Reformation of tissue balls from tentacle explants of coral Goniopora lobata: selforganization process and response to environmental stresses Qiongxuan Lu, Tao Liu, Xianming Tang, Bo Dong & Huarong Guo
In Vitro Cellular & Developmental Biology - Animal ISSN 1071-2690 In Vitro Cell.Dev.Biol.-Animal DOI 10.1007/s11626-016-0095-0
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Author's personal copy In Vitro Cell.Dev.Biol.—Animal DOI 10.1007/s11626-016-0095-0
Reformation of tissue balls from tentacle explants of coral Goniopora lobata: self-organization process and response to environmental stresses Qiongxuan Lu 1 & Tao Liu 1 & Xianming Tang 2 & Bo Dong 1,3,4 & Huarong Guo 1,3
Received: 13 April 2016 / Accepted: 30 August 2016 / Editor: Tetsuji Okamoto # The Society for In Vitro Biology 2016
Abstract Coral has strong regeneration ability, which has been applied for coral production and biodiversity protection via tissue ball (TB) culture. However, the architecture, morphological processes, and effects of environmental factors on TB formation have not been well investigated. In this study, we first observed TB formation from the cutting tentacle of scleractinia coral Goniopora lobata and uncovered its inner organization and architecture by confocal microscopy. We then found that the cutting tentacle TB could self-organize and reform a solid TB (sTB) in the culture media. Using chemical drug treatment and dissection manipulation approaches, we demonstrated that the mechanical forces for bending and rounding of the cutting fragments came from the epithelial cells, and the cilia of epithelial cell played indispensable roles for the rounding process. Environmental stress experiments showed that high temperature, not CO2-induced Electronic supplementary material The online version of this article (doi:10.1007/s11626-016-0095-0) contains supplementary material, which is available to authorized users. * Bo Dong
[email protected] * Huarong Guo
[email protected] 1
Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China
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Hainan Academy of Ocean and Fisheries Sciences, Haikou 570203, China
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Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China
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Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China
acidification, affected TB and sTB formation. However, the combination of high temperature and acidification caused additional severe effects on sTB reformation. Our studies indicate that coral TB has strong regeneration ability and therefore could serve as a new model to further explore the molecular mechanism of TB formation and the effects of environmental stresses on coral survival and regeneration. Keywords Coral . Goniopora lobata . Tissue ball . High temperature . Ocean acidification
Introduction Coral reefs are one of the most well-known ecosystems and possess a great biological diversity and productivity in our planet (Moberg and Folke 1999). The reef-building corals often contain symbiotic zooxanthellae within their tissues which offer the corals nutrients by photosynthesis; in return, these algae benefit from their hosts’ metabolites (Tremblay et al. 2012). The symbioses are considered as the main reasons for the thriving of coral reefs (Stanley and Van De Schootbrugge, 2009). However, these ecosystems are under unprecedented challenges from the climate changes. Rapidly increasing rate of CO2 emissions is one of the distinguished characteristics (Hoegh-Guldberg et al. 2007), which results in the warming and acidification of seawater. Consequently, the symbiotic relationship between corals and zooxanthellae (Berkelmans et al. 2004; Pandolfi et al. 2011) and the calcification of corals are disrupted, and finally leading to the bleaching or death of corals. Recent studies have confirmed the association of reduced calcification rate in some reefbuilding corals with ocean acidification (OA) (Doney et al. 2009; Fujita et al. 2011). However, data on the combined impact of high temperature and OA on the growth and
