The ‘Queen Conch Aquaculture: Hatchery and Nursery Phases User Manual’ was designed and written by Florida Atlantic University Harbor Branch Oceanographic Institute Research Professor, Megan Davis* and Science Communicator and Designer, Victoria Cassar.
This Manual is a deliverable of the Saltonstall-Kennedy NOAA Fisheries grant (NA19NMF4270029) ‘Development of a Fishers Operated Pilot-Scale Queen Conch (Strombus gigas) Hatchery and Nursery Facility for Sustainable Seafood Supply and Restoration of Wild Populations in Puerto Rico’, Megan Davis, PI (FAU Harbor Branch), Raimundo Espinoza, Co-PI (Conservación ConCiencia) and Carlos Velazquez, Collaborating Organization (Naguabo Fishing Association).
The manual includes the science and art of growing queen conch (S. gigas) that Davis has developed over her 40-year career designing, implementing and operating experimental size aquaculture facilities, as well as production-scale facilities, in Florida and throughout the Caribbean. In addition, Robinson Bazurto provided advice on Chapter 4: Growing Microalgae.
This edition is written for the Puerto Rican fishers of the Naguabo Fishing Association who are learning to operate the Naguabo Queen Conch Hatchery and Nursery; however, the majority of the information presented in this manual can be applied to other queen conch hatchery and nursery projects to produce conch for sustainable seafood, conservation and restoration. This manual is also available in Spanish.
The queen conch, Strombus gigas, is deeply rooted in the way of life in the Caribbean region. It is one of the most commercially important fisheries and many island communities depend on it for sustenance and their livelihoods. Intensive fishing and habitat degradation, however, have caused conch populations to significantly dwindle.
The Queen Conch Resources Fishery Management Plan established a program to help rebuild conch populations in the U.S. Caribbean. For Puerto Rico this includes a minimum harvest size of 9 inches (22.9 cm) in shell length or 3/8 inches (9.5 mm) in lip thickness.
In Puerto Rico, daily bag limits are 150 conch per licensed commercial fisher or 300 per vessel and the closed season is during the peak reproductive months (August 1 to October 31) in jurisdictional waters (0 - 9 nm). Conch harvest has been prohibited in the U.S. Exclusive Economic Zone (EEZ) off of Puerto Rico since 1997. The Puerto Rico Department of Natural and Environmental Resources (DNER) manages the state conch fishery and the Caribbean Fisheries Management Council manages the federal conch fishery. In Puerto Rico the conch, locally known as ‘carrucho’, is one of the main fisheries species with most of the conch consumed locally with little export. The annual landings are 300,000 to 350,000 pounds (DNER 2016 - 2017) and fishers receive $8 to $12 per pound.
In addition to their socio-economic importance, queen conch play a crucial ecological role. Most people associate a beautiful pink shell or a culinary dish to the name queen conch, but rarely do people know about the animal that lives inside. Queen conch are a marine gastropod, or snail, and are herbivorous. Using their proboscis, or snout, they graze upon microscopic algae and other epiphytes that grow on seagrass blades and the sandy bottom. Queen conch adults aggregate in ‘herds’ in seagrass meadows and sand flats to reproduce during the summer months. They lay egg masses which hatch into veligers that drift in the ocean currents for three to four weeks and then settle as benthic snails in the seagrass. It takes approximately four years for the queen conch to become mature with a well-developed flared lip.
The first step to culturing conch in a hatchery is to collect sections of egg masses from the wild. A full egg mass has approximately 500,000 eggs - much more than what is needed in the hatchery. This is why only a 1/4 or less of each egg mass is collected. An egg mass is one long, coiled strand.
To collect egg mass sections, it is necessary to bring a field kit on the boat containing the following:
- 5-gallon bucket with lid
- Snorkeling gear
- Data sheet with clipboard
- Pencil with eraser, and sharpener
- Refractometer (to measure salinity)
- Resealable bags (like Ziploc) numbered 1 to 6 (quart size; freezable)
Note: A permit is required to collect egg masses from the wild
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.
Once the eggs have hatched, there will be thousands of larvae, also known as veligers, swimming in the larval tanks. Larval rearing is the most intensive part of the queen conch culturing process.
This chapter contains how to successfully rear larvae using specific techniques, and is also where the art of rearing queen conch will come into play. By paying close attention to the animals, an innate understanding about their needs will occur over time.
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.
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.
- Eye dropper
- Depression slides
- Dissecting microscope
- Ocular micrometer
- Wash bottle
- Sieve container
- Data sheet and clipboard
- Pencil, eraser, and sharpener
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:
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.
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.
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:
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.
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.
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.
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.
Around day 18 - 21, veligers should start showing signs of competency, but will not go through metamorphosis without a natural cue - such as the presence of their food. In the wild, this cue are the epiphytes comprised of benthic diatoms that cover seagrass blades, macroalgae, and sand. Epiphytes trigger veligers to settle into seagrass beds, their juvenile habitat.
When veligers are metamorphically competent, they have a ‘swim-crawl’ behavior. They still have their lobes and can continue to swim or drift but they can also use their foot to test the substrate to see if it is the right place to settle.
This chapter will demonstrate how to guide veligers through this key transformation.
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.
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.
a) If 680 veligers (v) in one larval tank are ready for metamorphosis, and if those 680 are concentrated in a bucket filled with 10 liters of seawater, then the density would be 68 veligers per liter: 680 v / 10 L = 68 v / L
b) Considering that in real life the density in this larval tank will not be exactly 10 v/L, it will be necessary to take three 50-ml subsamples using a 50-ml tri-beaker from the bucket. These samples will provide a more accurate density count of the veligers.
Subsample 1: five veligers are counted in the beaker
Subsample 2: three veligers are counted in the beaker
Subsample 3: four veligers are counted in the beaker
Thus, on average, there are four veligers per 50 ml (0.05 L): 4 v / 0.05 L = 80 v / L = 800 v / 10 L Therefore, the larval tank was holding 800 veligers, and so is the bucket with the concentrated veligers.
c) If each metamorphosis tray can hold 420 veligers (p. 37) and there are 800 veligers total, then half of the bucket contents should be put into one tray and the other half in another: 800 v / 2 trays = 400 v and 10 L in the bucket / 2 = 5 L of the bucket per tray
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.
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.
As mentioned in Chapter 2: Larval Rearing, conch veligers are considered zooplankton and they feed on phytoplankton - also known as microalgae - during their entire larval cycle.
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'.
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.
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.
Lag phase: Lasts a few hours and occurs right after inoculant is transfered to a new vessel.
Exponential growth phase: During the first 4 - 5 days, the culture should be experiencing rapid cell division as the cells grow logarithmically in the available media and vessel space. The culture must be harvested and transferred right before peak density.
Stationary phase: Following peak density, cell division begins to slow down and plateau because the media has been used up. The cells have low nutritional value in this phase.
Crash phase: As the cells are starved of nutrients, the cell density decreases rapidly. During this phase, recovery is near impossible. The culture turns pale brown and smells sour. If a crash occurs unexpectedly early on, it may be due to contamination, low pH, light levels, and/or low oxygen.
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.
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.
Cleaning techniques for:
Glassware (test tubes, flasks, and pipettes): Use a small test tube or flask brush to wash vessels and caps with liquid alconox, rinse with fresh water, and dip in a mild muriatic acid solution (10 ml acid / L of freshwater.) Rinse well with freshwater and let dry, ideally for a day. Before use, rinse with seawater. After inoculation, keep the used Pasteur pipettes in a mild muriatic acid solution, rinse with fresh water and let dry. Note: For glassware, the muriatic acid solution can be kept and reused for one week.
Plastic and Fiberglass (carboys and suntubes): Pour about half a liter of mild muriatic acid solution (10 ml acid / L of freshwater) into carboys and swirl around. If necessary, use a carboy brush to clean out any remaining algae. Pour out, rinse well with fresh water, and let dry. The same can be done for suntubes using two liters of the mild acid solution and a mop to distribute the solution. Before use, rinse with seawater.
Sterilization techniques for:
Glassware (test tubes and flasks): Using a 700-watt microwave with a turn table, microwave loosely capped test tubes and flasks with their contents of seawater and media for 8 - 10 minutes per 1 - 1.5 L of liquid. For example, when test tubes and small flasks are all microwaved together, this can add up to 1 L. Let sit for 24 hours prior to inoculating with microalga. Glass pipettes in reusable bags can be microwaved for 10 minutes.
Plastic and Fiberglass (carboys and suntubes): Fill carboys and suntubes with seawater, chlorinate at 5 ppm (5 ml household chlorine per 1 L of seawater), cover and leave overnight. Dechlorinate with vitamin C (1 ml vitamin C solution / ml of chlorine used). It is advised to prepare vitamin C stock solution ahead of time: 165 grams of vitamin C per one liter of freshwater. After vitamin C is added, turn on aeration in carboys and/or suntubes. Wait 30 seconds. Using a pool chlorine test kit make sure all of the chlorine is gone.
To begin and/or continue a culture, vessels must be prepared to receive microalgal inoculant in the following order:
Test Tubes and Flasks (glassware):
Clean (acid solution)
Fill vessels with seawater and media
Let sit for one day
Add microalgal inoculant
Carboys and Suntubes (plastic/fiberglass):
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
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 ℉).
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 is the capacity of the water to resist changes in pH. It should be 200 - 250 ppm.
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.
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.
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.
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:
Wipe working surfaces and hands with 70% alcohol. Minimize any movement of air in the area where the transfers are taking place.
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).
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.
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.
Turn off the burner and transfer all new vessels to the incubator and shelves in the algae area.
All vessels that are empty and their caps should be properly cleaned (see p. 49).
Remove all materials from working area and wipe surfaces with 70% alcohol.
Transferring Inoculants to Carboys and Suntubes:
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.
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.
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.
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.
- Compound microscope (70X, 100X, 400X, 1000X)
- Hemocytometer with cover slip
- Pasteur pipettes
- Pipette bulb
- Small beaker (25 ml)
- Wipes to clean glassware
- 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.
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).
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.
Once the conch reach 3 - 4 mm in shell length, they are moved to the nursery. The tanks here are set up in a recirculating system, meaning the water is reused over and over again. The small conch will be grown on sand and will be cryptic during the first couple of months. Gradually, they will grow large enough to be handled with fingers. This is the longest phase for growing conch onshore and takes approximately one year.
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:
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.
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.
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.
The following glossary terms are in large part defined in the context of conch aquaculture.
Acclimation: The process in which an individual organism adjusts to a change in its environment (such as altitude, temperature, humidity, photoperiod, or pH), allowing it to maintain performance across a range of environmental conditions.
Agar plate: A Petri dish that contains agar, a jelly-like substance obtained from red algae, as a solid growth medium used to culture microorganisms such as microalgae.
Aperture: The main opening in gastropod shells, where the foot and head of the animal emerges for locomotion and feeding.
Apex: The pointed tip of the shell. It is the oldest part of the shell where the first whorl, or spiral, begins.
Beak: A projecting structure of the larval conch shell that wraps over the aperture and ends in a point.
Benthic: Anything associated with, or occurring on, the bottom of a body of water like an ocean, lake, and stream. The benthos are the bottom-dwelling plants and animals found on or in the sediments.
Buccal mass: Is the mouth part of molluscs that is first seen in the larvae once they are competent. It forms the proboscis, or snout, which is used for grazing.
Chitosan: Is made from treating the hard outter skeleton (exoskeleton) of shrimp and other crustaceans with an alkaline substance. It is used in pharmaceuticals and is used to flocculate microalgal cells for feeding conch juveniles in aquaculture.
Cilia: The hairlike structure along the edges of the lobes of veligers. Moves in a wavelike motion to propel veligers and are also used to capture food particles and oxygen exchange.
Competency / Competent: Larvae are considered competent when they are morphologically and physiologically ready to undergo metamorphosis.
Cryptic: Camouflage of an animal in their environment.
Ctenidium: A comblike structure, which is a respiratory organ (gill) in a mollusc. First seen in the larvae once they are competent. The gill will replace the cilia that aided the veliger with oxygen exchange.
Cue: A smell, chemical, temperature or other external factor that triggers a change such as metamorphosis.
Culture: The maintenance of plantlife or sealife in conditions suitable for growth. Aquaculture is the rearing of aquatic plants and animals specifically.
Diatom: Diatoms are a major group of phytoplankton found in the oceans, waterways and soils of the world. Their cell walls are made of silica, a glasslike material.
Dissecting microscope: This microscope is also known as a stereo microscope and is designed for low magnification observation of a sample.
GLOSSARY - CONTINUED
Downwelling system: Is used in aquaculture tanks to move water from the surface downwards through a screen tray.
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'.
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.
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.
Hemocytometer: A counting-chamber device originally designed for counting blood cells, which can be used to count microalgal cells.
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.
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.
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.
Nursery: A place where young plants and organisms, like juvenile conch, are grown to a certain size.
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.
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.
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.
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.
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.
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.
Veliger: The larval stage of certain molluscs that have ciliated lobes for swimming and feeding.
Whorls: A pattern of spirals, like that of a snail shell.
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.
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'.
1 inch = 2.54 centimeter
1 meter = 3.3 feet
1 meter = 100 centimeters
1 centimeter = 10 millimeters
1 millimeter = 1,000 micrometers
Celcius - Fahrenheit
C × 1.8 + 32 = F
F - 32 × 0.556 = C
0o C = 320??? F
28o C = 82o F (Ideal temperature for raising conch.)
Abbreviations and Symbols: