这项研究由台湾科学技术部资助，资助号为 MOST 111-2611-M-291-005 和 MOST 111-2811-M-291-001。

1. 在非原生境水产养殖池中悬挂珊瑚群落
<ol><li>在养殖罐上放置一个缺口杆（长 x 宽 x 高：75 cm x 1 cm x 3 cm），以下称为“吊杆”，以准备悬挂珊瑚群落。 注意：本实验中使用的吊杆是定制的，但只要一根带有突出螺钉（即充当槽口）的简单 PVC 管就足够了，只要它可以稳定地放置在培养罐的顶部并且足够坚固以容纳珊瑚。</li>
	<li>测量一条钓鱼线（参见 材料表...

Pocillopora acuta 的异位培养和有效饲喂技术

对温度和摄食对珊瑚繁殖影响的初步评估揭示了在不同处理条件下培养的菌落之间的繁殖产量和时间差异。此外，研究发现，在相对凉爽（24°C）和温暖的温度（28°C）下，将卤虫无节幼体喂食到珊瑚群落似乎有效。这些综合研究结果突出了这些直接技术在非原生境水产养殖系统中繁殖硬质珊瑚（以P. acuta为例）的喂养和培养的适用性。
在繁殖产量方面，?...

由于气候变化导致的高温压力，全球许多珊瑚礁生态系统正在丧失和退化1,2。珊瑚白化（即珊瑚-藻类共生的破坏3）在过去4 中被认为是相对罕见的，但现在发生得更频繁5，预计到本世纪中后期，许多地区将发生年度白化 6,7。白化事件之间过渡期的缩短会限制珊瑚礁的复原力8.高温胁迫对珊瑚群落的直接影响（例如，组织损伤9;能量消耗10）与珊瑚礁规模的间接影响有着内在的联系，其中繁殖/招募能力的降低尤其令人关注11。这刺激了一系列应用研究，例如，积极原位增强招募（例如，珊瑚礁播种12）、扩大珊瑚恢复的新技术 13，以及模拟繁殖线索以诱导迁地系统中的繁殖14。与这些积极干预措施相辅相成的是最近认识到在高温胁迫下珊瑚异养摄食的优势15，以及探索食物供应在繁殖中可能发挥的作用16。
众所周知，异养喂养会影响珊瑚的性能17，并且与珊瑚生长增加18,19 以及热阻和弹性 20,21 特别相关。然而，异养的好处在珊瑚物种中并不普遍22，并且可能因食用的食物类型 23 以及光照水平而有所不同24。在珊瑚繁殖的背景下，异养喂养显示出不同的结果，据报道，异养喂养后繁殖能力较高25 和较低26。异养摄食对珊瑚在一系列温度范围内繁殖的影响很少被评估，但在温带珊瑚Cladocora caespitosa中，发现异养对于较低温度条件下的繁殖更为重要27。可能需要更好地了解温度和摄食对繁殖产量的作用，以确定特定珊瑚礁（例如，与高食物供应相关的珊瑚礁28）在气候变化下是否具有更高的招募能力。
与繁殖产量类似，温度和摄食对珊瑚繁殖时间的影响仍然研究不足，尽管繁殖与非生物/生物条件的同步是在变暖的海洋中招募成功的重要考虑因素29。在实验室30进行的珊瑚热调节研究中，较高的温度已被证明会导致更早的繁殖，并且在31季节从天然珊瑚礁收集的珊瑚中也观察到了这一点。然而，有趣的是，最近在 非原生境 流通系统中养殖了 1 年的喂养珊瑚中观察到了相反的趋势（即，在冬季温度较低的月球周期中发生繁殖，在夏季温度较高的月球周期中发生后期）32。这一对比结果表明，在与丰富的能量资源相关的条件下，繁殖时间可能偏离典型模式。
不同温度情景下的长期对照实验有助于更好地了解异养对硬质珊瑚繁殖的影响。然而，在多个繁殖周期的非原生境条件下维持繁殖珊瑚群落可能具有挑战性（但参见以前的研究32,33）。本文描述了在流通式水产养殖系统中主动摄食（食物来源：卤虫无节幼体）和长期养殖育雏珊瑚（Pocillopora acuta）的简单而有效的技术;然而，应该指出的是，所描述的所有技术也可用于循环水产养殖系统。为了证明这些技术，对在“喂养”和“未喂养”处理下保持在24°C和28°C的珊瑚群落的繁殖产量和时间进行了初步比较。选择这些温度分别近似于台湾南部冬季和夏季的海水温度30,34;没有选择更高的温度，因为促进长期迁地养殖，而不是测试珊瑚对热应激的反应，是该实验的主要目标。此外，量化了喂养前后卤虫无节幼体的密度，以比较两种温度处理下异养喂养的可行性。
具体来说，从台湾南部国立海洋生物博物馆和水族馆研究设施的流通罐中获得24个P. acuta菌落（平均总线性延伸±标准差：21.3厘米±2.8厘米）。Pocillopora acuta 是一种常见的珊瑚物种，它既具有广播产卵，又具有典型的育雏繁殖策略35,36。这些珊瑚的亲本群落最初是在大约2年前从出口礁（21.931°E，120.745°N）收集的，用于另一项实验32。因此，本实验中使用的珊瑚群落是在非原生境养殖条件下饲养的;具体来说，菌落暴露于环境温度和 12 小时：12 小时光：暗循环，量子为 m−2·s−1，每周喂食两次卤虫无节幼体。我们认识到，这种长期的异地培养可能影响了菌落对本实验中处理条件的反应。因此，我们想强调的是，这里的主要目的是通过展示一个应用的例子来说明如何有效地使用所描述的技术来异地养殖珊瑚，其中评估了温度和喂养对珊瑚繁殖的影响。
珊瑚菌落均匀分布在六个流通系统培养罐中（罐内长x宽x高：175 cm x 62 cm x 72 cm;罐光状态：12 h：12h 光：暗循环，250 μmol量子m-2·s-1）（图1A）。其中三个罐中的温度设定为28°C，其他三个罐中的温度设定为24°C;每个储罐都有一个记录器，每 10 分钟记录一次温度（参见材料表）。使用冷却器和加热器独立控制每个水箱的温度，并使用流量马达保持水循环（参见材料表）。每个菌池中一半的菌落（n = 2 个菌落/池）每周喂食两次卤虫无节幼体，而其他菌落则不喂食。每次饲喂持续 4 小时，在两个独立的特定温度饲喂罐中进行。在饲养过程中，将所有菌落移入饲养罐，包括未饲喂的菌落，以标准化在罐之间移动菌落的潜在压力效应。饲喂和未饲喂处理中的菌落使用特定温度饲养罐内的网状框架放置在自己的隔室中，以便只有饲养条件下的菌落才能获得食物。每天上午09：00，通过计算一夜之间释放到幼虫收集容器中的幼虫数量，评估每个群落的珊瑚繁殖产量和时间。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Artemia cysts&#160;</td>
			<td>Supreme plus</td>
			<td>NA</td>
			<td>Food source&#160;</td>
		</tr>
		<tr>
			<td>Chiller</td>
			<td>Resun</td>
			<td>CL650</td>
			<td>To cool down water temperature if needed</td>
		</tr>
		<tr>
			<td>Conductivity portable meter</td>
			<td>WTW</td>
			<td>Cond 3110</td>
			<td>To measure salinity</td>
		</tr>
		<tr>
			<td>Enrichment diets</td>
			<td>Omega</td>
			<td>NA</td>
			<td>Used in Artemia cultivation</td>
		</tr>
		<tr>
			<td>Fishing line</td>
			<td>Super</td>
			<td>Nylon monofilament</td>
			<td>To hang the coral colonies</td>
		</tr>
		<tr>
			<td>Flow motors</td>
			<td>Maxspect</td>
			<td>GP03</td>
			<td>To create water flow</td>
		</tr>
		<tr>
			<td>Heater 350 W</td>
			<td>ISTA</td>
			<td>NA</td>
			<td>Heaters used in tanks</td>
		</tr>
		<tr>
			<td>HOBO pendant temperature logger</td>
			<td>Onset Computer</td>
			<td>UA-002-08</td>
			<td>To record water temperature</td>
		</tr>
		<tr>
			<td>LED lights</td>
			<td>Mean Well</td>
			<td>FTS: HLG-185H-36B</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Light portable meter</td>
			<td>LI-COR</td>
			<td>LI-250A</td>
			<td>Device used with light sensor to measure light intensity in PAR</td>
		</tr>
		<tr>
			<td>Light sensor</td>
			<td>LI-COR</td>
			<td>LI-193SA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Plankton net 100 &#181;m mesh size</td>
			<td>Omega</td>
			<td>NA</td>
			<td>To collect larvae and artemia&#160;</td>
		</tr>
		<tr>
			<td>Primary pump 6000 L/H</td>
			<td>Mr. Aqua</td>
			<td>BP6000</td>
			<td>To draw water from tanks into chiller</td>
		</tr>
		<tr>
			<td>Propeller-type current meter</td>
			<td>KENEK</td>
			<td>GR20</td>
			<td>Device used with propeller-type detector to measure flow rate</td>
		</tr>
		<tr>
			<td>Propeller-type detector</td>
			<td>KENEK</td>
			<td>GR3T-2-20N</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000-C&#160;</td>
			<td>To count the number of artemia&#160;</td>
		</tr>
		<tr>
			<td>Temperature controller 1000 W</td>
			<td>Rep Park</td>
			<td>O-RP-SDP-1</td>
			<td>To set and maintain water temperature</td>
		</tr>
	</tbody>
</table>

effective techniques for the feeding and ex situ culture of a brooding scleractinian coral, pocillopora acuta

气候变化正在影响全球珊瑚的生存、生长和招募，预计未来几十年珊瑚礁生态系统的丰度和群落组成将发生大规模变化。对这种珊瑚礁退化的认识促使了一系列新的研究和基于恢复的积极干预措施。通过建立健全的珊瑚养殖规程（例如，在长期试验中改善健康和繁殖）和提供稳定的亲鱼供应（例如，用于恢复项目），非原生境水产养殖可以发挥支持作用。在这里，以常见且经过充分研究的珊瑚Pocillopora acuta为例，概述了育雏硬质珊瑚的喂养和迁地培养的简单技术。为了证明这种方法，将珊瑚群落暴露在不同的温度（24°C与28°C）和摄食处理（喂食与未喂食）下，并比较了繁殖产量和时间，以及在两种温度下将卤虫无节幼体喂食珊瑚的可行性。不同菌落的繁殖产量差异很大，在温度处理之间观察到不同的趋势;在24°C下，投喂的菌落比未投喂的菌落产生更多的幼虫，但在28°C培养的菌落中发现相反的情况。 所有菌落在满月前繁殖，仅在28°C处理的未喂养菌落和24°C处理的喂养菌落之间发现繁殖时间的差异（平均月球繁殖日±标准差：分别为6.5±2.5和11.1±2.6）。在两种处理温度下，珊瑚群落都有效地以卤虫无节幼体为食。这些拟议的饲养和养殖技术侧重于以具有成本效益和可定制的方式减少珊瑚压力和促进繁殖寿命，在流通和循环水产养殖系统中具有广泛的适用性。

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.

作者聚焦：通过探索气候变化抵抗力、异地水产养殖和繁殖策略来推进珊瑚研究 

育雏硬质珊瑚（Pocillopora acuta）的饲养和迁地培养的有效技术

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.

所描述的方案允许 （1） 比较不同摄食和温度处理之间单个珊瑚群落的繁殖产量和时间，以及 （2） 评估 卤虫 无节幼体在不同温度下摄食的可行性。在此，对研究结果进行了简要概述，但由于该实验的短期性质（即只有一个繁殖周期）和使用适应 异地 条件的珊瑚群落，因此对温度和喂养对珊瑚繁殖的影响的广泛解释应谨慎。
在我们的监测期间（2022 年 9 月）?...

Watch this Scientific Journal Video about Advancing Coral Research by Exploring Climate Change Resistance, Ex Situ Aquaculture, and Reproduction Strategies at JoVE.com

气候变化正在影响全球珊瑚礁生态系统。来自 非原生境 水产养殖系统的珊瑚可以帮助支持恢复和研究工作。本文概述了可用于促进非 原生 境育雏硬质珊瑚长期维持的喂养和珊瑚养殖技术。

Climate change is affecting the survival, growth, and recruitment of corals globally, with large-scale shifts in abundance and community composition ...

我们的实验室有兴趣了解气候变化、珊瑚的抵抗力和复原力，并探索迁地水产养殖的作用。具体来说，我们的目标是在实验室中培育珊瑚，用于恢复和研究工作。在过去的几年里，我们看到了珊瑚迁地养殖的重大进展，包括能够诱导繁殖，以及在广播产卵和育雏珊瑚中完成整个珊瑚生命周期。在实验室中维持育雏珊瑚的长期繁殖通常非常具有挑战性，繁殖通常会在几个月后停止。然而，我们发现，如果我们在中宇宙池中一起培养菌落并为它们提供补充食物，它们可以继续每月产生幼虫。总的来说，我们对摄食和温度如何独立影响珊瑚繁殖有很好的了解，但它们的相互作用影响仍然相对不足。例如，与较低温度相比，异养在温暖的温度下可能具有不同的益处或局限性。使用我们描述的协议，我们可以在X2水产养殖系统中有效地长期喂养和培养蜂群，并最终可以在蜂群水平上跟踪繁殖，以帮助推断异养对繁殖的影响，以及在变暖的海洋中招募。

育雏硬质珊瑚（Pocillopora acuta）的饲养和迁地培养的有效技术

Abstract