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survival of corals is limited and the knowledge gap regarding the physiological response of corals needs to be filled (Castillo et al. 2014). Establishment of coral cell lines would be helpful for us to investigate the coral’s physiological response to environmental stresses at cellular levels. However, no coral cell lines are available up to now (Gates et al. 1992). Like other cnidarians (such as hydra), the in vitro cultured coral tissue fragments (i.e., explants) also have strong power of regeneration and could develop into new polyps and even whole individuals (Kramarsky-Winter and Loya 1996; Vizel et al. 2011). Kramarsky-Winter and Loya (1996) observed the formation of polyps and mouths from the in vitro cultured tissue fragments of coral Fungia granulose (Kramarsky-Winter and Loya 1996). Later, successful development of new individuals from coral explants was reported in several coral species including F. granulosa, Oculina patagonica, and Dipsastraea favus (Vizel et al. 2011). During these regeneration processes, an early larval-like stage, called tissue ball (TB), was observed prior to the formation of polyps or individuals. Interestingly, this TB-like structure could also be derived from the dissociated coral cell aggregates (CAs), which were prepared by chemical (Kopecky and Ostrander 1999; Domart-Coulon et al. 2004; Lecointe et al. 2013) or mechanical (Nesa and Hidaka 2008; Nesa and Hidaka 2009; Vizel et al. 2011; Feuillassier et al. 2014; Gardner et al. 2015) dissociation methods. Although these coral CAs have not been reported to develop into new polyps or individuals, these CAs share some common features with TBs such as continuously rotating, spherically structured, covered with ciliated epidermal cells and enclosed with endodermal cells and zooxanthellae (Kramarsky-Winter and Loya 1996; Kopecky and Ostrander 1999; Berkelmans et al. 2004; Nesa and Hidaka 2008; Nesa and Hidaka 2009; Vizel et al. 2011; Lecointe et al. 2013; Feuillassier et al. 2014; Gardner et al. 2015). These TBs or CAs are easy to prepare in the laboratory and thus provide us a rapid and useful tool to study the mechanism of biomineralization (Gardner et al. 2015; Huete-Stauffer et al. 2015) and symbiosis of cnidarian–dinoflagellate, in alternation to coral cell lines and individuals. The in vitro formation of TBs mainly depended on the corals’ innate potentials, though the environmental conditions are also important (Sabine et al. 2015). The explants of the solitary coral F. granulosa could not only be induced into new polyps but also maintained in the undeveloped stage by regulating the culture conditions of light and temperature (Vizel et al. 2011). This emphasized the important role of the proper environmental conditions in the coral regeneration processes. However, the mechanism underlying these biological processes is not well understood. To this end, investigation of the morphology and behavior of the TBs, which represent the unique characteristic of the coral explant regeneration during culture periods, and examination of the TBs’ response to
different environmental stresses are essential to fill the knowledge gap regarding coral physiology. Up to date, studies on the response of coral CAs and TBs to increasing thermal stress have been reported (Nesa and Hidaka 2008; Nesa and Hidaka 2009), but the combined impacts of high temperature and low pH stresses on the coral TBs are still unclear. In this study, we first described morphological process of scleractinian corals (Goniopora lobata) TB formation and its cellular characteristics using confocal microscopy. Then, we cut the tentacle TB and found that the cutting pieces of TB could self-organize and reform a solid TB (sTB) in our culture system. To explore the mechanisms of sTB formation, we further investigated the effects of intrinsic and environmental factors on sTB formation and found that the epithelial cells play dominant roles in sTB formation. Thermal stress and combination of thermal stress and low acidification influenced the sTB formation. Our work reveals the cellular characteristics and processes of coral sTB reformation. We therefore propose that the sTB reformation could be a model system to study the cellular and molecular mechanisms of TB formation and its response to the environmental stresses.
Material and Methods Corals The individuals of scleractinian corals G. lobata were collected from the coast of Hainan province, China and were kept in coral aquarium at ambient temperature 26°C and at pH 8.0 with a salinity of 34 ppt. Corals were maintained at 150 μmol photons m−2 s−1 lights, which provided by LED lights in a 12:12 h light/dark cycle. The animals were acclimatized in aquarium system at least 1 wk prior to experiments. Preparation of the tentacle TBs and solid TBs As the coral’s polyps were in full expansion, the polyp heads, which tipped with 24 stinging tentacles, were cut off using scissor and then were collected into sterile glass Petri dishes. After the polyp heads were rinsed by filtered sea water (FSW) 2–3 times, the tentacles were separated from the dissected polyp heads by fine scalpels and placed into new glass Petri dishes. The tentacles were then cut into small fragments using scissor and were incubated into new glass Petri dishes containing 50-ml FSW. The cut tentacle fragment culture was maintained under 26°C and 100 μmol photons m−2 s−1 lights, which was provided by the incubator-equipped LED lights in a 12:12 h light/dark cycle. About 12–24 h after being cultured, the tentacle-cutting TBs in diameter between 0.1 and 1 mm were formed. To identify the TB inner structure, an Olympus Fluoview 1000 confocal laser-scanning microscope (CLSM) equipped with a ×10 (NA 0.8) and ×60 water-immersion
