepieces called an ocular micrometer. Here is information to help fill out these three sections.

While looking through the microscope, consider the following:

SIGNS OF HEALTHY & UNHEALTHY LARVAE

Golden gut typical for younger veligers (healthy).

Dark gut typical for older veligers (healthy).

Round, fullly extended lobes (healthy).

Stunted lobes and pale guts (unhealthy).

MEASURING SHELLS WITH MICROMETER

The next step is to determine the shell length of five individuals. Conch veligers, however, are tiny and even at the end of their larval cycle they are only about the size of a pinhead (1,000 µm = 1 mm). It would be impossible to measure them with a regular ruler, this is why an ocular micrometer is used. The micrometer disk fits into the eyepiece of the dissecting microscope for use with the dominant eye.

MEASURING SHELLS WITH MICROMETER

Although the veligers do not look like adult queen conch yet, they do in fact have a tiny shell, the same one they will keep their entire lives. When looking at an adult conch, the very tip of their apex is the initial larval shell from which it grows more and more whorls. As the veligers develop over the course of their larval cycle their shells will grow from 1.5 whorls (Stage 1) to 4 whorls (Stage 5).

The illustrations below show how to measure shell length, from apex to siphonal canal. Stage 1 and Stage 2 veligers sometimes need to be measured from their apex to their beak due to their shape and the way they lay on the slide. This will still give an accurate measurement.

To measure the shell of veligers, pipette a few individuals using the eye dropper. This commotion causes their lobes to retract into their shell, making the veligers easier to measure. One by one, rotate the eyepiece with the micrometer to position it to measure the shell length. Repeat on five veligers. In this image you can see some veligers are marked with a red line as an example of how to measure newly-hatched veligers.

LARVAL DEVELOPMENT: OVERVIEW

Now that shell development has been described, this next section will discuss the morphology of veligers from the moment they hatch (Stage 1) to when they are ready to metamorphose (Stage 5). Conch larvae are considered zooplankton, microscopic marine animals that live in the water column. To accomodate this environment and lifestyle they have lobes which allow them to swim, feed, and breathe until they settle and become benthic snails.

VELIGER DEVELOPMENT

SHELL DEVELOPMENT

LARVAL FOOT DEVELOPMENT

The veliger foot has two parts: the propodium (front of foot) and the metapodium (back of foot), which has an operculum (larval, and later on adult clawlike structure). The foot can be used as an indicator organ for determinig the stage of larval development and has many functions during the larval cycle such as:

FOOT DEVELOPMENT

Illustrations from D'Asaro 1965

LARVAL DEVELOPMENT: DETAILED

STAGE 1 DAY 1

Newly-hatched veligers have a distinct, elongated beak, two velar lobes, and a light-colored gut. Although they hatch with a yolk reserve they are planktotrophic and will need to feed on phytoplankton. The black eye spots and orange pigments on the foot are visible. Shell length is 300 - 350 µm and the shell has 1.5 whorls.

STAGE 2 DAY 4

The shell has an elongated beak that projects over the aperture. The velar lobes have indented to form 4 lobes. Siphonal canal is visible. The foot has expanded and the orange pigments multiply near the bottom of the foot. Phytoplankton makes the veliger digestive glands look golden. Shell length is 430 - 470 µm and the shell has 2 whorls.

STAGE 3 DAY 6 - 10

Cleavage of the third pair of lobes completed by day ten. The digestive area has expanded and is golden brown with phytoplankton. The shell continues to have an elongated beak. The shell length is 510 - 670 µm and the shell has 2.5 whorls.

STAGE 4 DAY 12 - 16

The 6 lobes have elongated and the shell beak has mostly receded. The digestive area is dark brown. The foot has greatly expanded and the adult operculum claw is visible. Some of the orange foot pigments have turned to dark geen spots on the metapodium. Some veligers exhibit swim-crawl behavior. The shell length is 750 - 910 µm and the shell has 3 whorls.

STAGE 5 DAY 18 - 21

The eyes of competent veligers are at the base of their tentacles. Pigments on the foot have changed from orange to dark green. Ctenidium (gill) and osphradium are visible. Buccal mass is developing. The digestive area is dark brown to green. The larval shell has no beak, and has reached terminal larval shell length of 1,000 - 1,200 µm. The shell has 4 whorls. Many veligers exhibit swim-crawl behavior.

