quantifying artemia nauplii density pre- and post-feeding of pocillopora acuta

Ex Situ Culture of &lt;em&gt;Pocillopora acuta&lt;/em&gt; and Effective Feeding Techniques

Many coral reef ecosystems globally are being lost and degraded as a result of high-temperature stress driven by climate change1,2. Coral bleaching (i.e., the breakdown of the coral-algal symbiosis3) was considered relatively rare in the past4 but is now occurring more frequently5, with annual bleaching expected to occur in many regions by&#160;mid to late century6,7. This shortening of the interim period between bleaching events can limit the capacity for reef resilience8. The direct impacts of high-temperature stress on coral colonies (e.g., tissue damage9; energy depletion10) are intrinsically linked to indirect impacts at the reef-scale level, of which a reduction in reproductive/recruitment capacity is of particular concern11. This has spurred a range of applied research exploring, for example, the active in situ enhancement of recruitment (e.g., reef seeding12), new technologies for scaling-up coral restoration13, and the simulation of reproductive cues to induce reproduction in ex situ systems14. Complementary to these active interventions are the recent recognition of the advantages of heterotrophic feeding in corals under high-temperature stress15 and the exploration of the role that food provision may play in reproduction16.
Heterotrophic feeding is known to influence the performance of corals17 and has been specifically linked to increased coral growth18,19, as well as thermal resistance and resilience20,21. Yet, the benefits of heterotrophy are not ubiquitous among coral species22 and can differ based on the type of food being consumed23, as well as the level of light exposure24. In the context of coral reproduction, heterotrophic feeding has shown variable results, with observations of higher25 as well as lower26 reproductive capacity following heterotrophic feeding being reported. The influence of heterotrophic feeding on coral reproduction across a spectrum of temperatures is rarely assessed, yet in the temperate coral Cladocora caespitosa, heterotrophy was found to be more important for reproduction under lower temperature conditions27. A better understanding of the role of temperature and feeding on reproductive output is likely needed to determine whether specific reefs (e.g., reefs associated with high food availability28) possess a higher capacity for recruitment under climate change.
Similar to reproductive output, the effect of temperature and feeding on reproductive timing in corals remains relatively understudied, despite the synchronization of reproduction with abiotic/biotic conditions being an important consideration for recruitment success in a warming ocean29. Warmer temperatures have been shown to result in earlier reproduction in coral thermal conditioning studies conducted in the lab30, and this has also been observed in corals collected from natural reefs across seasons31. Yet, interestingly the opposite trend was recently observed in fed corals cultured over the course of 1 year in an ex situ flow-through system (i.e., reproduction occurred earlier in the lunar cycle at cooler winter temperatures and later in the lunar cycle at warmer summer temperatures)32. This contrasting result suggests that reproductive timing may stray from typical patterns under conditions associated with abundant energetic resources.
Long-term controlled experiments under different temperature scenarios could contribute to a better understanding of the influence of heterotrophy on reproduction in scleractinian corals. Maintaining reproducing coral colonies under ex situ conditions for multiple reproductive cycles, however, can be challenging (but see previous research32,33). Herein, straightforward and effective techniques for the active feeding (food source: Artemia nauplii) and long-term culture of a brooding coral (Pocillopora acuta) in a flow-through aquaculture system are described; yet, it should be noted that all the techniques described can also be used in recirculating aquaculture systems. To demonstrate these techniques, a preliminary comparison of the reproductive output and timing of coral colonies held at 24 &#176;C and 28 &#176;C under &#34;fed&#34; and &#34;unfed&#34; treatments was conducted. These temperatures were chosen to approximate seawater temperatures in winter and summer, respectively, in southern Taiwan30,34; a higher temperature was not chosen because the promotion of long-term ex situ culture, rather than testing coral response to thermal stress, was a primary goal of this experiment. Further, the density of Artemia nauplii before and after the feeding sessions was quantified&#160;to compare the feasibility of heterotrophic feeding at both temperature treatments.
Specifically, 24 colonies of P. acuta (mean total linear extension &#177; standard deviation: 21.3 cm &#177; 2.8 cm) were obtained from flow-through tanks at the research facilities of the National Museum of Marine Biology &#38; Aquarium, southern Taiwan. Pocillopora&#8203;&#160;acuta is a common coral species that possesses both a broadcast spawning, but&#160;typically brooding reproductive strategy35,36. The parent colonies of these corals were originally collected from the Outlet reef (21.931&#176;E, 120.745&#176;N) approximately 2 years earlier for another experiment32. Consequently, the coral colonies used in the present experiment had been reared for their entire lives under ex situ culture conditions; specifically, the colonies were exposed to ambient temperature and a 12 h:12 h light: dark cycle at 250 &#181;mol quanta m&#8722;2&#183;s&#8722;1 and were fed Artemia nauplii twice per week. We recognize that this long-term ex situ culture could have affected how the colonies responded to the treatment conditions in this experiment. We, therefore, would like to emphasize that the primary aim here is to illustrate how the described techniques can be effectively used to culture corals ex situ by demonstrating an applied example wherein the effects of temperature and feeding on coral reproduction were assessed.
Coral colonies were evenly distributed across six flow-through system culture tanks (tank interior length x width x height: 175 cm x 62 cm x 72 cm; tank light regime: 12 h:12h light:dark cycle at 250 &#181;mol quanta m&#8722;2&#183;s&#8722;1) (Figure 1A). The temperature in three of the tanks was set at 28 &#176;C, and the temperature in the other three tanks was set at 24 &#176;C; each tank had a logger that recorded the temperature every 10 min (see the Table of Materials). The temperature was independently controlled in each tank using chillers and heaters, and water circulation was maintained using flow motors (see the Table of Materials). Half of the colonies in each tank (n = 2 colonies/tank) were fed Artemia nauplii twice per week, while the other colonies were not fed. Each feeding session was 4 h in duration and was conducted in two independent temperature-specific feeding tanks. During feeding, all the colonies were moved into the feeding tanks, including the unfed colonies, to standardize the potential stress effect of moving the colonies between the tanks. The colonies in the fed and unfed treatments were positioned in their own compartment using a meshed frame within the temperature-specific feeding tanks so that only the colonies in the fed condition received food. The coral reproductive output and timing were assessed for each colony daily at 09:00 A.M. by counting the number of larvae that had been released into the larval collection containers overnight.