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objective lens (NA 1.4) were used. Confocal images were processed by Photoshop. The solid TB (sTB) was prepared by cutting the tentacle TBs into defined ratio (1:8, 1:3, 2:3) of its original size using hooked glass needles under inverted microscope. They were then cultured with FSW media in an illumination incubator with the same culture condition as above. About 150 min later, sTB was observed to form in the media. In order to explore the role of endoderm on sTB reformation, the 1/3 partial of TB with or without endoderm, which was removed by hooked glass needles under inverted microscope, was cultured in individual glass Petri dishes containing 50-ml FSW for 150 min. The process of TB formation and sTB reformation was recorded by a Nikon Ts 100 inverted microscope equipped with a CCD camera. A circularity (C) was used to quantitative description of both processes. Circularity was calculated from the image data automatically by a plugin from ImageJ (NIH, http://rsbweb.nih.gov/ij/), which C was calculated as: S C ¼ 4π 2 L where S was the area of the TB, and the L was the perimeter of the TB. Temperature and acidification treatments To assess the effect of temperature on TB formation and sTB reformation, the process was exposed to varied culture temperature (26, 28, 30, and 32°C). For each experiment, 250–300 cut tentacle fragments or 5–10 1/3 partial of TB were incubated into individual glass Petri dishes containing 50-ml FSW, which would then be transferred to the incubators with temperatures at 26, 28, 30, and 32°C respectively. The FSW was used to acclimatize to experimental temperatures for 1 d before experiments. To estimate the effect of pH on the TBs formation and sTB reformation, the CO2-induced acidification was applied for the acidification experiments by setting up the incubator with three CO2 partial pressures, 380, 800, and 2800 ppm, which corresponding to pH 8.0, pH 7.7, and pH 7.3, respectively. For each experiment, 250–300 cut tentacle fragments or 5–10 1/3 partial of TB were incubated into individual glass Petri dishes containing 50-ml acidified FSW (pH 8.0, pH 7.7, and pH 7.3), which would then be transferred to the incubators with temperature at 26°C. The FSW used for acidification experiments was acclimatized to corresponding CO2 partial pressures for 4 d to reach the destined pH before experiments. The TB formation and sTB reformation were also exposed to 32°C/pH 7.3 and 26°C/pH 8.0 to investigate the effect of combination of high temperature and acidification on these processes. For each experiment, 250–300 cut
tentacle fragments or 5–10 1/3 partial of TB were incubated into individual glass Petri dishes containing 50-ml FSW (pH 8.0) or acidified FSW (pH 7.3), which would then be transferred to the incubators with temperatures at 26 or 32°C. The effect of temperature and acidification treatment on TB formation was evaluated by calculating the C of individual TB. Drug and Ca2+-free ASW treatment To identify the function of the ciliary motility on sTB reformation, the 0.1-mM sodium orthovanadate (Na3VO4, Aladdin, Shanghai, China) solution in FSW was used. The 1/3 partial of TBs was cultured in 50 ml FSW or 0.1 mM Na3VO4 solution for 150 min. To explore the role of Ca2+ on sTB reformation, the 1/3 partial of TBs were cultured in the Ca2+-free artificial seawater (CaFASW) or artificial seawater (ASW) for 150 min. The CaFASW was prepared by mixture of 23.0 g NaCl, 0.763 g KCl, 1.89 g MgSO 4 .7H 2 O, 3.0 g Na 2 SO 4 , 10.45 g MgCl2.6H2O, 0.25 g NaHCO3, and 0.026 g StCl2, in 1-L dH2O. ASW was prepared by adding 1.56 g CaCl2 into 1 L of Ca2+-free ASW as the control. The evaluation of the effect of both the 0.1 Mm Na3VO4 and Ca2+-free ASW treatment on sTB reformation was based on their morphological change. Morphometric analysis and statistics All of the experiments were replicated at least three times to ensure repeatability. The circularity, perimeter, and area of TB were determined autonomously with ImageJ. The circularity was expressed as the mean plus standard deviation (SD). Student’s two-sided unpaired t test with equal variance was used to assess the statistical significance through the SPSS software (version 18.0; SPSS Inc., Chicago, IL, USA).