METAMORPHOSIS DAY 21

The metamorphosed conch has lost its velar lobes and crawls with its foot. The eyes have partially migrated up the tentacles and the proboscis is used for grazing. The shell goes from smooth whorls to ridged whorls. The shell length two days after settlement is 1,300 - 1,600 µm.

LARVAL DEVELOPMENT SUMMARY

This figure is based on development during a typical 21-day larval cycle from hatch to metamorphosis. The larval cycle length varies depending on conditions such as temperature, density of the culture, and feeding quantity.

CRITICAL NOTES

Improper handling: It is extremely important to handle veligers gently during water changes and when the larvae are in the tanks. If the water change is done too fast the veligers may land roughly on the sieve, which can cause breakage of the shells. Veligers will then need to spend time repairing their shells rather than growing their shells larger. If the veligers are kept on the water change sieve too long, the aeration in the tank is too fast, and/or the veligers are overfed this will cause stress and they will secrete mucus chains.

Inadequate food supply: Veligers will begin to feed on microalgae the next morning after hatching (day 1), however, veligers will not fully begin to utilize this food source until day three or four. By day four the embryonic food in the yolk cells in the stomach has decreased completely and by day ten the albumen cells have decreased completely. Sufficient microalgae early in development will make the veligers more robust for the later stages. Therefore, it is recommended that veligers are fed starting on day one.

Correct amount of food supply: It is important to follow the Microalgae Daily Feeding Table (p. 31) to determine how much microalgae to feed the veligers each day. Feeding amounts are also determined based on veliger density, development stage, and how much food they have in their digestive gland. Too much food will cause mucus chains, which can cause clumping of the veligers.

Contamination: Bacteria and protozoans (ciliates) will proliferate under unhealthy conditions, which can be caused by not removing the hatched egg mass soon enough or introducing a contaminated egg mass to a tank, too much food or poor-quality microalgae, and not removing debris or dead veligers during water changes. Additionally, beginning with the swim-crawl stage, the veligers will tend to spend time at or near the bottom of the tank. It is important to keep the bottom clean or these late stage veligers will come in contact with metabolic waste. Keep the aeration higher to keep the late stage veligers in suspension.

Disease vs. toxicity: Disease is caused by an organism, typically a bacterial infection such as Vibrio. The larvae will grow normally but show a high mortality usually between day seven and ten. Bacteria can be introduced from the microalgae, aeration system, or from the seawater. Toxicity usually occurs from the system such as new tanks and piping. It could also come from the air or water. The larvae do not grow and mortality is high, usually starting on day four. Both of these situations can cause 100% mortality also known as a crash of the larval batch.

WATER CHANGE

The water in the larval tank should be changed every other day, starting on day 2, to allow the tank surfaces to be cleaned. This removes bacteria films and prevents harmful bacteria and protozoans to proliferate in the tanks. During a water change the veligers are siphoned from the tank into a sieve (10″ dia × 12″ h) with the appropriate mesh size (p. 15), to cull out the stunted and dead veligers.

Water changes also allow larval density to be adjusted. For example, if the density is double the recommended amount, the water in the tank should be siphoned only half-way to collect the veligers needed. The other half should be transferred into another tank with lower density or be released into the wild.

Only start a water change once the Larval Rearing Data Sheet (p. 16) is filled out because the status of the larval culture must first be determined such as larval density and microscope observations of the vital signs of the veligers.

FEEDING VELIGERS

Veligers hatch with yolk reserves, and will begin feeding on single-celled microalgae six to eight hours after hatching. Therefore, to increase larval survival and vigor, the veligers should be fed the morning after they hatch. From then on, feed veligers daily and it is recommended to feed them shortly after water changes as this stimulates them to swim and be active feeders. See Chapter 4: Growing Microalgae for more details.

MICROALGAE DAILY FEEDING TABLE

This feeding table is used to determine how much microalgae to feed veligers in each 68-L larval tank.

FROM VELIGER TO S N A I L:

Competent veligers wait for a cue to trigger settlement.

Natural cues found in seagrass and sandy substrate habitat are used to trigger metamorphosis.

A subset of larvae are presented this cue in the hatchery setting.

The swimming larvae metamorphose into bottom-dwelling snails. (Image by LeRoy Creswell)

MORPHOLOGICAL AND BEHAVIORAL CHANGES

As the conch go through metamorphosis, many changes occur in their method of movement, feeding, and respiration. During these changes the veligers use a lot of energy, therefore, it is important to make sure that they are well fed and cared for prior to metamorphosis. For instance, a few days prior to competency, veligers are fed the lipid-rich diatom Chaetoceros gracilis to give them extra energy. Here are the key changes the conch go through as they transition from larvae to benthic snails.