Author Spotlight: Advancing Coral Research by Exploring Climate Change Resistance, Ex Situ Aquaculture, and Reproduction Strategies 

Effective Techniques for the Feeding and Ex Situ Culture of a Brooding Scleractinian Coral, Pocillopora acuta

Our lab is interested in understanding climate change, resistance, and resilience in coral and exploring ex-situ aquaculture's role. Specifically, we aim to cultivate coral in a lab that can be used for restoration and research efforts. Over the past few years, we have seen major advances in coral ex-situ aquaculture, including being able to induce reproduction, as well as complete the entire coral life cycle in both broadcast spawning and brooding corals.Maintaining the long-term reproduction of brooding corals in the lab is usually quite challenging, with reproduction typically stopping after a few months. However, we found that if we culture the colonies together in mesocosm tanks and provide them with supplemental food, they can continue to produce larvae each month. In general, we have a good understanding of how feeding and temperature independently affect coral reproduction, but their interactive effects remain relatively understudied.For example, heterotrophy may have different benefits, or limitations at warmer compared to cooler temperatures. Using the protocols that we have described, we can effectively feed and culture the colonies long-term in X2 aquaculture systems, and can ultimately track the reproduction at the colony level to help infer the influence of heterotrophy on reproduction, and recruitment in a warming ocean.

Watch this Scientific Journal Video about Quantifying Artemia Nauplii Density Pre- and Post-Feeding of Pocillopora acuta at JoVE.com

Quantifying Artemia Nauplii Density Pre- and Post-Feeding of Pocillopora acuta

To quantify Artemia Nauplii used for coral feeding experiments, collect the samples at two time points. At each time point, use three syringes to collect 20 milliliters of water from the feeding container's surface, middle layer, and bottom layer. Transfer the collected water sample into individual 500-milliliter beakers.Next, add 180 milliliters of hot water, approximately 60 degrees Celsius, to the beaker to achieve a 1 to 10 dilution. Add two milliliters of water sample from the beaker into each well of a six-well plate. Next, using a stereo microscope, set to 6.5 times magnification.Count the number of Artemia Nauplii in each well. Density measurements of Artemia Nauplii before and after coral feeding sessions showed that the initial assessment at T0 showed no difference in both temperature treatments. After two and four weeks of culture, the Artemia Nauplii density was lower after feeding in both temperature treatments.There was no difference in the pre-feeding density between temperature treatments, or the post-feeding density between temperature treatments at any of the three time points assessed.

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76 Views.  National Museum of Marine Biology & Aquarium. To quantify Artemia Nauplii used for coral feeding experiments, collect the samples at two time points. At each time point, use three syringes to collect 20 milliliters of water from the feeding container's surface, middle layer, and bottom layer. Transfer the collected water sample into individual 500-milliliter beakers.Next, add 180 milliliters of hot water, approximately 60 degrees Celsius, to the beaker to achieve a 1 to 10 dilution. Add two milliliters of water sample from the bea...