Results In vitro formation of tissue balls from the cut coral tentacles and its architecture It was well known that when the coral is partially broken, the remaining coral has the ability to generate a new one. To investigate whether the small piece of broken coral could organize into new tissue, we cut down the tentacles from the head of polyps of scleractinia coral G. lobata (Fig. 1a–c), and the broken tentacle explants were further dissected into irregular pieces with the size of 0.1–0.2 cm diameters in Petri dish containing culture media (Fig. 1d). Then we found that each small cutting piece could form an intact spherical tissue ball (TB) (0.02–0.1 cm in diameter) in the culture media after 12 to 24 h (Fig. 1e). On the surface of spherical TB, high-density cilia were found (Fig. 1e’) and they swung either in
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Figure 1 . Formation of tentacle tissue balls (TBs) from scleractinian coral Goniopora lobata. (a-a’) The blooming of scleractinian coral G. lobata with full tentacle expansion (a). White dashed line cycle in (a) indicates one polyp head. Inset shows close view of one dissected polyp head (a’). (b–e) Formation of spherical tentacle TBs in vitro. b The schematic procedure for tentacle TBs formation. The tentacles (c) were
cut from the dissected polyp head and further cut into irregular pieces (d). The cut tentacle pieces were then cultured in the filtered seawater (FSW) media. Spherical TB (e) with high-density cilia (indicated by the white arrow in e’) in the Petri dish. Scale bars represent 1 cm in a, a’, and c and 500 μm in d, d’, e, and e’.
clockwise or anticlockwise direction (see ESM Video S1) and pushed the TBs rotating in culture media (see ESM Video S2). To explore the mechanisms of TB formation, we examined the inner structure of TBs utilizing confocal microscopy and found that coral tissue (green, auto-fluorescence) and zooxanthellae (magenta, auto-fluorescence) co-existed in the wall of formed TBs, and the center of TBs was cell-free cavity (Fig. 2a). Three distinct coral layers were presented in TBs (Fig. 2a’). In the middle section of confocal picture, it clearly
showed that ectoderm, tightly packed by high-density column epithelial cells, was localized at the TBs surface (Fig. 2a’). While endoderm was composed of endothelial cells, the endosymbiotic zooxanthellae was localized at the inner part of the TBs (Fig. 2a^). Between ectoderm and endoderm, a mesoglea layer was presented (white arrow in Fig. 2a’), in which extracellular matrix (ECM) was filled. The above observations suggest that the cutting broken coral tentacles could form TBs in culture media. The inner structure of TBs is similar to the living coral.
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Figure 2 . The architecture and organization of tentacle TBs. (a–a^) The middle section of confocal picture shows tentacle TB was composed of coral tissue (green, auto-fluorescence) and zooxanthellae (magenta, autofluorescence). The interested areas in a (white dashed line rectangles) are displayed in a’ and a^ at higher magnification. The tentacle TBs contain three layers: ectoderm (Ec), mesoglea (Me), and endoderm (En) (a’). The
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The effects of temperature and acidification on TB formation The effects of temperature and acidification on the TBs formation were studied. To evaluate the process of TBs formation at different temperatures and acidification conditions, we took the video and calculated the circularity of the cut tentacles at different time points (Fig. 3a) based on a formula from imageJ (Fig. 3b). The results showed that all the cutting pieces of tentacles formed round TBs after 24 h in the culture media at 26, 28, 30, and 32°C (Fig. 3c). The final shapes of TBs formed at 26, 28, and 30°C were similar. There was no significant difference in their circularity at 24 h (Fig. 3c). However, the circularity of TBs formed at 32°C was significantly lower than that of others (P < 0.001) and the surface of TBs formed at 32°C was more irregular than others (Fig. 3c). The effect of CO2-induced acidification on the formation of TBs was also studied. The explants in culture were exposed to pH 8.0 (normal culture condition), pH 7.7, and pH 7.3 filtered seawater (FSW), which had been acclimatized for 4 d in the CO2 incubator setup under CO2 pressure (pCO 2 ) of 380 (control), 800, and 2800 ppm,
Reformation of solid tissue balls from coral tentacle tissue balls As described above, the cutting coral tentacles were able to form a spherical TB under experimental conditions. The TBs showed a similar organization and architecture with living coral tissues. We therefore assume that the TBs have the ability to regenerate. To verify this idea, we further cut the formed TBs into different sizes, for example 1/3 part of TBs and observed that they indeed could reform a solid tissue ball (defined as sTB) after 150 min under the culture condition
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Figure 3 . The effects of temperature and acidification on TB formation. (a) Time sequential pictures from a timelapse movie showing tentacle TB formation. (b) The formula used from imageJ for circularity calculation. The numbers shown in a are corresponding to the circularity of the tentacle TB at 0, 12, and 24 h, respectively. (c) The shape of tentacle TBs formed at 26, 28, 30, and 32°C after 24 h. The circularities of TBs formed at experimental temperatures are shown in the right panel. The colorful bar represents the area of cutting tentacle pieces. The black dots indicate the outlier data point of TB circularity. (d) The final shape of tentacle TBs formed at three CO2-induced acidification pH 8.0, 7.7, 7.3 and the combination of high temperature 32°C and pH 7.3 respectively. Scale bars represent 500 μm in a, c, and d.
respectively. The data showed that there was no detectable difference on the TBs shape and circularity at different acidification (Fig. 3d). Since high temperature affects TBs formation, we further examined the TB formation at both lower acidification (pH 7.3) and high temperature (32°C) and found that no further effect was detected. The circularity after combination of lower acidification and high temperature condition was similar to that of pH 8.0 and 32°C. Together, CO2-induced acidification has no effects on the coral TBs formation, but high temperature does, under laboratory condition.
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(Fig. 4a, b). Utilizing the light microscopy, we observed the detailed processes of sTB formation: After being cut, the partial piece of TB stretched out and presented a curved shape. Then it started to bend inward from two ends, it experienced a rounding process, and finally enclosed into a spherical ball at 150 min (Fig. 4a, and also see ESM Video S3). The circularity of sTBs increased continuously at the first 70 min (Fig. 4b). At a later stage, it increased slowly. Like the tentacle TBs, the sTB was composed of three layers of coral and the endosymbiotic zooxanthellae. The difference between the tentacle TB and reformed sTB was that the center of sTB had no cavity, but was filled by endothelial cells. The outer surface of these reformed sTBs was assembled smoothly and covered by highdensity cilia (Fig. 4c). Effects of cutting size, endoderm, and cilia motility on sTB reformation The effects of intrinsic factors including cutting size, endoderm, and cilia motility on the sTB reformation were examined (Fig. 5). As shown in Fig. 5a, both the 2/3 cutting tentacle TB and 1/8 one could reform sTBs within 150 min. However, the sTB reformation from bigger cutting pieces (2/3 TB) experienced a bending process at the first
90 min (Fig. 5a) and then a rounding process by centripetal contraction (defined as type 1). While for the small cutting piece (1/8 TB), the bending process was not observed; it rather rapidly rounded up after splitting (defined as type 2). The results indicate that the cutting size of TBs does not affect their ability to reform a new sTB, but different sizes of cutting TB use distinct types of sTB reformation strategies. The cutting pieces are composed of epithelial cell tightly packed ectoderm and relatively loosely organized endoderm. To elucidate the mechanic force for cutting tentacle TB bending and rounding, we made epithelial cell by only cutting TB and removing endoderm and its symbiotic zooxanthellae and the mesoglea layer together. As shown in Fig. 5b, the ectoderm-only piece still reformed a sTB within 150 min, suggesting that endoderm and mesoglea layers are not required for sTB reformation and the mechanical force for the bending and rounding of cutting tentacle TB comes mainly from epithelial cells. Cilia are important organelles playing important role during organogenesis and regeneration. In epidermal cells of coral TBs, we found high-density cilia (Fig. 1d’, e’) on the surface, which swung continuously during TB and sTB
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Figure 4 . Reformation of a solid tissue ball (sTB) from a tentacle TB. (a) Time sequential pictures show the processes of sTB reformation. The 1/3 cutting partial TB stretches out at first and presents a curved shape and then it bends inward from two ends. After about 70 min, it experienced a rounding process and finally enclosed into a spherical ball at 150 min. (b)
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The circularities of sTB (data are shown by mean ± SD, n = 32) at different time point. (c) The carton depicted the reformation of sTB. The cutting tentacle TBs are cavity structures, while the sTB showed a solid one. Scale bars represent 100 μm in a.
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Figure 5 . Effects of cutting size, endoderm, and cilia motility on sTB reformation. (a) sTB reformation from different cutting tentacle TB sizes. 2/3 cutting tentacle TB reform into sTB via bending and rounding processes indicated by type 1 and 1/8 one experiences rounding process indicated by type 2, respectively. (b) sTB reformation from endoderm free cutting tentacle TB. (c) sTB reformation under the treatment of 0.1 mM Na3VO4. Scale bars represent 100 μm in a– c.