TRANSFORMATIONS

LARVAE: Six elongated lobes.

SWIM-CRAWL: Lobes and foot are visible.

SWIM-CRAWL: Lobes and foot are visible.

BENTHIC SNAIL: Lobes absorbed, snout and foot are visible.

PREPARING THE METAMORPHOSIS CUE

Seaweed extract of the red macroalga Laurencia poitei or a small dose of hydrogen peroxide are both reliable cues that trigger metamorphosis in the hatchery setting. These cues should induce approximately 75% of the veligers. The success of metamorphosis for a batch of larvae is dependent on the uniformity of the veligers in the larval culture. Therefore, it is important to cull the slow growers during water changes. Also, testing a small group of veligers before inducing the whole tank of larvae will ensure that the culture is ready for metamorphosis.

To determine the potency of the extract, a test set is done:

Before any new batch of Laurencia extract is used on the large-scale, the dosage is determined by placing 25 metamorphically competent veligers in three different extract concentrations: 7, 10, 15 ml of Laurencia extract / L of seawater for four hours.

This test set can be done in small 50- or 100-ml polypropylene tri-beakers. After removing the conch from the extract, percent metamorphosis is determined using a dissecting microscope. A minimum of 60% metamorphosis is considered effective to select a given dosage of Laurencia extract for use on the large-scale. The test set only needs to be done once for each new batch of extract.

PREPARING FOR METAMORPHOSIS

Veligers need to go from a swimming stage to a benthic stage and this is a big transition for them. If the veligers are not developed enough, or too developed, they will not be able to complete metamorphosis and will die. There is only about a five day window when they can be successfully induced, typically beginning around day 21.

Transferring correct amount of veligers to metamorphosis trays:

Assuming that there are 10 veligers / L in the larval tanks, there would be 680 veligers per larval tank since the larval tanks are 68 L.

Thus, only two trays out of the four will be used for inducing metamorphosis for this one larval tank. If there is another larval tank with veligers ready for metamorphosis, the same procedure can be done and the veligers can be added to the remaining two trays.

INDUCING METAMORPHOSIS

CARING FOR METAMORPHOSED CONCH

Following metamorphosis, the conch will look like tiny snails under the microscope and will grow rapidly over the next three to four weeks. The conch are maintained on screen trays in the metamorphosis tanks until they reach an average size of 3 - 4 mm in shell length. Typically, 50% of the conch survive from the competent veliger stage to the post-metamorphosed stage (4 mm). To ensure good survival, growth, and development it is important to care for the conch daily. This includes observing and feeding the conch, and cleaning their environment which will all be recorded on the Metamorphosed Conch Observation Data Sheet.

FEEDING GUIDE FOR METAMORPHOSED CONCH

Each day the conch are fed flocculated Chaetoceros. Prior to feeding there are several observations that need to be taken into consideration: shell length, conch density, mortality, growth rate per day, and amount of left-over feed. Too much feed in the tanks can cause bacteria to flourish, not enough feed and the conch growth can be stunted. One sign that there is not enough feed, is observing the conch crawling up the sides of the trays in search of more food. The table below is a guide for feeding 1.0 mm to 4.0 mm conch. With proper feeding, they will grow approximately 0.18 - 0.22 mm per day.

Use the Metamorphosed Conch Feeding Data Sheet to keep track of the volume of flocculated algal cells needed to feed to the metamorphosed conch on a daily basis.

FEEDING NEWLY-METAMORPHOSED CONCH (0 - 2 weeks old)

Newly-metamorphosed conch are fed 6 × 10⁷ cells/conch/day of flocculated Chaetoceros and the feeding amount will increase over time. The concentration and volume of the flocculated microalgae to feed the conch needs to be determined from the cell count of the Chaetoceros culture prior to flocculation. Here is an example of the calculations used to determine how much to feed the newly-metamorphosed conch:

Assuming the Chaetoceros cell count is: 6 × 10⁶ cells/ml

Amount of conch per tray: 420

Conch feed needs: 6 × 10⁷ cells/conch/day

This is the total number of cells that are fed to the 420 conch in one tray for one day.

Knowing that a 95-L suntube of microalga before flocculation has a cell count of 6 × 10⁶ cells/ml:

This is the amount of cells in a 95-L suntube. All of these cells have been concentrated through the flocculation process and now fit into a 1-L container.

