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Rapid shifts in thermal reaction norms and tolerance of brooded coral larvae following parental heat acclimation

Lei Jiang | Cheng-Yue Liu | Guoxin Cui | Lin-Tao Huang |
Lei Jiang | Cheng-Yue Liu | Guoxin Cui | Lin-Tao Huang |
Xiao-Lei Yu | You-Fang Sun | Hao-Ya Tong | Guo-Wei Zhou |
Xiao-Lei Yu | You-Fang Sun | Hao-Ya Tong | Guo-Wei Zhou |
Xiang-Cheng Yuan | Yi-Si Hu | Wen-Liang Zhou Manuel Aranda |
Xiang-Cheng Yuan | Yii-Si Hu | Wen-Liang Zhou Manuel Aranda |
Pei-Yuan Qian | Hui Huang
Pei-Yuan Qian | Hui Huang
CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute
of Oceanology (SCSIO), Chinese Academy of Sciences, Guangzhou, China
Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou, China
Department of Ocean Science and Hong Kong Branch (HKB) of the Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), The Hong
Kong University of Science and Technology (HKUST), Hong Kong, China
Biological and Environmental Sciences and Engineering Division, Red Sea Research Center, King Abdullah University of Science and Technology, Thuwal, Saudi
Arabia 阿拉伯 CAS-HKUST Sanya Joint Laboratory of Marine Science Research, Key Laboratory of Tropical Marine Biotechnology of Hainan Province, Sanya Institute of
Ocean Eco-Environmental Engineering, SCSIO, Sanya, China
Sanya National Marine Ecosystem Research Station, Tropical Marine Biological Research Station in Hainan, Chinese Academy of Sciences, Sanya, China
University of Chinese Academy of Sciences, Beijing, China

Correspondence 通信

Manuel Aranda, Biological and
Environmental Sciences and Engineering Division, Red Sea Research Center, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia. Email: manuel.aranda@kaust.edu.sa
Pei-Yuan Qian, Department of Ocean Science and Hong Kong Branch (HKB) of the Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), The Hong Kong University of Science and Technology (HKUST), Hong Kong, China.
Email: boqianpy@ust.hk 电子邮件: boqianpy@ust.hk
Hui Huang, CAS Key Laboratory of Tropical Marine Bio-resources and Ecology; Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology (SCSIO), Chinese Academy of Sciences, Guangzhou 510301, China.
黄晖,中科院热带海洋生物资源与生态重点实验室;中国科学院南海海洋研究所广东省应用海洋生物学重点实验室,中国广州 510301。
Email: huanghui@scsio.ac.cn
电子邮件: huanghui@scsio.ac.cn
Funding information 资金信息
National Key Research and Development Program, Grant/Award Number: 2021 YFF0502804 and 2021YFC3100504;
国家重点研发计划,资助/奖励编号:2021 YFF0502804 和 2021YFC3100504;

Abstract 摘要

Thermal priming of reef corals can enhance their heat tolerance; however, the legacy effects of heat stress during parental brooding on larval resilience remain understudied. This study investigated whether preconditioning adult coral Pocillopora damicornis to high temperatures and could better prepare their larvae for heat stress. Results showed that heat-acclimated adults brooded larvae with reduced symbiont density and shifted thermal performance curves. Reciprocal transplant experiments demonstrated higher bleaching resistance and better photosynthetic and autotrophic performance in heat-exposed larvae from acclimated adults compared to unacclimated adults. RNA-seq revealed strong cellular stress responses in larvae from heat-acclimated adults that could have been effective in rescuing host cells from stress, as evidenced by the widespread upregulation of genes involved in cell cycle and mitosis. For symbionts, a molecular coordination between light harvesting, photoprotection and carbon fixation was detected in larvae from heat-acclimated adults, which may help optimize photosynthetic activity and yield under high temperature. Furthermore, heat acclimation led to opposing regulations of symbiont catabolic and anabolic pathways and favoured nutrient translocation to the host and thus a functional symbiosis. Notwithstanding, the improved heat tolerance was paralleled by reduced light-enhanced dark respiration, indicating metabolic depression for energy
珊瑚礁珊瑚的热预处理可以提高它们的耐热性;然而,亲代育雏期间的热应力对幼体恢复能力的遗留影响仍未得到充分研究。本研究调查了将大疣梭子蟹成体置于高温环境中 是否能使其幼体更好地适应热应激。结果表明,热适应成体繁殖的幼虫共生体密度降低,热性能曲线发生变化。对等移植实验表明,与未适应热应激的成虫相比,适应热应激的成虫的抗漂白能力更强,光合作用和自养性能更好。RNA-seq发现,来自热适应成虫的幼虫具有强烈的细胞应激反应,这可能有效地将宿主细胞从应激中解救出来,细胞周期和有丝分裂相关基因的广泛上调就是证明。就共生体而言,在来自热螯合成虫的幼虫体内检测到了采光、光保护和碳固定之间的分子协调,这可能有助于优化高温条件下的光合作用和产量。此外,热适应导致共生体分解代谢和合成代谢途径的相反调节,有利于营养物质向宿主转移,从而实现功能性共生。尽管如此,在耐热性提高的同时,光照增强的暗呼吸也减少了,这表明能量代谢受到抑制

