Maintaining larvae from brooding species
For brooded larvae, settlement may begin soon after release, so potentially larvae can immediately be introduced to conditioned settlement substrata or areas of reef if direct enhancement is being carried out (see section 5.9). However for some Atlantic brooding species it has been shown that it is beneficial to maintain larvae in a separate clean tank for at least two days before attempting to settle them12. This is because, for certain species, at time of
release there is a significant amount of both lipids and mucus associated with the larvae. It is necessary to wash away the mucus and lipids by doing regular water changes otherwise they will become a source of energy for
unwanted bacteria in the settlement tanks. During this period, collected larvae should be maintained in clean filtered seawater at ambient temperature at densities of not more than 300 larvae per litre of seawater, with water changes done at least once each day (see below for water change techniques). Conversely, for larvae of some common Indo-Pacific species (e.g. Pocillopora damicornis) settlement can occur very soon after release and larvae will settle readily on almost any surface. For this reason, when working with P. damicornis it is advisable to make sure that your holding tanks and settlement tanks are thoroughly cleaned to remove biolfilms that will encourage unwanted larval settlement and to have your conditioned settlement substrates ready as soon as larvae are released.
As brooded larvae contain zooxanthellae you should provide enough light for photosynthesis. Shaded sunlight should be adequate, however if larval cultures are maintained outdoors it may be difficult to control temperature in the culture tanks while still providing sufficient light. For this reason it is advisable for you to maintain your culture of brooded larvae in a temperature controlled room (with temperatures set close to that of ambient sea water) with an artificial light source. Ideally you should use a high intensity actinic lamp with strong emissions in the short wavelength region of the spectrum, peaking at 420 nm (e.g. Coral Sun®Actinic 420
T5-HO).
Maintaining embryos and larvae from broadcasters
at protected sites close to the shore. This method has the advantage that temperature can be buffered by the surrounding sea water, furthermore water exchange can occur if mesh windows are built in to a floating tank. However, floating ponds are subject to inclement weather. If mesh screens are not incorporated for water exchange then it is necessary to cover the ponds to keep off rain water that will change the surface salinity. You will also need to clean mesh screens using scuba to prevent clogging by fouling organisms and sediment. In Okinawa, Japan, floating ponds have been used to rear coral larvae successfully1, 13-14. The
ponds are constructed of vinyl sheet and are connected by floating rafts. Water is sprayed against the walls of the pond by means of a hose attached to the upper part of the pond, with holes made at intervals, supplied by a submerged pump. This prevents larvae sticking to the walls of the pond during rearing and promotes water exchange.
During the rearing phase it is necessary to assess larval health and readiness of the larvae to settle (see Box 5.4). embryos in the rearing tanks until swimming larvae have
developed. During this period, which is usually around 2–5 days, it is essential that you maintain a healthy environment for the larvae. You should keep rearing tanks shaded using a net that reduces 40–60% of direct sunlight and protect from rain showers as sudden reductions in salinity will increase larval mortality. Checking and maintaining water temperature within a normal range is critical. Ideally, you should not allow temperatures to rise above normal ambient levels (which will vary from one location to another) and this can be done by increasing shading above the tank and carrying out regular water changes as necessary.
During the larval rearing phase you should check embryos and larvae at least daily by examination under a stereo dissecting microscope to assess health and status (see Figure 5.4). An important consideration at this stage is handling of embryos. During the early stages, developing embryos must be treated extremely carefully so that dividing cells are not ‘broken’. Rough handling of embryos during the cell division stage, i.e. from 1 hour post spawning until embryos are ‘ball shaped’ (see Figure 5.4), will result in many of the embryos not completing development or being smaller than normal. Once you have carefully transferred the embryos to the rearing tank you should leave them in static water without aeration until embryos have a rounded ‘ball shape’ (usually 24–36 hours after fertilisation). Gentle aeration can be introduced after this and should be gradually increased each day as larvae become more robust.
You should check water quality daily and carry out water changes after 24 hours if water quality has deteriorated or temperature has risen in the tank. Signs that water quality has deteriorated include cloudiness and the appearance of white foam on the water surface. Foam and floating scum can be removed using polyethylene plastic wrap (e.g. Glad wrap™ or Saran wrap™) by placing sheets on the water surface and allowing any scum to stick to the wrap before removing. However, in most cases such treatments are unable to reduce mortality once water quality has deteriorated due to bacterial propagation.