How many ml of flocculated microalga from the 1-L container need to be fed to the conch in each metamorphosis tray?

Thus, 44 ml of the 1 L flocculated microalga must be used to feed the conch in one metamorphosis tray.

Therefore, 352 ml of the 1 L flocculated microalga is needed to feed the conch in all eight metamorphosis trays, if both tanks are full of conch.

FEEDING POST-METAMORPHOSED CONCH (2 - 4 weeks old)

Two to four weeks after metamorphosis, feed amount increases up to 20 × 10⁷ cells/conch/ day. Here is an example of the calculations used to determine how much to feed the post-metamorphosed conch that are 3 - 4 weeks old after metamorphosis.

Assuming the cell count is: 6 × 10⁶ cells/ml

Conch per tray (assuming a 50% survival rate: 420 × 0.50 survival) = 210 conch per tray

Conch feed needs: 20 × 10⁷ cells/day

This is the total number of cells needed to feed the 210 conch in one tray for one day.

Knowing that a 95-L suntube of microalga before flocculation has a cell count of 6 × 10⁶ cells/ml:

This is the amount of cells in a 95-L suntube. All of these cells have been concentrated through the flocculation process and now fit into a 1-L container.

What is the new cell count (cells/ml) in the concentrated 1-L (1,000 ml) container?

Thus, 74 ml of the 1 L flocculated microalga must be used to feed the conch in one metamorphosis tray.

Therefore, 592 ml of the 1 L flocculated microalga will be needed to feed the conch in all eight metamorphosis trays, if both tanks are full of conch.

UNDERSTANDING THE LAYOUT AND VESSELS

To have a steady supply of food for the conch veligers, a section of the hatchery is designated for growing microalgae. There are four types of vessels in the microalgae room: test tubes, flasks, carboys, and suntubes (from smallest to largest). Each play an important role in the inoculation sequence known as ‘scaling up'.

VESSELS AND ACCESSORIES

SCALING UP OVERVIEW

The process of ‘scaling up’, is more formally described as a progressive batch culture. It begins with single-cells which multiply exponentially into millions of identical cells as they are transfered to progressively larger vessels. This is how both Isochrysis and Chaetoceros are grown. Each of these microalgal species are grown as monocultures meaning they are grown separately.

The Isochrysis monoculture is used to feed conch veligers throughout the entire larval cycle.

The Chaetoceros monoculture is used to feed conch veligers from Stage 4 (usually around day 16) through Stage 5. Chaetoceros is also grown in a progressive batch culture sequence.

To feed metamorphosed conch, repeat the same process with the Chaetoceros culture, and grow it even further up to the 95-L suntubes. Finally, flocculate the microalga (see p. 58).

EXPONENTIAL GROWTH CURVE

The microalgal monocultures follow the growth curve depicted below starting with the lag phase. If left alone, it will reach a stationary phase (determined by the size of the vessel and the amount of media available), and will eventually crash.

It is important to understand this growth curve because the optimal time to harvest the microalgae is during the exponential growth phase when it is at its highest nutritional value. Harvest microalgae to either feed veligers or as part of scaling up for the progressive batch culture.

INOCULATION: DETAILED

Microalga grown in each culture vessel serve as the inoculant for the next larger vessel, until the quantity of cells required for feeding is reached in the exponential growth phase.

The general rule for scaling up is to use 10% inoculant for smaller vessels and 5 - 7% for larger vessels. This level of inoculation results in faster exponential growth and the cultures are less prone to contamination. Inoculation begins with a pure stock culture, which is used to make the backup stock and the working stock.

PRODUCTION SCHEDULE

The microalgal production schedule is determined by knowing the maximum amount of microalgae needed to feed the veligers and metamorphosed conch. It is advised to culture extra in case of slow growth or crashes. Cultures should begin at least three weeks before bringing in the first egg masses, and are grown in a repetitive cycle throughout the hatchery phase.

VESSEL CLEANLINESS AND STERILIZATION

When beginning the microalgal cultures and when transferring inoculant from vessel to vessel, it is important to always be thinking about cleanliness and sterilization.

