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No matter what method is used to harvest gametes, the methods for fertilisation, maintaining embryos, and rearing the larvae until they are ready to settle are similar. Once you have collected all gamete bundles (methods 2 or 3 above) and transferred them to a separate container, the level in the container should be topped up to a known volume (100 litres makes subsequent calculations easier). Fertilisation will take place in this tank, therefore it is important that clean, filtered seawater is used (Box 5.3) and that the temperature is close to ambient seawater temperature. Temperatures higher than 4°C above normal ambient temperature markedly reduce fertilisation success in some Acropora species and we recommend that temperature does not deviate more than 1°C from ambient.

Gamete bundles will break apart and the water should become white and almost opaque as a result of the released sperm. It is generally accepted that sperm densities between 106_{to 10}7_{sperm per ml are most}

suitable. In practice sperm density seems to have little effect on fertilisation rates providing that gamete bundles from several colonies are mixed in a relatively small volume container (e.g. 50 to 100 litres). Egg density at this stage is also not critical. More than 1 million eggs can be fertilized in a single 100 L polycarbonate tank. If colonies have been isolated or divided into separate tanks then gametes from the separate batches should also be fertilised in separate fertilisation tanks.

As soon as the all of the gamete bundles have been transferred to the fertilisation tank(s) and the bundles have broken apart, you need to estimate the number of eggs you have. This is necessary to determine the volume of water required for stocking during the larval rearing phase (see below). Eggs are usually very buoyant so to estimate egg density, it is important to distribute the fertilising eggs in the container as evenly as possible by vigorously stirring using a large plastic spoon, paddle or plunger. One person should constantly stir while another takes several small samples from different parts of the tank using plastic screw top sampling tubes. You should take at least five 15-ml samples from a 100 litre tank to get an average density estimate and count the eggs immediately under a stereo microscope. Using the protocol in Example 5.1 (right), you can then estimate of the total number of eggs.

Significant numbers of eggs are fertilised within the first few

minutes and fertilisation increases steadily for up to an hour after gametes are mixed together. Therefore the cross- fertilised gamete mixture should be left for between 15 to 30 minutes (not longer than one hour) with gentle stirring approximately every 5 minutes to prevent oxygen depletion. Once fertilisation has occurred it is important to ‘clean’ the eggs to remove excess sperm. Excessive numbers of sperm can lead to a problem known as polyspermy (where many sperm attempt to fertilise one egg). Furthermore, breakdown of excess sperm in the fertilisation or rearing tank will lead to a reduction in water quality and high mortality of embryos. Cleaning can be achieved by draining the fertilisation tank onto a plankton mesh sieve (100 µm) from the bottom via either a plug or a tube siphon and carefully re-filling with clean sea water several times. An alternative and convenient method if draining is not possible is to scoop eggs from the surface of the fertilisation tank and transfer them to another similar sized tank containing clean filtered sea water using the same method as used to scoop from the spawning tank (i.e. a clean cup is held just below the water meniscus to collect as many eggs and as little water as possible).

After the excess sperm have been removed, you need to gently transfer the eggs to a rearing tank at a suitable stocking density (see how to calculate in Example 5.2). After about 2 hours you should take a sample of the culture (approx. 50 ml) and check under a stereo-microscope for evidence of fertilisation (Figure 5.4). If sufficient numbers of fertilised embryos are present (>80% of eggs are dividing), the initial stages of larval rearing have been successful and you can proceed to rear the embryos through to larvae as described in section 5.8. It is important that there are not many unfertilized eggs in the culture as these will rapidly deteriorate and damage the water quality, killing other normal embryos. If fertilisation levels are low (<60%) it may be worth considering abandoning the culture. The fertilisation work should be completed within four hours of spawning.

Example 5.1 Estimating eggs numbers

Total number of eggs = 10.0 x 100,000†= 1 million †_{for a 100 litre fertilisation tank}

Sub-sample (15 ml) 1 2 3 4 5 Average Number of eggs 150 eggs

per 15 ml 100 200 170 130 150 per 15 ml Number of eggs 10.0 eggs

5

Establishing a suitable stocking density

It is essential that you stock the embryos at the correct density. If kept at too high a density there will be high mortality. The optimal volume will depend on various factors such as water temperature, water quality and frequency of water changes. Tank surface area is also important in the early stages of larval rearing because buoyant eggs will tend to aggregate at the surface of the tank. High survival of larvae can be attained with relatively little effort when embryos are kept at a stocking density of 300 propagules per 1 litre volume and 40 propagules per 1 cm2_surface

area of seawater (covering about 10–20% of the water surface area). You can make rearing tanks of any suitable material, e.g. fibre-glass, polycarbonate or PVC. If the work is being done on a budget or in remote locations, any available uncontaminated water-tight container can be used, e.g. inflatable paddling pools which are available from department stores or toy shops.

A 100 litre polycarbonate fertilisation tank containing eggs, sperm and embryos (J. Guest).

A 100 litre polycarbonate fertilisation tank containing gamete bundles being filled to a known volume. Notice the cloudiness of the water which is from the high sperm density (J. Guest).

Example 5.2 Estimating suitable stocking density Tank volume (litres) 100 500 1000 1500 2000 Min. tank surface area (m2_{) 0.08 0.38 0.75 1.13 1.50} Min. tank diameter (cm)† _{31 69 98 120 138} Total number of propagules (eggs, embryos or larvae)†† _{30,000 150,000 300,000 450,000 600,000}

†Minimum diameter is for a circular tank.

††Stocking density of 300 propagules per 1 litre volume or 40 propagules per 1 cm2_{surface area of sea water.}

Figure 5.4. Embryo developmental stages for a typical Acropora coral (based on Okubo and Motokawa 200710_{). 1) An unfertilised egg (or} oocyte) with distinct spherical shape and opaque appearance; 2) about 2 hours after fertilisation occurs, embryos divide into two cells (known as blastomeres) by a process called cleavage; 3) at about 3 hours embryos divide again to become four cells; 4) cell division continues to produce 8 cells at about 4 hours; 5) 16 cells at about 5 hours and until the embryo consists of many cells 6) this stage is known as the morula and occurs after about 6 hours; 7) after about 7 hours the embryo is known as a ‘prawn chip’ because of its distinctive shape; 8) then after about 10 hours the embryo has a distinctive bowl shape; 9) and by 36 hours the embryo becomes completely ‘ball shaped’ and at this stage is considerably more robust and can withstand water changes (see section 5.8); 10) larvae gradually become more motile and elongated in shape until a ‘cigar shaped’ larva is formed at around 96 hours. (Photos 1–5, 7 and 9: C. Boch (A. digitifera); Photos 6 and 8: N. Okubo; Photo 10: M. Omori (A. tenuis).)

Box 5.3

Ideally, seawater used from fertilisation onwards should be as clean as possible to reduce the possibility of bacterial growth and increase the survivorship of the larvae. You should use seawater that has been filtered through at least a 10 µm filter bag and if possible a 0.2 µm filter. Where possible you should use UV-treated, filtered water as this kills unwanted bacteria. Filters are available from aquarium suppliers and you may need to seek expert help to set up of the aquarium facilities required for larval rearing. It is essential that you practise good husbandry and cleanliness during larval rearing. All materials used in the rearing and fertilisation tanks, should be thoroughly cleaned with freshwater before use to prevent introduction of bacteria. Detergents should be avoided during cleaning as they may be toxic to coral larvae. It is sometimes common practice to sterilise tanks prior to use with dilute household bleach (1%) to kill unwanted bacteria. If bleach has been used to clean tanks it is important that tanks are rinsed several times with freshwater then left to dry in the sun for one day. It is also important that everyone involved in handling coral eggs and larvae has clean hands free of sunscreens, insect repellents and other creams. For longer culturing of brooded larvae, antibiotics (e.g. Rifampicin at 1 g per 10 litres of seawater) have been used successfully to prevent bacterial fouling. Note, however, that larvae reared in seawater containing Rifampicin need to be rinsed in normal filtered seawater for several hours before you attempt to settle them.

5.7 Collecting and rearing larvae from brooding