During water changes, you should treat embryos and larvae as gently as possible. You can do water changes by siphoning water from the rearing tank onto a submerged 100 µm mesh sieve or net. Larvae that are trapped on the sieve or net should remain submerged as the tank is being emptied and should be carefully replaced by ‘backwashing’ them into the tank. You can also use a two-sided sieve (see right) to do water changes. With this method, the sieve is placed in the tank so that embryos or larvae remain in the tank during the water exchange. Once the rearing tank is half empty it should be topped up with clean filtered sea water. Using these methods it is possible to do partial or full water changes.
Rearing tanks for embryos and larvae are typically kept on land; however it is possible to rear larvae in floating ponds
A floating rearing pond containing fertilised embryos in Akajima, Japan (M. Omori).
Floating ponds used for larval rearing in Akajima, Japan (M.Omori). An example of a two sided sieve that can be used for convenient water changes (J. Guest).
Large inflatable pools (4000 litres) being used as rearing tanks in Palau. Note that rearing pools are covered by a roof and shade netting to protect embryos from excessive sunlight and rain (J. Guest).
5
Box 5.4Assessing settlement competency of coral larvae from broadcast spawnersThe time needed for the larvae to develop to a stage when they are ready to settle varies considerably among species and locations and is dependent on environmental factors such as temperature; therefore it is important that you visually assess levels of settlement competency during the larval development period. An effective method is to track a sub-sample of developing larvae held in smaller volume containers. You should remove approximately 4 litres of seawater containing larvae from the rearing tanks immediately after stocking. This sub-sample can be kept in clean plastic bottles (e.g. four 1 litre bottles) which can be left floating in the rearing tank (to buffer temperature). You should take samples (~400 ml) of larvae from one of these bottles at 12 hours post fertilisation by pouring into a clean cup; repeat this at 24 hours and subsequently every 24 hours until high levels (>80%) of larvae are ready to settle.
From the cup containing the sample of larvae, place approximately 20 larvae into each of 6 sterile replicate plastic wells or medicine cups containing about 10–20 ml of UV-treated, 0.2 µm filtered seawater. Plastic laboratory 6-well culture plates are ideal, however disposable plastic cups can also be used. You should flush the larvae well with clean seawater (ideally 0.2 µm filtered) before adding them to the culture wells. In seawater alone, settlement rates of larvae will be very low, therefore it is necessary to add an inducer for settlement and metamorphosis. The presence of certain species of crustose coralline algae (CCA), particularly Hydrolithon spp. and Peyssonnelia spp., has been shown to induce metamorphosis in a number of coral species15-17.
A chip of CCA approximately 5 mm x 5 mm in size should be scraped from the surface of a larger piece of CCA that you have collected from the reef. You should thoroughly clean the chips using a soft brush while flushing with filtered seawater. Place one CCA chip in each well or cup. Alternatively, you can use a small piece of coral rock or dead coral that has been immersed in seawater for more than 2 months (so that crustose coralline algae is attached). You should keep the wells or cups indoors on a stable surface free from vibrations where
temperature throughout the day remains within the normal range of ambient seawater temperature for the locality (i.e. not in an air conditioned room or close to working machinery). You should check each well or cup under the dissecting microscope after 24 hours and count the number of larvae in one of the following four conditions: 1) attached, 2) metamorphosed, 3) alive but not attached, 4) dead. Larvae are recorded as settled when they are either attached to the substrate or have metamorphosed into a polyp. The average percentage settlement can then be calculated and plotted on a graph (Figure 5.5). When the average settlement rate reaches at least 80% then you should introduce larvae from the main holding facility to settlement substrata for subsequent rearing or to areas of degraded reef. If only low levels of attachment and metamorphosis (e.g. <50%) are ever achieved, this may indicate that larvae are not healthy due to poor water quality in the rearing tanks.
Knowledge about spawning times is a pre-requisite for doing larval collection and rearing work.
Mixing gametes from three or more colonies will increase the chances of fertilisation success and will create higher genetic variation.
You should remove water containing excess sperm from the eggs about 15 to 30 minutes after fertilisation has commenced to prevent reductions in water quality (see section 5.6).
Developing embryos are very delicate from first cleavage until about 24–36 hours after fertilisation and so must be treated very gently during this period. Maintaining larvae at low densities (not more than 300 larvae per litre) in clean sea water is critical for the survival and health of the larvae.
Days after fertilisation % larvae settled 0 20 40 60 80 100 1 2 3 4 5 6 7 8 9 10 11 12
Assessing larval competency to settle. Small chips of crustose coralline algae (CCA) are placed in 12 ml culture wells and about 20 larvae are placed into each well with a disposable pipette (J. Guest).
Settled and metamorphosed larvae on a chip of crustose coralline algae (A. Morse).