PREPARING THE MEDIA

Microalgae will not grow on their own in the vessels. They need a growth media to encourage cells to multiply. The media functions as a fertilizer (nutrients) and is always introduced into the vessels before adding the microalgal inoculant. In nature, high concentrations of nutrients can cause algal blooms, which are not always desirable. In culture, these nutrients are in high concentrations to optimize microalgal cell growth. Here is an example of media that can be used:

OPTIMIZING CELL GROWTH

Temperature:

The hatchery as a whole should be kept at 27 ℃ (80 ℉) and certain vessels such as test tubes and small flasks should be kept in the incubator at 24 - 25 ℃ (75 - 77 ℉).

pH:

As microalgae grow, the pH of the cultures increase. It is best to start a culture with a pH of 7.9 - 8.0. When silicates are used to grow Chaetoceros, the pH typically goes up to 9.0. Muriatic acid drops should be added to bring the pH back down.

Alkalinity:

Alkalinity is the capacity of the water to resist changes in pH. It should be 200 - 250 ppm.

Salinity:

Full strength seawater from the ocean can be used (salinity 36), however, a slightly lower salinity (30 - 32) minimizes bacterial contamination from Vibrio.

Overall water quality:

The seawater used for microalgal cultures should be pre-filtered with mechanical filters (5 - 10 µm) and UV-sterilized to kill unwanted microalgae, bacteria, and other potential pathogens and contaminants.

Aeration:

Cultures that are in test tubes and flasks will not have a direct air source, therefore, they will need to be swirled once or twice a day. Carboys and suntubes have an airline, with no air stone, to circulate the microalgal culture. Place an air filter of 0.22 µm on each carboy and suntube culture to minimize bacteria entering the culture.

Light:

Microalgae should be grown with a diurnal cycle (12 hours light: 12 hours dark), however, 24 hours of light may be advantageous. Natural or artificial lighting can be used. LED lights work very well and can be placed on an automatic timer to come on in the morning around 8:00 AM and off at 8:00 PM.

Example of LED lights for growing microalgae.

Fluorescent lights can also be used to grow microalgae.

seawater filter and UV-sterilization system.

SAFELY TRANSFERRING INOCULANTS

The biggest threat to microalgal cultures is contamination. This is why vessels are always cleaned, sterilized, and kept covered. Transferring inoculants is when the cultures are at greatest risk of contamination, as lids are temporarily removed. Transfers must be done with attention to detail.

Another threat is improper growing conditions. Microalgae need certain amounts of light and air space for photosynthesis and respiration to take place. Vessels are never filled to their maximum volume to allow for this air exchange.

For example, 25-ml test tubes only contain 20 ml of liquid (seawater with media and inoculant) allowing for 5 ml of air space.

Transferring inoculants between test tubes and flasks:

1. Wipe working surfaces and hands with 70% alcohol. Minimize any movement of air in the area where the transfers are taking place.

2. Place all of the flasks and test tubes necessary for transfers on the working surface. Make sure receiving vessels are labeled with a piece of tape (species name + date of inoculation).

3. a. When transferring from test tube to test tube, ignite a small alcohol burner. Hold the glass pipette in the dominant hand, and the two test tubes in the other hand. Remove caps with ring finger, pinky, and thumb, with dominant hand, and do not put down on work bench. Flame the tip of the pipette as well as the neck of the test tubes. Draw 1 ml of inoculant from the transfer test tube with the pipette, flame necks of the two test tubes again, and place the contents of the pipette into the receiving test tube. Flame the necks of the test tubes and put the caps back on. If additional receving test tubes will be inoculated with the transfer test tube, repeat the process.

4. b. When transferring from test tube to small flask or small flask to large flask, ignite a small alcohol burner and hold one vessel in each hand. Remove cap from the transfer vessel (put down on the bench), and remove cap from the receiving vessel (keep in opposite hand). Flame the neck of each vessel by slowly rotating it into the flame. In one motion, pour the entire content of the smaller vessel into the receiving vessel. Flame its neck and close with cap.

5. Turn off the burner and transfer all new vessels to the incubator and shelves in the algae area.

6. All vessels that are empty and their caps should be properly cleaned (see p. 49).

7. Remove all materials from working area and wipe surfaces with 70% alcohol.

Transferring Inoculants to Carboys and Suntubes:

1. Always bring transfer vessels as close as possible to receiving vessels. A step stool might be needed when transferring flasks to carboys and carboys to suntubes.

2. When transferring from a 1-L flask to a carboy, remove flask cap, tilt the carboy cap back, and pour the entire flask inoculant into the carboy. This should all be done swiftly.

3. When transferring from a carboy to suntube, remove carboy cap, slide suntube lid slightly off to the side, pour 1/3 of the carboy inoculant into each suntube, and close suntube.