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formation. Since epithelial cell layer itself can form sTB, we wondered what the roles of cilia in the process of sTB reformation were. To verify their function, cilia motility was arrested by sodium orthovanadate (Na 3VO 4) treatment (Shapiro et al. 2014). The results showed that the bending process was not disturbed, but the rounding process was interrupted, resulting in a rough surface in the newly reformed sTB compared with the control (Fig. 5c). This result suggests cilia motility is essential for sTB formation. It further indicates that bending and rounding are two mechanically distinct processes for sTB reformation, and cilia motility is involved in rounding process. Effect of Ca2+ on the sTB reformation The above experiments showed that small piece of cutting TBs did not experience bending process as the bigger one during sTB reformation, indicating the bending mechanism comes from collective large group of epithelial cells. Calcium, which is required for maintenance of cell–cell connection and communications via E-cadherin molecule, plays indispensable roles in organizing
collective cell behaviors. To verify and examine the effect of Ca2+ on the sTBs’ reformation, the cutting tentacle TBs were incubated in Ca2+-free artificial seawater (ASW). After 150 min, the cutting pieces of tentacle TBs in Ca2+-free ASW culture media did not reform into sTBs (Fig. 6a). Neither bending nor rounding processes were observed. The epidermal cells tended to dissociate from the cutting pieces of TBs after 2 h. This result confirms that Ca2+ is indispensable for the sTBs’ reformation. Effects of temperature and CO2-induced acidification on the sTB reformation To investigate the effects of temperature on the sTB reformation, the cutting tentacle TBs were exposed to 26, 28, 30, and 32°C for 24 h in the normal FSW (pH 8.0) respectively. The reformation processes were monitored, and the changes of circularity were calculated. As shown in Fig. 7a, b, both the bending and rounding processes of cutting tentacle TBs were disrupted under the higher temperatures (30 and 32°C). The circularities of the reformed sTBs obviously decreased (Fig. 7b), and the surface of the reformed sTBs
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Figure 6 . Ca2+ is indispensable for sTB reformation. (a) 1/3 sTB reformation of the cutting pieces of tentacle TBs in artificial seawater (ASW) Ca2+-free culture media. (b) 1/8 sTB reformation of the cutting pieces of tentacle TBs in artificial seawater (ASW) Ca2+free culture media. Scale bars represent 100 μm in a and b.
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became much rougher than that of lower temperature groups (26 and 28°C), and more dissociated cells were found from the reformed sTBs (Fig. 7a). The effects of CO2-induced acidification on sTBs reformation were also investigated. We set up three pH gradients including pH 8.0 (normal seawater), pH 7.7, and pH 7.3 for the experiments. The cutting tentacle TBs were incubated at different pH values for 4 d to accumulate before the experiments. The results showed that CO2-induced acidification did not disrupt sTB reformation (Fig. 7c). The circularities at three pH values did not show significant difference, although the surface of the formed sTB at lower pH looked smoother than that at higher pH (Fig. 7c). Global warming and ocean acidification often occurred simultaneously under the climatic changes. Thus, the effect of combined exposure of high temperature (32°C) and acidification (pH 7.3) on the sTB reformation was also investigated. The results presented that the reformed sTBs were significantly disrupted under the combined condition of acidification at pH 7.3 and high temperature at 32°C (Fig. 7e, f). The result indicates that combination of acidification and high temperature causes additional influence on the coral sTB reformation than the single environmental factor.
Discussion In this study, we record that the explants of the scleractinian coral G. lobata was able to form intact TBs in vitro in culture media. Tissue ball-like structures, named coral cell aggregates (CAs), were first reported from ten colonial cnidarians in cell culture media (Frank et al. 1994). Later, these round-shaped structures from the primary culture of branching scleractinian
coral Acropora micropthalma and Pocillopora damicornis were named multicellular endothelial isolates (MEIs), and its structure was described (Kopecky and Ostrander 1999). Recently, in the culture of coral explants, the round-shaped structures were called tissue ball (TB) (Nesa and Hidaka 2009; Lecointe et al. 2013). Based on the literature, the CAs, MEIs, and TBs share similar external and internal structures, indicating that they are the common characteristics of cnidarian cell and explant in culture. However, the detailed inner structure of TB and dynamics of TB formation has not been well characterized. Utilizing auto-fluorescence of coral cells and confocal microscopy, we, for the first time, clearly showed that the scleractinia coral TB contains three distinct tissue layers: ectoderm, mesoglea, and endoderm. The column epithelial cells are tightly packed in ectoderm. At the outward surface of epithelial cells, high-density cilia are presented. Beneath the ectoderm is the endoderm, where zooxanthellaes are symbiotic inside of endothelial cells. The TB structure that we revealed is identical with that in living coral tissue (Kopecky and Ostrander 1999; Lecointe et al. 2013; Gardner et al. 2015). Intriguingly, we further found that in vitro cultured TB had strong regeneration ability, which was similar to living coral tissue. When the TBs were cut into small pieces, they could reform into a new solid TB autonomously (Fig. 8). Thus, a new model is established to study cellular and molecular mechanisms of coral TB formation and ecotoxicology. Using the model, we found that sTB’s formation strategies for different cutting sizes are distinct. For the larger cutting tentacle TB fragment, it reforms into sTB via bending and subsequently rounding processes, while for smaller ones, it forms sTB experiencing only rounding process. The biological mechanical force is mainly generated by actomyosin network, of which the activity is energy (ATP)
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Figure 7 . Effects of temperature and CO2-induced acidification on the sTB reformation. (a–b) The effects of temperature (26, 28, 30, and 32°C) on sTB reformation from the 1/3 cutting tentacle TBs. The circularity of the reformed sTBs at different temperatures and different time points are showed in b. (c–d) The effect of CO2-induced acidification on sTB reformation. The pH value of seawater is set up at pH 8.0, pH 7.7, and pH 7.3 respectively. The circularity at different temperatures and different
time points are shown in d. The circularities at different pH conditions at 150 min show no significant difference (p > 0.05). (e–f) The effect of combined exposure of high temperature (32°C) and acidification (pH 7.3) on sTB reformation. The circularities of sTBs are shown in f. The circularities at 150 min at pH 7.3 and 32°C are significantly lower than that at pH 8.0 and 26°C (p < 0.001). Scale bars represent 100 μm in a, c, and e.
dependent (Heisenberg and Bellaïche 2013). The bending process is energy dependent, and epithelial cells at the outer layer of cutting fragment provide the contractile force. The endosymbiotic zooxanthellae are believed to be the nutrient suppliers for their host endothelial cells, which could absorb photosynthetic products produced by zooxanthellae (Tremblay et al. 2012). The symbiotic zooxanthellae were the main energy source for the sTBs’ reformation in the oligotrophic FSW or ASW. However, our results showed that as zooxanthellae
contained endothelial layer were removed, the remaining ectodermal layer still maintained the ability to bend and finally reform a sTB (Fig. 8). This result suggests that the epidermal layer plays more important roles than the endodermal layer for bending processes. Epidermal cilia might exert its effect on these mechanical processes, which were thought to be the reason for their continuous rotations by ciliary flow. This vertical flow around the surface of coral TBs in culture would also result in active mass transport (Shapiro et al. 2014) and
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Figure 8 . The model of coral sTB reformation. The irregular cutting coral tentacles are able to form spherical TBs under the amiable temperature. However, under the higher temperature condition, the shape and the surface of the formed TBs are affected. The cutting partial TB could reform into sTB. Neither cutting size nor endodermal cells affect reforming process. However, inhibition of cilia motility causes moderate disruption. Ca2+ is absolutely required for the sTB reformation. High temperature (30 and 32°C) affected the sTB reformation moderately, while combination of thermal stress (32°C) and acidification (pH 7.3) caused severe disruption.
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then may prolong their maintenance in culture (Nesa and Hidaka 2009; Lecointe et al. 2013). Our results showed that the rounding process was disturbed when the cilia were arrested by chemical inhibitor. Based on these experimental evidences, we conclude that the mechanical force for bending comes from large group of collective epithelial cells and the motility of cilia on the surface of epithelial cell, which play key roles in the rounding process. TBs explant had been used as an experimental model system to study the coral physiology, symbiosis, and development (Kopecky and Ostrander 1999; Domart-Coulon et al. 2004; Nesa and Hidaka 2008; Nesa and Hidaka 2009; Vizel et al. 2011; Feuillassier et al. 2014; Gardner et al. 2015; HueteStauffer et al. 2015). Especially, those laboratory cultured TBs possess a potential application to investigate the effects of environmental stresses on coral survival and regeneration (Gardner et al. 2015). It has been reported that the thermal stress causes dysfunction of the coral cell adhesion, which further results in the cell detachment from the epidermal layer (Gates et al. 1992). TBs derived from Pavona divaricate exhibited that the coral cells may suffer the increased DNA damage under thermal stresses (Reynaud et al. 2003). The survival time of TBs at 31°C is shorter than at 26°C, and the zooxanthellae appear to secrete toxic substance under high temperature stress (Nesa and Hidaka 2009). However, it has not been reported that the TBs were in response to acidified culture media yet. Due to the unique morphology and the reformation ability of sTB, we suggest that the processes of sTB reformation are applicable to mimic the response of coral to environmental stresses. In this study, we showed the circularity of sTBs under the thermal stresses was significantly lower than that under the
room temperature, indicating that the sTB’s reformation was affected. However, the CO2-induced acidification showed no detectable influences on sTB formation (Fig. 8). Comparing the results between the temperature and acidification experiments, it seems to suggest that the thermal stress produces a more immediate threat than the acidification environment. This is consistent with the result from some of the field studies, which showed that the ocean acidification posed a less immediate threat than the ocean warming for the coral Siderastrea siderea (Castillo et al. 2014). However, some investigation indicated that the CO2-derived seawater acidification not only resulted in decreased calcification of coral Stylophora pistillata (Tambutté et al. 2015), but also caused morphological change of the coral skeleton to a more porous and potentially fragile phenotype. These results suggest that different coral species might react differently to OA (Kroeker et al. 2013; Castillo et al. 2014). The threats for coral reef survival mainly come from the combination of ocean warming (OW) and OA. Using replicated microcosms that simulate future nature conditions, it has been found that the temperatures and seawater pCO2 concentrations reach highest during summer, which were very likely to have serious impacts on coral reef ecosystems (Dove et al. 2013). However, in some scleractinian corals, the calcification rate could be declined (Reynaud et al. 2003), no effects under the interactive stresses of ocean acidification and elevated temperature (Muehllehner and Edmunds 2008). This suggests that corals may exhibit great variability in response to climate change. We examined the impact of the combination of high temperature and lower acidification on sTBs reformation in the culture system. Our results showed that the combination
Author's personal copy REFORMATION OF TISSUE BALLS FROM TENTACLE EXPLANTS OF CORAL
stress (32°C + pH 7.3) causes severe disruption of sTB formation than the single stress (Fig. 8), indicating that combination of high temperature and acidification produces additional stress on coral growth and survival. However, the underlying mechanisms need further exploration, although the complexity of OW and OA combination on the coral reefs is still far way to conclude that our results suggest sTB reformation could be potentially useful to investigate these fundamental biological questions and the underlying mechanisms. Acknowledgments We thank Xin Liang from Tsinghua University (Beijing, China) for generously providing technical guidance on circularity measure and calculation; and Mavis Adusei-Fosu for critical reading of the manuscript. This work is supported by National Natural Science Foundation of China (Grant No. 31472274 and 31172391), Regional Demonstration of Marine Economy Innovative Development Project (No.12PYY001SF08), National High-tech R&D Program of China (863 Program; Grant No. 2012AA10A402), and open funds of Institute of biodiversity and evolution, Ocean University of China (Grant No. 201362017). B. Dong is supported by Taishan Scholar Program of Shandong Province. This work is supported by National Natural Science Foundation of China (Grant No. 31472274 and 31172391), Regional Demonstration of Marine Economy Innovative Development Project (No.12PYY001SF08), National High-tech R&D Program of China (863 Program; Grant No. 2012AA10A402), and open funds of Institute of biodiversity and evolution, Ocean University of China (Grant No. 201362017). B. Dong is supported by Taishan Scholar Program of Shandong Province. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Author contributions Q.L. performed all the experiments except confocal images capture, which were done by B.D. Q.L. and B.D. prepared all the figures. T.L. and X.T. provide regents and materials. H.G., B.D., and Q.L. analyzed the data, wrote the main manuscript text, and designed experiments. All authors reviewed the manuscript. Compliance with ethical standards Conflict of interest All authors declared no conflict of interest.
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