Lei Jiang and Cheng-Yue Liu contributed equally to this work.
National Natural Science Foundation of China, Grant/Award Number: 42106115 and 41906097; Science and Technology Planning Project of Guangdong Province, China, Grant/Award Number: 2020B212060058
国家自然科学基金,批准/授予号:42106115 和 41906097;广东省科技计划项目,批准/授予号:2020B212060058
Handling Editor: Pim Bongaerts saving. Our findings suggest that adult heat acclimation can rapidly shift thermal tolerance of brooded coral larvae and provide integrated physiological and molecular evidence for this adaptive plasticity, which could increase climate resilience. However, the metabolic depression may be maladaptive for long-term organismal performance, highlighting the importance of curbing carbon emissions to better protect corals.
处理编辑:Pim Bongaerts我们的研究结果表明,成体热适应可以迅速改变育雏珊瑚幼虫的热耐受性,并为这种适应性可塑性提供了综合的生理和分子证据,这可以提高气候适应能力。然而,新陈代谢抑制可能会对生物体的长期表现产生不良影响,这凸显了遏制碳排放以更好地保护珊瑚的重要性。


acclimation, coral larvae, heat tolerance, metabolic depression, parental preconditioning, transcriptome


Reef-building corals, the framework builders of coral reef ecosystems, are symbiotic cnidarians that form an obligate mutualistic relationship with dinoflagellates from the family Symbiodiniaceae (LaJeunesse et al., 2018). The algal symbionts translocate the majority of their photosynthetic products to sustain the energy demand of their coral host, and in return, the host nourishes symbionts with inorganic nutrients to fuel photosynthesis (Yellowlees et al., 2008). Tropical reef corals often live close to their upper thermal limits and are adversely affected by marine heat waves associated with climate change (Hoegh-Guldberg et al., 2017). During prolonged warming conditions, reef corals expel their algal symbionts, resulting in bleaching and mortality which have contributed significantly to the global degradation of coral reefs over the past decades (Hughes et al., 2018). It is therefore imperative to assess the ability of corals to acclimatize and/or adapt to ocean warming in this era of rapid climate change.
珊瑚礁生态系统的框架构建者--造礁珊瑚是一种共生的刺胞动物,与共生藻科的甲藻形成一种强制性的互生关系(LaJeunesse 等人,2018 年)。藻类共生体将其大部分光合产物转运到珊瑚宿主,以维持其能量需求,而作为回报,宿主则用无机营养物质滋养共生体,以促进光合作用(Yellowlees 等人,2008 年)。热带珊瑚礁珊瑚通常生活在其热上限附近,并受到与气候变化相关的海洋热浪的不利影响(Hoegh-Guldberg 等人,2017 年)。在长期变暖的条件下,珊瑚礁珊瑚会排出它们的藻类共生体,导致白化和死亡,这在过去几十年中极大地加剧了全球珊瑚礁的退化(Hughes 等人,2018 年)。因此,在这个气候变化迅速的时代,评估珊瑚适应和/或适应海洋变暖的能力势在必行。
Despite the consensus that the genetic adaptation of reef corals might not keep pace with the current rate of ocean warming due to their long generation time, it has been increasingly recognized that acclimatization through phenotypic plasticity may to some extent buffer against the immediate impacts of ocean warming on reef corals (Hackerott et al., 2021; Torda et al., 2017; Voolstra et al., 2021). Several studies demonstrated that thermal priming and environmental hardening can enhance the acclimatization and resistance of reef corals to thermal stress (Brown et al., 2015; DeMerlis et al., 2022; Hackerott et al., 2021; Yu et al., 2020), either through symbiont shuffling or transcriptional reprogramming (Barshis et al., 2013; Kenkel & Matz, 2016; Silverstein et al., 2015). Furthermore, numerous recent studies showed that adult preconditioning can mitigate and even fully offset the negative effects of climate change stressors on their offspring in a wide range of marine taxa, suggesting that transgenerational and/or developmental plasticity may also serve as key acclimatory mechanisms by which marine organisms can persist under climate change (Donelson et al., 2018; Maboloc & Chan, 2021; Wong et al., 2018; Yin et al., 2019).
尽管人们一致认为,由于珊瑚礁珊瑚的世代较长,其遗传适应性可能跟不上当前海洋变暖的速度,但人们越来越认识到,通过表型可塑性实现的适应性可在一定程度上缓冲海洋变暖对珊瑚礁珊瑚的直接影响(Hackerott等人,2021年;Torda等人,2017年;Voolstra等人,2021年)。多项研究表明,通过共生体洗牌或转录重编程(Barshis 等人,2013 年;Kenkel & Matz,2016 年;Silverstein 等人,2015 年),热引诱和环境硬化可增强珊瑚礁珊瑚对热应力的适应性和抵抗力(Brown 等人,2015 年;DeMerlis 等人,2022 年;Hackerott 等人,2021 年;Yu 等人,2020 年)。此外,最近的许多研究表明,在多种海洋类群中,成体预调节可以减轻甚至完全抵消气候变化压力因素对其后代的负面影响,这表明跨代和/或发育可塑性也可能成为海洋生物在气候变化下持续生存的关键适应机制(Donelson等人,2018年;Maboloc和Chan,2021年;Wong等人,2018年;Yin等人,2019年)。
Corals are sessile invertebrates with a pelagic larval phase which plays a vital role in population replenishment and recovery following disturbances. In an era of rapid climate change, it is of great interest to know if parental thermal acclimation/adaptation could improve larval heat resilience. Previous studies on broadcast spawning corals have shown strong maternal and paternal contributions to larval heat tolerance (Dixon et al., 2015; Howells et al., 2016, 2021). On the other hand, studies on the effects of adult stress exposure on offspring performance in brooding corals reported inconclusive results. For instance, preconditioning adult brooding corals to high temperature and/or either led to larval metabolic acclimation and improved settlement and survivorship of new recruits (Galanto et al., 2022; Putnam & Gates, 2015; Putnam et al., 2020), exerted negligible or negative influence on larval traits and tolerance to warmer temperatures (Bellworthy et al., 2019; McRae et al., 2021; Wong et al., 2021). Recently, parental preconditioning and transgenerational plasticity were proposed to be promising mechanisms of evolutionary rescue in coral reef conservation (Torda et al., 2017; Voolstra et al., 2021). However, it remains unclear to what extent heat acclimation of brooding corals could promote larval resilience. More importantly, our understanding of the physiological and molecular mechanisms underpinning this plasticity remains limited. Such information is of paramount importance to predict the adaptive capacity and evolutionary trajectory of reef corals facing global climate change.
珊瑚是一种无梗无脊椎动物,其幼虫处于浮游阶段,在受到干扰后的种群补充和恢复过程中发挥着至关重要的作用。在气候变化迅速的时代,了解亲本的热适应/适应能力是否能提高幼体的抗热能力是非常有意义的。之前对直播产卵珊瑚的研究表明,母体和父体对幼虫的耐热性有很大的贡献(Dixon 等人,2015 年;Howells 等人,2016 年,2021 年)。另一方面,关于成体压力暴露对育雏珊瑚后代表现的影响的研究却没有得出结论。例如,将成体育雏珊瑚预处理在高温和/或 条件下,要么会导致幼虫代谢适应,提高新成员的定居和存活率(Galanto 等人,2022 年;Putnam 与 Gates,2015 年;Putnam 等人,2020 年),要么对幼虫性状和对较高温度的耐受性影响微乎其微或产生负面影响(Bellworthy 等人,2019 年;McRae 等人,2021 年;Wong 等人,2021 年)。最近,有人提出亲本预调节和跨代可塑性是珊瑚礁保护中很有希望的进化拯救机制(Torda 等人,2017 年;Voolstra 等人,2021 年)。然而,目前仍不清楚育雏珊瑚的热适应在多大程度上能促进幼虫的恢复能力。更重要的是,我们对支撑这种可塑性的生理和分子机制的了解仍然有限。这些信息对于预测珊瑚礁珊瑚面对全球气候变化的适应能力和进化轨迹至关重要。
In the present study, we investigated the impact of thermal priming in adult coral Pocillopora damicornis on the heat tolerance of their brooded larvae. P. damicornis is a hermaphrodite species with a predominantly brooding reproductive mode (Fan et al., 2002). Here, we simulated a thermal priming event in adult P. damicornis that coincided with the larval development within the parent polyps. Although this design, similar to most prior studies, could not distinguish between parental effects and developmental acclimation of larval offspring (Donelson et al., 2018), it is ecologically relevant and does not affect the main objective of studying the acclimatory mechanism of corals to cope with future warming. We first examined the influence of thermal preconditioning on adult performance, timing of larval release, larval quantity and quality, and determined the effects of adult exposure on larval thermal performance curves (TPC). We then conducted a reciprocal transplant experiment to test the potential interaction between adult and offspring temperatures on larval physiology and analysed the transcriptomes of the host and symbionts after
在本研究中,我们调查了大鳃珊瑚成虫的热引诱对其育雏幼虫耐热性的影响。P. damicornis 是一种雌雄同体物种,主要采用育雏繁殖模式(Fan 等人,2002 年)。在此,我们模拟了大菱鲆成虫的热引诱事件,该事件与幼虫在亲体息肉中的发育过程相吻合。虽然这种设计与之前的大多数研究类似,无法区分亲本效应和幼虫后代的发育适应(Donelson 等人,2018 年),但它与生态相关,并且不影响研究珊瑚应对未来变暖的适应机制这一主要目标。我们首先考察了热预处理对成体性能、幼虫释放时间、幼虫数量和质量的影响,并确定了成体暴露对幼虫热性能曲线(TPC)的影响。然后,我们进行了一次相互移植实验,以检验成虫和后代温度对幼虫生理的潜在相互作用,并分析了成虫和后代温度变化后宿主和共生体的转录组。

reciprocal exposure to unravel the molecular signatures of thermal acclimation and phenotypic plasticity.

2 材料和方法

2.1 | Experimental design
2.1 实验设计

Eight adult colonies of . damicornis diameter) were collected at depth on Luhuitou reef, Sanya, Hainan Island, China (N18 ) on 24 September 2020. P. damicornis from Sanya release free-swimming zooxanthellate larvae following a lunar cycle, with release beginning near the new moon, peaking around the first quarter moon, and ending before the full moon (Zhang, 2015). Gametogenesis and brooding usually overlap in P. damicornis, and larval development is rapid, because the time required for larval growth before release is approximately 3-4weeks (Permata et al., 2000; Stoddart & Black, 1985).
2020 年 9 月 24 日,在中国海南岛三亚鹿回头珊瑚礁(N18 ,采集到 8 个 . damicornis 直径)的成体。三亚的 P. damicornis 按月周期释放自由游动的聚氧贝壳幼体,释放从新月附近开始,在第一个季月前后达到高峰,在满月前结束(Zhang,2015 年)。P. damicornis的配子发生和育雏通常是重叠的,幼虫发育迅速,因为释放前幼虫生长所需的时间约为3-4周(Permata等人,2000年;Stoddart和Black,1985年)。
Colonies were immediately transported to the Tropical Marine Biological Research Station and reared in independent 20-L tanks that received flowing sand-filtered seawater and natural sunlight. Four colonies were randomly assigned to either the control treatment , mean or heat treatment , mean , Figure 1a). Seawater temperature in the control and heat treatment was regulated using chillers (Haili, HS90A, China) and digital heaters (Chaning, CN-008-1000 W, China), respectively. The greater temperature variation in the control treatment was due to the lower accuracy of temperature control of the chillers (Figure S1).
菌落被立即运送到热带海洋生物研究站,并在 20 升的独立水箱中饲养,水箱中的海水经过流沙过滤, ,并接受自然阳光照射。四个菌落被随机分配到对照处理 , 平均 或加热处理 , 平均 , 图 1a)。对照处理和加热处理的海水温度分别由制冷器(海利,HS90A,中国)和数字加热器(长宁,CN-008-1000 W,中国)调节。对照处理的温度变化较大,这是因为制冷机的温度控制精度较低(图 S1)。
Temperature in the control treatment represented the mean summer ambient temperature on the reefs, while temperature in the heat treatment was about above the bleaching threshold of local coral communities (Li et al., 2012). The heat ramping commenced on 25 September, which was five days before the full moon and close to the end of larval release in September. Seawater temperature in heat treatment was increased from at a rate of per day to and maintained thereafter for about 1 month until 27 October (Figure S1). Hence, the time course of adult priming encompassed the development duration of larvae that were expected to release in October.
对照处理的温度代表了珊瑚礁夏季的平均环境温度,而加热处理的温度比当地珊瑚群落的白化阈值高出约 (Li 等人,2012 年)。升温从 9 月 25 日开始,此时距满月还有 5 天,接近 9 月份幼虫释放的结束时间。加热处理中的海水温度从 以每天 的速度升至 ,此后维持约 1 个月,直至 10 月 27 日(图 S1)。因此,成虫启动的时间过程包含了预计在 10 月份释放的幼虫的发育期。
During the predicted planulation period in October, three colonies from each treatment were observed to release larvae and larvae were collected as described in Jiang et al. (2020). The number of larvae released by each colony was counted daily and normalized to the colony ecological volume (Levy et al., 2010). On 21 October (lunar day 5) near the peak release, larvae released by the three adults from each treatment were pooled in order to incorporate the maximum number of cohorts and focus on the average effects of thermal acclimation (Wong et al., 2018). Larvae from these two pools were randomly assigned for larval traits and TPCs characterization and the reciprocal transplant experiment (Figure 1b,c).
在 10 月份的预测繁殖期,观察每个处理的三个蜂群释放幼虫的情况,并按照 Jiang 等人(2020 年)的方法收集幼虫。对每个蜂群每天释放的幼虫数量进行计数,并将其归一化为蜂群生态体积(Levy 等,2010 年)。在 10 月 21 日(农历 5 月 5 日)接近释放高峰时,将每个处理的三只成虫释放的幼虫集中起来,以纳入最大数量的群落,并关注热适应的平均效应(Wong 等人,2018 年)。这两个池中的幼虫被随机分配用于幼虫性状和 TPCs 表征以及对等移植实验(图 1b、c)。

2.2 | Larval traits upon release and adult responses
2.2 | 幼虫释放后的性状和成虫的反应

On 12 October 2020, 100 larvae from each parental pool were haphazardly selected and photographed under a stereomicroscope. The length and width of each larva were measured using ImageJ, and larval volume was calculated following Isomura and Nishihira (2001). Four groups of 10 larvae from each adult pool were dark adapted for , and the maximum photochemical efficiency of photosystem (PS) II was measured using a Diving-PAM fluorometer (Walz, Germany) following Putnam et al. (2008). Briefly, one group of 10 larvae within a drop of seawater was loaded onto the tip of the 8 -mm-diameter probe. Both the measuring light intensity and gain were adjusted to seven to ensure optimal initial fluorescence. Furthermore, eight groups of 20 larvae were randomly sampled from each adult group and stored at for symbiont density quantification. Larvae were homogenized in of filtered and UV-sterilized seawater (FSW), centrifuged at for , and the pellet was resuspended in of FSW. The number of algal symbionts was counted using a haemocytometer with six replicate counts per sample, and the symbiont densities were expressed as average cells per larvae.
2020 年 10 月 12 日,从每个亲本池中随机挑选 100 只幼虫,在体视显微镜下拍照。使用 ImageJ 测量每条幼虫的长度和宽度,并按照 Isomura 和 Nishihira(2001 年)的方法计算幼虫体积。将每个成虫池中的四组 10 只幼虫进行黑暗适应 ,并按照 Putnam 等人(2008 年)的方法,使用 Diving-PAM 荧光仪(Walz,德国)测量光系统(PS)II 的最大光化学效率 。简言之,将 一滴海水中的一组 10 只幼虫装入直径为 8 毫米的探针顶端。测量光强和增益均调至 7,以确保最佳的初始荧光。此外,从每组成虫中随机抽取 8 组共 20 只幼虫,保存在 ,用于共生体密度定量。幼体在 过滤和紫外线灭菌海水(FSW)中匀化,在 离心 ,然后将沉淀重新悬浮在 的 FSW 中。使用血细胞计数器对藻类共生体的数量进行计数,每个样本重复计数 6 次,共生体密度以每条幼虫的平均细胞数表示。
Acute ramping heat experiments were conducted on four groups of 35 larvae randomly sampled from each parental pool, during which net photosynthesis rate and light-enhanced dark respiration rate (LEDR) were measured to establish larval TPC. A customed respirometry system was established, consisting of four transparent glass vials with optical oxygen sensors, a water bath , a magnetic stirrer, and a 4-channel oxygen meter (FireStingO2, Pyroscience). The oxygen sensors were two-point calibrated using air-equilibrated seawater ( oxygen) and a zero solution. Each group of 35 larvae was placed in a beaker filled with FSW and sequentially exposed to six temperatures . Larvae were illuminated with T5 fluorescent bulbs (Giesemann) providing an irradiance of mol photons . After incubation at each temperature for and LEDR rates of the eight samples were measured within . Each group of 35 larvae was transferred to a vial containing temperatureequilibrated FSW, and seawater within the vials was well mixed at a stirring rate of . Larvae gently swirled and remained intact and alive throughout the measurement under this stirring rate. After the respirometry measurement at one temperature, larvae were transferred back to the beaker and exposed to the next temperature for before the next respirometry measurement. was measured as the rate of production over in the light, after which the light was turned off, and LEDR was immediately measured as the consumption rate over another . Seawater concentrations within the vials were recoded every 10 s. and LEDR rates were calculated using the least-squares linear regressions of concentrations plotted against time and expressed as nanomoles of per min per larvae. Gross photosynthesis ) was obtained by adding to LEDR.
从每个亲本池中随机抽取四组共 35 只幼虫进行急性升温实验,在此期间测量净光合速率 和光增强暗呼吸速率 (LEDR) 以确定幼虫 TPC。建立了一套定制的呼吸测量系统,包括四个 带有光学氧气传感器的透明玻璃瓶、一个水浴 、一个磁力搅拌器和一个四通道氧气测量仪(FireStingO2,Pyroscience 公司)。氧传感器使用空气平衡海水 ( oxygen) 和零 溶液进行两点校准。每组 35 只幼虫被放置在一个装有 FSW 的 烧杯中,并依次暴露在六种温度下 。幼虫用 T5 荧光灯(Giesemann)照明,辐照度为 摩尔光子 。在每个温度下孵化 后,在 内测量 8 个样本的 LEDR 率。每组 35 只幼虫被转移到装有温度平衡 FSW 的 小瓶中,小瓶中的海水以 的搅拌速率充分混合。在此搅拌速率下,幼虫轻轻旋转,并在整个测量过程中保持完整和存活。在一个温度下进行呼吸测量后,幼虫被移回烧杯,并在下一个温度下进行 ,然后再进行下一次呼吸测量。 ,测量值为光照下 的产生率超过 ,然后关闭光照,LEDR 立即测量,测量值为 的消耗率超过另一个 。小瓶中的海水 浓度每 10 秒重新编码一次。 和 LEDR 率的计算采用 浓度与时间的最小二乘法线性回归,并以每只幼虫每分钟的纳摩尔 表示。总光合作用 ) 是通过将 加入 LEDR 而得到的。
Control 控制
Parental acclimation (b)
父母适应性 (b)
FIGURE 1 (a) Experimental design showing the adult acclimation to control and heat treatments and larval reciprocal transplant experiment; the potential shift in thermal performance curves and interaction between parental and offspring temperatures due to prior thermal preconditioning; (d) rates of adult net photosynthesis ), light-enhanced dark respiration and dark respiration (LEDR and ), gross photosynthesis , and ratio of to ; (e) symbiont densities and maximum photochemical efficiency ; (f) planulation timing and the number of larvae released by control and heat-acclimated adults, with the grey rectangle showing the larvae collected on lunar day 5 for subsequent larval experiments; (g) larval volume, symbiont density and of freshly-released larvae. Data are mean and 3 for adult physiological traits and larval release, respectively; and 8 for larval volume, and symbiont densities, respectively). Asterisks indicate significant differences between two adult groups at -values determined by Student's -test or Mann-Whitney U test [Colour figure can be viewed at wileyonlinelibrary.com]
图 1 (a) 实验设计显示成虫适应对照和热处理以及幼虫相互移植实验; ,由于先前的热预处理,热性能曲线可能发生变化,亲代和子代温度之间也可能发生相互作用;(d) 成虫净光合作用速率 )、光增强暗呼吸和暗呼吸速率 (LEDR 和 )、总光合作用速率 ,以及 之比;(e) 共生体密度和最大光化学效率 ;(f) 对照组和热适温成虫的刨坑时间和释放的幼虫数量,灰色矩形表示在农历第 5 天收集的幼虫,用于随后的幼虫实验;(g) 刚释放幼虫的幼虫体积、共生体密度和 。成虫生理特征和幼虫释放的数据分别为 和 3 的平均值;幼虫体积、 和共生体密度的数据分别为 和 8 的平均值)。星号表示两个成虫组在 -值 上的显著差异,由学生的 -检验或曼-惠特尼 U 检验确定[彩图可在 wileyonlinelibrary.com 上查看]。
At the end of larval release, a branchlet was cut from each colony, glued onto a ceramic base, and allowed to recover overnight. were measured on dark-adapted branchlets using a Diving-PAM. Adult branchlets were placed in chambers with a stir bar to measure and LEDR as described above. Furthermore, dark respiration of branchlets was measured after 1-h dark adaptation. Adult were calculated by adding to LEDR, and the ratio of to was calculated as a proxy for autotrophy. A ratio of suggests the capacity for autotrophy, that is, the net productivity of algal symbionts could satisfy holobiont dark respiration. After respirometry, adult branchlets were stored at to quantify symbiont densities and skeletal surface area. Coral tissue was airbrushed into FSW, and the tissue slurry was homogenized and centrifuged at for . The resulting symbiont pellets were resuspended in FSW for symbiont density quantification as aforementioned. Adult metabolic rates and symbiont densities were normalized to the skeletal surface area, which was measured using a JTscan-MS
在幼虫释放结束时,从每个群落中切下一个 小枝,粘在一个陶瓷基座上,并让其过夜恢复。 ,使用 Diving-PAM 对暗适应小枝进行测量。将成年小枝放入带有搅拌棒的 室中,如上所述测量 和 LEDR。此外,小枝的黑暗呼吸 是在黑暗适应 1 小时后测量的。成体 的计算方法是将 与 LEDR 相加,并计算 的比率,作为自营养的代表。 之比表明具有自养能力,即藻类共生体的净生产力可以满足全生物体的黑暗呼吸。呼吸测定后,将成体小枝保存在 ,以量化共生体密度和骨骼表面积。将珊瑚组织气刷到 FSW 中,然后将组织浆液匀浆并在 下离心 。将得到的共生体颗粒重新悬浮在 FSW 中,如上所述进行共生体密度定量。成体代谢率和共生体密度根据骨骼表面积进行归一化处理,骨骼表面积使用 JTscan-MS 测量。

high precision 3D scanner (Jeatech) following the manufacturer's instructions.
高精度 3D 扫描仪(Jeatech)。

2.3 | Larval reciprocal transplant experiment
2.3 幼虫相互移植实验

To test the interaction between parental temperature ( ) and offspring temperature (To) on larval physiology, larvae from each parental pool were directly exposed to either ambient temperature , mean or high temperature , mean SD, Figure 1c and Figure S2). This typical reciprocal transplant design generated four treatments: (1) larvae from control and unacclimated adults raised at (U29), (2) larvae from control and unacclimated adults raised at (U32), (3) larvae from heat-acclimated adults raised at (A29), and (4) larvae from heat-acclimated adults raised at (A32). Coral larvae were incubated under these four treatments for five days, an ecologically relevant duration that could reliably detect the physiological effects of increased temperature on P. damicornis larvae (Cumbo et al., 2013).
为了检验亲本温度( )和子代温度(To)对幼虫生理的交互作用,将每个亲本池中的幼虫直接暴露于环境温度 ,平均值 或高温 ,平均值 SD,图 1c 和图 S2)。这种典型的对等移植设计产生了四种处理:(1)在 (U29)培养的对照组和非恒温成体的幼虫;(2)在 (U32)培养的对照组和非恒温成体的幼虫;(3)在 (A29)培养的热恒温成体的幼虫;(4)在 (A32)培养的热恒温成体的幼虫。珊瑚幼虫在这四种处理条件下孵化五天,这是一个与生态相关的持续时间,可以可靠地检测温度升高对 P. damicornis 幼虫的生理影响(Cumbo 等人,2013 年)。
Temperature treatments were created in eight 50-L tanks filled with fresh water. Four tanks were controlled at and the other four at using digital temperature controllers connected to titanium heaters (Weipro). Two 1-L beakers with of FSW were submerged into each tank, and 150 larvae from either adult group were transferred to each 1-L beaker for the sampling and analyses of physiology and transcriptomes (Figure S2). Furthermore, two glass vials, each containing 30 larvae from one of the two parental pools, were allocated to each tank to assess larval survivorship (Figure S2). The larval incubation system was illuminated with T5 fluorescent bulbs (Giesemann) on a 12:12 h light-dark cycle from 07:00 AM to 7:00 PM with a mean light intensity of photons . The light intensity was representative of that at the collection depth of adult colonies (Lei Jiang, personal observations), although the ecological relevance still depends on larval position in the water column. Seawater within beakers and glass vials was completely exchanged with temperature equilibrated FSW at 10:00 PM every day, and we used a mesh during seawater exchange to ensure that all larvae were retained and then transferred back to their corresponding container.
在八个装满淡水的 50 升水箱中进行温度处理。使用连接到钛加热器(Weipro)的数字温度控制器,将四个水箱的温度控制在 ,另外四个水箱的温度控制在 。将两个装有 FSW 的 1-L 烧杯浸入每个水槽中,并将来自任一成虫组的 150 只幼虫转移到每个 1-L 烧杯中,以进行生理和转录组的取样和分析(图 S2)。此外,两个 玻璃瓶分别装有来自两个亲本池之一的 30 只幼虫,被分配到每个池中以评估幼虫存活率(图 S2)。幼虫孵化系统采用 T5 荧光灯(Giesemann)照明,光暗周期为 12:12 小时,从早上 7:00 到晚上 7:00,平均光强为 photons 。光照强度代表了成体采集深度的光照强度(蒋磊,个人观察),但生态相关性仍取决于幼体在水体中的位置。烧杯和玻璃瓶中的海水每天晚上 10:00 与温度平衡的 FSW 完全交换,我们在海水交换过程中使用了 网眼,以确保保留所有幼虫,然后将其转移回相应的容器中。
At the end of the 5-day exposure, the number of larvae within glass vials were counted and those missing were considered dead. Larval mortality was calculated as the number of dead larvae divided by the initial number of 30 . At 10:00 AM on day 5 , one group of 10 larvae were sampled from each 1-L beaker. The effective photochemical efficiency was immediately measured on those light-adapted larvae using a Diving-PAM fluorometer, and then larvae were dark-adapted for to measure . Maximum excitation pressure over PSII and nonphotochemical quenching (NPQ) were calculated using the following equations: and (Iglesias-Prieto et al., 2004). Furthermore, one group of 35 larvae was sampled from each 1-L beaker, and rates of , LEDR, , and were measured and calculated as aforementioned (Figure S2). Following respirometry, these larvae were preserved for symbiont density quantification as described above. Larval bleaching rates were assessed as the relative change in symbiont density after five days exposure relative to the initial average.
在 5 天暴露结束时,对 玻璃瓶中的幼虫数量进行计数,未计数的幼虫视为死亡。幼虫死亡率的计算方法是:死亡幼虫数除以初始幼虫数 30。第 5 天上午 10:00 时,从每个 1 升烧杯中取样,每组 10 只幼虫。立即使用 Diving-PAM 荧光仪测量这些光适应幼虫的有效光化学效率 ,然后将幼虫进行暗适应 ,以测量 。PSII 和非光化学淬灭(NPQ)的最大激发压力是通过以下公式计算得出的: (Iglesias-Prieto 等人,2004 年)。此外,从每个 1-L 烧杯中抽取一组 35 只幼虫,按照上述方法测量和计算 、LEDR、 的速率(图 S2)。在进行呼吸测定后,这些幼虫被保存起来,以便按上述方法进行共生体密度定量。幼虫漂白率是以暴露五天后共生体密度相对于初始平均值的相对变化来评估的。

2.4 RNA-Seq and transcriptome analysis
2.4 RNA-Seq 和转录组分析

On completion of the incubation, one group of 50 swimming larvae were sampled from each 1-L beaker and snap-frozen in liquid nitrogen for transcriptome analysis (Figure S2). Total RNA was extracted using a Trizol reagent kit (Invitrogen), and RNA quantity and quality were assessed on an Agilent 2100 Bioanalyser and checked using RNase free agarose gel electrophoresis. One of the four U32 samples was discarded because of poor RNA quality. For each sample, of total RNA was processed (including mRNA enrichment via polyA+ selection) using the NEB Next Ultra RNA Library Prep Kit (NEB) to construct cDNA libraries. Sequencing of paired-end reads on a total of 15 samples was performed on Illumina Novaseq 6000 at Gene Denovo Biotechnology Co., Ltd.
培养结束后,从每个 1 升烧杯中抽取一组 50 只游动幼虫,在液氮中速冻,用于转录组分析(图 S2)。使用 Trizol 试剂盒(Invitrogen 公司)提取总 RNA,在 Agilent 2100 生物分析仪上评估 RNA 的数量和质量,并使用无 RNase 琼脂糖凝胶电泳进行检测。四个 U32 样本中有一个因 RNA 质量不佳而被放弃。使用 NEB Next Ultra RNA 文库制备试剂盒(NEB)处理 总 RNA(包括通过 polyA+ 选择富集 mRNA),构建 cDNA 文库。 成对末端读数的测序是在 Gene Denovo Biotechnology Co., Ltd. 的 Illumina Novaseq 6000 上进行的。
Raw data were trimmed using FASTP to remove low-quality reads, -content reads, and reads containing adapters and poly-A tails (Chen et al., 2018), and then subjected to rRNA filtering using the short reads alignment tool Bowtie2 (Langmead & Salzberg, 2012). The remaining high-quality clean reads were first mapped to P. damicornis genome using HISAT2.2.4 (Cunning et al., 2018; Kim et al., 2015). The mapping rate ranged from to , and these reads were assembled using StringTie version 1.3.1 in a reference-based approach to construct the coral transcriptomes (Pertea et al., 2015). The remaining unmapped reads were assembled de novo using the Trinity program (Grabherr et al., 2011), and then aligned against a constructed database of Symbiodiniaceae genomes and transcriptomes (Table S1) using BLASTN (E-value cutoff of . . Those transcripts significantly aligned to Symbiodiniaceae references were labelled as symbiont genes. Redundancy of symbiont transcripts was removed using CD-HIT-EST (Huang et al., 2010) with a threshold of at least sequence similarity. Details of raw reads quality control, mapping, and information on coral and symbiont transcriptomes are provided in Table S1. Transcripts were functionally annotated using BLAST with a cutoff E-value of based on the following databases: Swiss-Prot, NCBI nonredundant (Nr) protein, EuKaryotic orthologous groups (KOG), gene ontology (GO), and KEGG orthologue database (KO).
使用 FASTP 对原始数据进行修剪,以去除低质量读数、 -content 读数以及含有适配体和 poly-A 尾部的读数(Chen 等,2018 年),然后使用短读数比对工具 Bowtie2(Langmead & Salzberg,2012 年)进行 rRNA 过滤。剩余的高质量洁净读数首先使用 HISAT2.2.4 (Cunning 等,2018;Kim 等,2015)映射到 P. damicornis 基因组。映射率从 不等,这些读数使用 StringTie 1.3.1 版进行组装,采用基于参考的方法构建珊瑚转录组(Pertea 等人,2015 年)。剩余的未映射读数使用 Trinity 程序(Grabherr 等人,2011 年)从头组装,然后使用 BLASTN(E 值截断为 .与 Symbiodinceae 参考文献明显对齐的转录本被标记为共生体基因。共生转录本的冗余采用 CD-HIT-EST(Huang 等,2010 年)去除,阈值为至少 序列相似性。表 S1 提供了原始读数质量控制、绘图以及珊瑚和共生体转录组信息的详细信息。使用 BLAST 对转录本进行功能注释,E-value 临界值为 ,基于以下数据库:Swiss-Prot、NCBI 非冗余(Nr)蛋白质、EuKaryotic orthologous groups (KOG)、gene ontology (GO) 和 KEGG orthologue database (KO)。
Differentially expressed genes (DEGs) were identified using DESeq2 with thresholds of an adjusted -value of and an absolute (fold change) value of (Love et al., 2014). Four pairwise comparisons were designated in the DEG analyses: versus U32, U29 versus A29, A29 versus A32 and U32 versus A32. The short time-series expression miner (STEM) program was used to cluster DEGs into profiles based on the expression trend across treatments (Ernst & Bar-Joseph, 2006). GO functional enrichment analyses were
使用 DESeq2 鉴定差异表达基因(DEGs),阈值为 的调整 - 值和 的绝对 (折合变化)值(Love 等人,2014 年)。在 DEG 分析中指定了四个配对比较: 与 U32、U29 与 A29、A29 与 A32 和 U32 与 A32。使用短时序列表达挖掘程序(STEM)根据不同处理间的表达趋势将 DEGs 聚类为剖面图(Ernst & Bar-Joseph, 2006)。GO功能富集分析

performed on the STEM-defined profiles to identify the enriched GO terms (Yu et al., 2012).
对 STEM 定义的图谱进行分析,以确定富集的 GO 术语(Yu 等人,2012 年)。
To identify the symbiont genotypes within larvae, we conducted a local BLAST search of the symbiont transcriptomes against a database containing sequences of three molecular markers (PSII protein D1, mitochondrial cytochrome b, and elongation factor 2) from the eight genera of Symbiodiniaceae downloaded from GenBank.
为了确定幼虫体内的共生体基因型,我们对共生体转录组进行了局部 BLAST 搜索,并将其与从 GenBank 下载的、包含三个分子标记(PSII 蛋白 D1、线粒体细胞色素 b 和伸长因子 2)序列的数据库进行了比对。

2.5 | Data analysis
2.5 数据分析

Acute TPC of metabolic traits was fitted for each larval group using four functions: second-order polynomial, Gaussian, GaussianGompertz and Sharpe-Schoolfield. Using the Akaike information criterion (AIC) approach, the second-order polynomial was selected as the best-fit function to describe the relationship between temperature and larval metabolism based on the lowest AIC score. AIC is a mathematical method that estimates the relative amount of information lost by a given model, and therefore the model with the lowest AIC score is considered as the best-fit. Curve fitting and model selection were performed using the nls package in R. The optimal temperatures and maximal performance rates were determined from the parameterized functions. The critical thermal maximum was defined as the temperature at which larval metabolic rates were reduced to only of the maximal performance. of and