CELL COUNTS

Hemocytometers are typically used for counting human blood cells and are perfect for counting microalgal cells. It is important to count microalgal cells to ensure that the culture is healthy and to determine how much of the culture is needed to feed to veligers and metamorphosed conch.

A hemocytometer is a thick glass slide with a mirrored surface which has precisely etched grids defining a known volume (Fig. A). A special cover slip is placed on top of the mirrored surface and a drop of the algal sample is added (Fig. B). The sample is drawn under the coverslip by capillary action.

Materials Needed:

• - Compound microscope (70X, 100X, 400X, 1000X)

• - Hemocytometer with cover slip

• - Pasteur pipettes

• - Pipette bulb

• - Small beaker (25 ml)

• - Wipes to clean glassware

• - Hand-counter

• - Data sheet

• - 70% alcohol

WHAT TO LOOK FOR:

Isochrysis galbana (Motile, 5 - 7 µm)

• The culture should look rich golden brown in color.

• About 70 - 80% of the algal cells should be moving in a helical (spiral) movement. If a large portion of the cells is not moving, take another sample. If nothing changes, there is an issue with the culture. Select another culture to observe for cell counts and feeding.

• There should be minimal clumping of the microalgal cells. If there is a lot of clumping there may be a bacterial contamination. Bacterial cells are very small so it is unlikely to see them. This is why it is important to note symptoms such as clumping.

• Protozoans, like ciliates, can be similar in size to algal cells, and seen in the culture. It is alright to have a small number, however, if there are a lot in the culture they may contaminate the larval tank and compete with the veligers for food. They dart and move differently than Isochrysis.

Chaetoceros gracilis (Non-motile, 8 - 10 µm)

• The culture should look rich golden brown in color.

• The cells should be dividing. This will look like two or more cells latched together.

• Chaetoceros does not move, so if a lot of movement is observed, then some other contaminants are present, such as ciliates.

• There should be minimal clumping in the culture. If there is a lot of clumping then there may be a bacterial contamination and it should not be fed to veligers. If bacteria contamination is found in the suntube, but the health of the cells look good overall, it should be fine to flocculate the culture and feed to the metamorphosed conch.

COUNTING CELLS FOR FEEDING

In order to accurately count microalgal cells, focus on five specific squares marked in blue below.

In this example, all of the ‘checked-off’ cells that are within the blue border, including the cells on the border, are counted. If two thirds or more of the cell is outside of the border then it is not counted. Below, 90 cells were counted.

Multiply this result by five and then multiply by 10,000 to calculate how many cells are in 1 ml of the culture:

90 cells × 5 counts = 450 cells

450 × 10,000 = 4.50 × 10⁶ cells/ml

There are 4,500,000 algal cells in 1 ml (approximately one drop) of this culture. Typical cell counts are 4.0 to 8.0 × 10⁶ cells/ml for Isochrysis, and 3.0 to 6.0 × 10⁶ cells/ml for Chaetoceros.

Close-up of the central square grid of a hemocytometer with Isochrysis cells counted.

FEEDING VELIGERS

How many ml of microalgal culture are needed to feed a (68-L) tank full of veligers? As the veligers grow larger they will require more food. The quantity of microalgal cells needed to feed veligers is therefore determined by the age of the veligers. Refer to the Microalgae Daily Feeding Table (p. 31) and the Microalgae Feeding Data Sheet below to find out how much to transfer to the larval tank.

Example of calculations:

• The size of the laval tank is 68 L or 68,000 ml (A).

• - According to (B) 5,000 algal cells are needed per ml of tank water on day 1 of the larval cycle.

• - Using the hemocytometer it was determined that the Isochrysis cell count was 6 × 10⁶ cells/ml (C)

• - To determine how many cells are needed for each larval tank (D), multiply column (A) times (B).

• - To determine the amount of microalgal culture to feed the larval tank (E), divide column (D) by (C).

Sequence from cell counts to feeding veligers. (Vector by Graphics RF)

FLOCCULATION: FOOD FOR METAMORPHOSED CONCH

The conch are now benthic grazers, therefore, they are unable to feed on planktonic microalgae. A process known as flocculation is used to cause floating particles, such as the microalga Chaetoceros, to clump together and settle out in a thick mass that the conch can graze upon with their proboscis.

Microalga before (left) and after floccuation (right).

A 95-L suntube which is kept in the microalgae area.

JUVENILE STAGES AND GROWTH RATE

After metamorphosis, the conch will be 1.0 - 1.3 mm in shell length and look more like a tiny sea snail. The conch will grow at an ideal rate of 0.22 mm per day which means it will take about one year to reach 80 mm (3.2 in) in shell length (80 mm / 0.22 mm per day = 363 days). This is the size that can be used for restoration of seagrass beds or for growout in sea pens to produce sustainable seafood. Throughout the nursery phase, different terminology is used to describe which part of this year-long process the conch are in based on their shell length:

NURSERY SYSTEMS

The post-metamorphosed conch (3 - 4 mm; 0.12 - 0.16 in) are placed into fiberglass tanks at 1,700 conch / m² (160 conch / ft²). The tanks are set up to have a thin layer of elevated coral aragonite sand substrate on top of a window screen to allow for good water flow.

Three pairs of juvenile tanks stacked on top of each other on a shelving unit. This system is located outdoors next to the hatchery and is protected from the weather with a fiberglass roof. The roof has a transparent section that allows sunlight to enter to help promote natural diatom growth used to supplement the feed that the conch receive. Each tank is 1.5 m² (16 ft²; 96″ L × 24″ W × 12″ D) and contains four screen trays filled with sand. (Image by Raimundo Espinoza)

Each tank has four screen trays filled with sand for the early juveniles (4 - 40 mm). Eventually the juveniles will be moved into the tank directly with a sand bed on the bottom. The conch will stay in this system from when they are 40 mm to 80 mm in shell length.

The seawater comes into each tray with a small hose (f) and circulates through the sand using a downwelling action (a, b, c). For the raised sand bed, the small hoses that went into each tray will now be used over the length of the tank. The seawater leaves the tank through an external standpipe and into a sump (d) that is filled with biofiltration media and then the water is pumped (e) back into the tanks. There will be three tanks on one recirculating system, therefore, there will be two recirculating systems in the nursery.

STOCKING DENSITY AND FEEDING TABLE

This table is based on the conch juveniles growing 0.22 mm per day with a survivorship of 75% from 4 mm to 80 mm shell length. Use this table as a reference when determining stocking density based on the size of the conch and daily feeding rate per conch.

ADJUSTING STOCKING DENSITY

PREPARING THE GEL DIET

During the nursery phase, conch are fed daily with a gel-based diet. The gel diet can be prepared in bulk ahead of time and stored in the freezer for several months. Here are the ingredients and steps to prepare the gel diet.

A single nursery tank is 1.5 m² and the stocking density for 80 mm conch is 150 conch per m².

150 conch per m² × 1.5 m² = 225 conch per tank 225 conch per tank × 2 g of feed per conch = 450 g

Therefore, the daily feeding for a nursery tank containing 225 juvenile conch (80 mm) would be 450 grams (∼ 50 - 70 cubes).

The maximum amount of gel diet daily that would be needed to feed six nursery tanks stocked at full capacity with juvenile conch 80 mm in shell length would be:

Therefore, the mixture on the previous page is approximately enough for one day of feeding all six tanks for this size and number of juvenile conch.

JUVENILE OBSERVATIONS

GLOSSARY - CONTINUED

Downwelling system: Is used in aquaculture tanks to move water from the surface downwards through a screen tray.

E

Egg mass: The female conch lays crescent-shaped egg masses in the sand. Each egg mass is comprised of a long sticky strand that is covered in sand and contains thousands of eggs.

Embryo: The early stage of development of a multicellular organism. It is the part of the life cycle that begins just after fertilization and continues through the formation of body structures, such as tissues and organs.

Epiphytes: Benthic diatoms and other organisms that grow on the surface of seagrass, macroalgae, sand, and rocks. The term is derived from Greek epi meaning ‘upon’, and phyton, meaning ‘plant'.

F

Filter: A porous device that mechanically removes impurities or particles from liquid such as seawater.

Flocculation: The aggregation of cells, that were once in suspension, by the addition of an agent such as chitosan.

G

Gastropod: A mollusc of the class Gastropoda, such as a snail, slug, or whelk. Most gastropods have a single spiral shell into which the body can be withdrawn.

Genetic diversity: The total number of genetic characteristics in the genetic makeup of a species. It serves as a way for populations to adapt to changing environments.

H

Hemocytometer: A counting-chamber device originally designed for counting blood cells, which can be used to count microalgal cells.

I

Incubation: The phase of keeping eggs in favorable environmental conditions until hatch.

Inoculation: Is used for the continuous production of microalgae using the batch culture technique.

Inoculant: A small volume of a dense microalgal culture which is typically transfered into a larger vessel.

L

Larva(e): For marine animals, the larval stage starts after hatch and ends at metamorphosis. Larvae are usually planktonic and spend most of their time in the water column.

Lobes: Protrusions that are characteristic in queen conch larvae. They are used for locomotion, respiration, and feeding.

M

Macroalga(e): Unlike microalgae, macroalgae are visible without a microscope. They are also known as seaweed.

Mantle: A layer of tissue in molluscs which secretes the shell.

Media: Nutrient-rich substance used for cultivation of microorganisms.

GLOSSARY - CONTINUED

Metapodium: The posterior portion of the foot of some molluscs.

Motile: Capable of motion.

Monoculture: The cultivation of a single crop such as a single plant or algal species.

Morphology: The study of the form and structure of organisms.

N

Nursery: A place where young plants and organisms, like juvenile conch, are grown to a certain size.

O

Ocular micrometer: A glass disk that fits in a microscope eyepiece that has a ruled scale. It is used to measure the size of magnified objects.

Oligotrophic: A term used to describe environments of water with relatively low nutrient levels.

Operculum: A structure resembling a lid or a small door that protect gastropods while inside of their shell.

Osphradium: An olfactory organ in certain molluscs, linked with the respiratory organ. The main function of this organ is thought to be used for testing incoming water for silt and possible food particles.

P

Phytoplankton: A group of free-floating microscopic algae that drift with the water currents. They form an important part of the ocean food web. Derived from the Greek phyton, meaning ‘plant’, and planktos, meaning ‘wanderer’ or ‘drifter'.

Planktotrophic: Refers to the development of larvae that must feed on plankton in order to develop to metamorphosis.

Proboscis: An elongated sucking mouthpart that is typically tubular and flexible.

Propodium: The anterior portion of the foot of a mollusc.

R

Rear: To raise and care for animals in a particular manner or place until fully grown or until a certain development stage.

Recirculating system: A type of aquacuture system, which operates by filtering the water from the fish or conch tanks so that the water can be reused. This dramatically reduces the amount of water used and helps to control water quality.

Restocking: The raising of animals in aquaculture to release them into a river, lake, or ocean to supplement existing populations or to create a population where none exists.

S

Settlement: Refers to when some planktonic organisms, such as conch, find a place in the benthic zone to settle for metamorphosis.

Silica: An oxide of silicon with the chemical formula SiO₂, most commonly found in nature as quartz and in various living organisms such as diatoms, sea sponges, and hydroids. It is one of the components of glass.

GLOSSARY - CONTINUED

Siphonal canal: A semi-tubular extension of the aperture of the shell.

Siphon hose: A device that involves the flow of liquids through tubes without a pump.

Sterilization: The process of making something free from bacteria. For example, microwaving glassware with media or using an alcohol burner for flaming vessels during microalgae transfers.

Substrate: The surface on which an organism lives.

Sump: A tank that is positioned below the culture tanks, which collects seawater in a recirculating system and returns the water back to the culture tanks with a pump.

T

Tentacles: A flexible, mobile, and elongated sensory organ present in many molluscs. They are receptive to touch, vision, and smell or taste of particular foods or threats. Examples of such tentacles are the eyestalks of various kinds of snails.

Test set: A group of smaller tests that are done to predict and achieve the desired outcome on a larger scale.

U

Upwelling system: When water rises up (upwells) to replace the water that was displaced at the surface. In aquaculture, the water in tanks can artificially create this upwelling motion.

UV-sterilized: A disinfection method that uses short wavelength ultraviolet light to kill unwanted microorganisms such as bacteria.

V

Veliger: The larval stage of certain molluscs that have ciliated lobes for swimming and feeding.

W

Whorls: A pattern of spirals, like that of a snail shell.

Y

Yolk reserve: Certain species of animals, usually those with short incubation periods, hatch with a yolk reserve. This gives them the energy they need for the first several hours of their lives until they are able to feed on their own.

Z

Zooplankton: Plankton consisting of small animals and the immature stages of larger animals. The word zooplankton is derived from the Greek zoon, meaning ‘animal’, and planktos, meaning ‘wanderer’ or ‘drifter'.

MEASUREMENTS

Volume:

Linear measurements:

Area:

Temperature:

Exponent examples:

Abbreviations and Symbols: