Cultivation and conservation of the soft coral Sarcophyton trocheliophorum: Light intensity and photoperiod regulation

doi:10.21203/rs.3.rs-1986464/v1

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Cultivation and conservation of the soft coral Sarcophyton trocheliophorum: Light intensity and photoperiod regulation

https://doi.org/10.21203/rs.3.rs-1986464/v1

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Coral reefs are one of the most diverse, productive ecosystems in the world, and light plays crucial role in its survival. Notably, the effects of light conditions on soft coral and its adaptive mechanism were unclear. Thus, the present study aimed to investigate and evaluate the effects of different light intensities (30, 80 and 130μmol m⁻² s⁻¹) and photoperiods (18D:6L, 12D:12L and 6D:18L) on cultivation of soft coral Sarcophyton trocheliophorum. During two 50-day of the experiments, we monitored the zooxanthellae density, Chl a content, enzyme activities (SOD, CAT and GST) and microbial diversity of S. trocheliophorum. Our study's outcomes found that, at the end of the experiment, the 80μmol m⁻² s⁻¹ light intensity group and 12D:12L photoperiod group both possessed the highest zooxanthellae density (2.54×10⁸ ± 0.14×10⁸cells g⁻¹ DW and 2.40×10⁸ ± 0.07×10⁸cells g⁻¹ DW, respectively), Chl a content (295.01 ± 14.13µg g⁻¹ DW and 287.78 ± 16.13µg g⁻¹ DW, respectively) and microbial diversity and relatively stable enzyme activities level. Besides, we speculated that the reason for the decline of zooxanthellae density Chl a content and microbial diversity under other light conditions might be that it induced light stress and caused oxidative damage. The main bacterial composition of S. trocheliophorum in different light conditions was similar at the phylum level, showing the stability of microbial community structure. Proteobacteria, Actinobacteria and Firmicutes were dominant under all light conditions, so we hypothesized that these bacteria phylum play a crucial role in coral growth and survival.

In conclusion, compared with the other treatments, 80μmol m⁻² s⁻¹ light intensity and 12D:12L photoperiod were more beneficial to the growth performance of S. trocheliophorum and could be recommended for its cultivation condition. Our study could provide helpful information for sustainable management plans for the cultivation and conservation of soft corals, which was especially important to the protection and restoration of degraded coral reefs.

Sarcophyton trocheliophorum

Light intensity

Light photoperiod

Zooxanthellae density

Enzyme activity

Microbial diversity

Coral reefs are one of the most bio-productive and diversified ecosystems globally, which provide habitat for many marine organisms and play an essential role in the whole marine ecosystem (Soares et al. 2020). However, because of ocean warming, ocean acidification, rising water temperatures and mass bleaching events (Hughes et al. 2018; Brunner et al. 2021), and other anthropogenic stressors like overfishing, tourism, and environmental pollution (Cinzia et al. 2018; Patterson et al. 2020), many coral reef ecosystems around the world are facing degradation or even loss (Oliver et al. 2018; Zhang et al. 2021). In a narrow sense, coral is usually defined as the “coral polyp”, which is a class of lower marine invertebrates belonging to Coelentera, Anthozoa, and Corallidae (Huang et al. 2019). Zooxanthellae is particularly important to corals and the symbiotic relationship between corals and zooxanthellae is of great ecological significance for the growth and survival of coral (Jaspers et al. 2019). Soft corals are dominant space-occupiers, critical structural components of coral reef communities, and contributors to coral reef biomass, which provides habitat and food for many marine organisms (France 2010; Satoe et al. 2012). In recent years, more and more attention has been paid to soft coral, not only because it is facing a significant threat with the degradation of the coral reefs ecosystem, but also because it is the hot biotechnological research subject of marine natural products and the trading subject of the marine aquarium trade (Blunt et al. 2004). Therefore, the optimization of soft coral propagation and production techniques deserve more research efforts targeting economically profitable soft corals, contributing to the conservation and restoration of the coral reef ecosystem.

Light as the most important environmental factor is divided into three essential elements: light spectrum, light intensity, and photoperiod. Light affects marine organisms' growth, energy conversion, feeding, and reproductive behavior, and the degree of influence varies with species (Fox et al. 2008). In a symbiotic system, light provides energy for inducing various physiological reactions of symbiosis (Liu et al. 2021). Symbiotic algae transfer a large amount of fixed carbon to the host through photosynthesis and provide energy for basic life activities of corals (Cheng et al. 2022). Apparently, different light conditions would have a significant effect on both survival, metabolism of symbiotic algae and the host coral.

As for symbiotic algae, Chlorophyll is necessary for photosynthesis and helps protect host corals and zooxanthellae from UV radiation. Different light conditions will influence Chlorophyll in zooxanthellae. Too strong light intensity makes zooxanthellae produce excess oxygen, and a high concentration of oxygen is harmful to corals. In this case, some zooxanthellae will separate from the host coral, or the zooxanthellae cells will discharge chlorophyll, which may eventually lead to the death of corals. Conversely, the too low light intensity will increase the Chlorophyll content in the zooxanthellae, which may lead to the change of coral color. And Rocha et al (2013a) recorded that low light intensity was more conducive to the growth of Sinularia flexibilis and increased density of its zooxanthellae. Moreover, the time of illumination is significant for the process and efficiency of photosynthesis in zooxanthellae, and photoperiod played an essential role in monitoring photosynthetic characteristics and growth performance of host-zooxanthellae symbiosis.

Light intensity and photoperiod will affect the photosynthesis of zooxanthellae and ultimately the growth and survival of corals. However, inappropriate light intensity and photoperiod can also disrupt the physiology of coral symbiosis by affecting the cell cycle, metabolism, and other physiological mechanisms (Rosenberg et al. 2017; Levy et al. 2020). Therefore, appropriate light intensity and photoperiod in coral cultivation are of vital importance.

Corals cannot grow and survive without zooxanthellae. In addition to zooxanthellae, light conditions also have a significant impact on the coral itself. Sakai et al (2020) reported that corals polyps' sensitivity to light intensity. Besides, changes in light conditions could cause a variety of biological stress responses in corals, accompanied by excessive production of reactive oxygen species (ROS) (Chen et al. 2019). Generally, in marine organisms the antioxidant system, SOD, CAT and GST have the function of scavenging reactive oxygen species and play an essential role in enhancing the defense ability of phagocytes and immune function (Verlecar et al. 2008). Thereby, different light conditions will induce the change of antioxidant enzyme activity, and the alterations of antioxidant enzyme activity were also used to detect the effect of light stress. For example, Levy et al (2006) ever recorded the activity of two antioxidant enzymes (SOD and CAT) in the coral Favia favus under different light periodicities.

Additionally, as the critical roles in nutrient cycling and metabolism, the microorganisms are a crucial part of coral, and the microbial communities are also sensitive to the change of light condition. Different light conditions could also cause the evolution of the microbial community diversity level and the microbial community structure, which are crucial to the health of host coral. Thus, the microbial community could be seen as an indicator of the health status of corals. Taken together, the light conditions are essential to coral cultivation, and the indicators of the symbiotic zooxanthellae, enzyme activity and the microbial community should be considered for the safe and productive cultivation of soft corals.

Sarcophyton Trocheliophorum, as an important component of the coral reef ecosystem, is widely distributed in the South China Sea. The related physiological mechanisms as S. trocheliophorum responses to different light conditions have remained largely unexplored. To provide data support and scientific basis for the reasonable artificial culture and conservation of soft coral in the South China Sea, different light intensities and photoperiods were first set to study S. trocheliophorum, including an index of symbiotic algae, related enzyme activity, microbial diversity in the present study. Essentially, the study aimed to explore the appropriate light intensity and photoperiod for S. trocheliophorum. Our study will provide baseline data concerning the optimal artificial light conditions for the cultivation and conservation of soft corals, which could benefit the protection and restoration of the whole coral reef ecosystem.

2.1. Experimental coral and culture system

Two colonies of S. trocheliophorum were collected from Sanya in the South China Sea. The collection strictly complied with the regulations on coral reef protection of Hainan Province. Then the colonies were transported to the Hainan Tropical Ocean University and stocked for 2 months in a recirculating system filled with natural seawater for acclimation. The mother colonies were stocked in a glass tank (432L water volume, 120cm×60cm×80cm), and the glass tank was connected to a 192L filter tank. The filter tank was equipped with a protein skimmer (APF-600 Deltec, China), a biological filter (composed of 10kg of live rock, cotton, and plastic filter), and a submerged pump (HQB-2500, SUNSUN, China) that supplied a flow of approximately 2000L h^− 1 to the mother colonies tank. The mother colonies' tank was equipped with a wavemaker (JVP-202A, SUNSUN, China) and illuminated with LED light (3W-3535, CREE, USA), producing a light intensity of 80µmol m^− 2 s^− 1 at the surface of the colonies with a 12D:12L photoperiod (light period: 7:00–19:00h) (Rocha et al. 2013b). The light intensity value was measured with a Digital Luxmeter (ZDS-10W-2D, ZIMO, China) with a submersible sensor.

During the whole experiment, the water quality parameters were maintained as follows: salinity 34, temperature 26℃, pH 8.2 ± 0.2, TN 0.05 ± 0.01mg L^− 1, NO₂⁻-N 0.03 ± 0.02mg L^− 1, NO₃⁻-N 1.0 ± 0.2mg L^− 1, PO₄³⁻-P 0.01 ± 0.01mg L^− 1.

2.2. Effect of light intensities and photoperiods on S. trocheliophorum

After two months of acclimation, the mother colonies were both fragmented into 50 similar-sized fragments (3cm×3cm per colony) by scalpel and scissors. The fragments were stocked for 20 days in the mother colonies tank to recover from fragmenting stress. Then the fragments were distributed among different experimental treatments for cultivation.

In different light intensity treatments, 45 coral fragments were selected randomly and divided averagely into three groups (L, M and H). The distances between each group and the LED lamp were about 100cm, 60cm and 30cm, respectively. The light intensities (± 5%) reaching the surface of fragments in different groups were 30µmol m^− 2 s^− 1 (L), 80µmol m^− 2 s^− 1 (M) and 130µmol m^− 2 s^− 1 (H), respectively. The photoperiod was 12D:12L.

In different photoperiod treatments, 45 coral fragments were selected and distributed equally to three groups (X, Y and Z). The three groups’ specific photoperiod were 18D:6L (10:00–16:00h) (X), 12D:12L (7:00–19:00h) (Y), and 6D:18L (4:00–22:00h) (Z), respectively, and the light intensity was 80µmol m^− 2 s^− 1.

The light intensity and photoperiod experiment were both carried for 50 days. Samples were taken at day 1 (D₁), day 5 (D₂), day 15 (D₃), day 30 (D₄) and day 50 (D₅) days of the light intensity and photoperiod experiment, respectively. Then the coral samples were frozen in liquid nitrogen immediately and stored at -80℃ for further analysis.

2.3. Sample analysis

2.3.1. Density determination of zooxanthellae

Coral samples were ground and homogenized with about 10ml phosphate buffer (PBS, PH = 7.4). The homogenate (9ml) was mixed with propionaldehyde (1ml). After standing for 3min, centrifuged at 4500g for 15min, the amount of zooxanthellae was counted with a hemacytometer. After taking out the supernatant, the remaining solid was freeze-dried for 24h and weight (g) was measured as the dry weight of the coral sample. The zooxanthellae density was defined as the number of zooxanthellae per unit weight of coral (cells g^− 1) (Rocha et al. 2013b).

2.3.2. Determination of chlorophyll a content

The coral sample was ground and homogenized with 10ml acetone (90% purity) and the extraction was performed at 4℃ for 24h in the dark. Then centrifuged at 4500g for 15min, supernatant was collected. The absorbance was measured with a spectrophotometer (UV-2800A, Unico, Shanghai) at 630nm, 647nm, 663nm and 750nm. The remaining solid was freeze-dried for 24h and weight (g).

Chlorophyll a (Chl a) was calculated by following formula of Jeffrey and Humphrey (1975):

$$\text{C}\text{h}\text{l} \text{a}= \left({\text{C}}_{1}\times \left({\text{A}}_{663}-{\text{A}}_{750}\right)+{\text{C}}_{2}\times \left({\text{A}}_{647}-{\text{A}}_{750}\right)+{\text{C}}_{3}\times \left({\text{A}}_{630}-{\text{A}}_{750}\right)\right)/\text{W}$$

In (1), C₁ = 11.85, C₂=-1.54, C₃=-0.08. Chl a is the content of chlorophyll a in the coral sample (µg g^− 1). A₇₅₀, A₆₆₃, A₆₄₇ and A₆₃₀ are absorbance values at different wavelengths. W is the dry weight of the coral sample (Rocha et al. 2013a).

2.3.3. Determination of enzyme activity

The enzyme activity was measured after accurately measuring weights of coral samples and the addition of NaCl at the ratio of (1:9 W/V). Then, the coral samples were placed into the grinder and ground into 10% homogenate quickly under ice bath condition. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) in the dilutions were determined by the kits (A001, A007 and A004, Nanjing Jiancheng, China). The BCA protein concentration kit determined the total protein content in coral samples (QS3202, solebao, China). The unit of enzyme activity was U/mg prot.

2.3.4. Analysis of microbial diversity

2.3.4.1. Sample collection and DNA extraction

Coral samples from different light conditions were collected during the experiment when the tentacles of S. trocheliophorum were fully extended. In the light intensity trial, L₁, L₂ and L₃ were taken from group L; M₁, M₂ and M₃ were taken from group M; H₁, H₂ and H₃ were taken from group H. In the photoperiod trial, X₁, X₂ and X₃ were taken from group X; Y₁, Y₂ and Y₃ were taken from group Y; Z₁, Z₂ and Z₃ were taken from group Z. DNA from each coral sample was extracted with the Column Marine Animals DNA Kit (Guangzhou, Dongsheng, China) according to the manufacturer’s instructions. DNA was eluted in the Tris-EDTA buffer solution and stored at -80℃ until use.

2.3.4.2. PCR amplification and high throughput sequencing

The bacterial variable regions 3 and 4 of the 16S rRNA gene were amplified using the primer pair: 338F (5'-gtactcctacgggaggcagca-3') and 806R (5'- gtggachtachvgggtwtctaat-3').

PCR products were run with 10µM primer 1µL, 2×Taq master Mix (Sangon Biotech Co., Ltd, Shanghai, China) 15µl, 20ng DNA and DNase free water to make a total volume of 30µL: denaturation at 94°C for 5min, 5 cycles of denaturation at 94°C for the 30s, annealing at 50°C for 30s and extension at 72°C for 60s, and a final extension at 72°C for 7min. Pooled samples were cleaned with Agencourt AMPure XP magnetic bead system (Beckman Coulter, Brea, CA, USA). The samples were then quantified by Qubit3.0 DNA (Life Technologies, CA, USA) and pooled in equimolar ratios. Finally, the high-throughput was sequenced using Illumina MiSeq (Sangon Biotech Co., Ltd. Shanghai, China).

2.4. Data analysis

The symbiotic zooxanthellae content, enzyme activity, and microbial community diversity data were statistically analyzed by Microsoft Excel software (Fylstra et al. 1998). One-way ANOVAs were used to evaluate the significant differences SPSS 22.0 (SPSS Inc, Chicago, IL, USA) software. α = 0.05 was taken as the significant level of difference. Descriptive statistics were expressed as mean ± SD.

3.1. Effects of different light intensity treatments on S. trocheliophorum

3.1.1. Zooxanthellae density

At the beginning of the experiment (D₁), there was no significant difference among the three light intensity groups in the zooxanthellae density of S. trocheliophorum (Fig. 1). However, zooxanthellae density decreased significantly in groups L and H during the experiment. No significant differences in zooxanthellae density were found in group M during the experiment. As a result, at the end of the experiment (D₅), the zooxanthellae density of group M ( 2.54×10⁸ ± 0.14×10⁸cells g^− 1 DW) was significantly higher than group L ( 2.12×10⁸ ± 0.12×10⁸cells g^− 1 DW) and H ( 2.04×10⁸ ± 0.09×10⁸cells g^− 1 DW) (P < 0.05) (Fig. 1).

3.1.2. Chl a content

At D₁, no significant differences in the content of Chl a were found in groups L, M and H (P > 0.05) (Fig. 2). Chl a content in group L and H decreased after D₁, but Chl a content in group M didn’t fluctuate significantly during the whole experiment (P > 0.05). At the end of the experiment (D₅), the Chl a content of group M (295.01 ± 14.13µg g^− 1 DW) was significantly higher than the group L (209.06 ± 8.09µg g^− 1 DW) and H (181.07 ± 15.23µg g^− 1 DW) (P < 0.05) (Fig. 2).

3.1.3. Enzyme activity

All enzyme activities in different light intensity (L, M and H), including SOD (Fig. 3a), CAT (Fig. 3b) and GST (Fig. 3c), showed no significant differences (P > 0.05) at the beginning of the experiment (D₁). In group M, the SOD, CAT and GST activities showed no significant (P > 0.05) results throughout the whole experiment (Fig. 3a, b, c). SOD activity in groups L and H increased significantly (P < 0.05) after D₁ and reached their peak values at D₂ (18.76 ± 0.44U/mg prot) and D₄ (20.23 ± 0.17U/mg prot) (Fig. 3a), respectively. CAT activity in groups L and H also increased significantly after D₁ (P < 0.05) and reached their peak values at D₃ (9.16 ± 0.12U/mg prot) and D₄ (9.28 ± 0.06U/mg prot) (Fig. 3b), respectively. GST activity in L and H increased significantly (P < 0.05) after D₁ and reached their peak values at D₃ (91.52 ± 0.541U/mg prot) and D₄ (103.55 ± 0.3.90U/mg prot) (Fig. 3c), respectively.

3.1.4. Diversity of microbial communities

In total, 340431 sequences were obtained. As shown in Table 1, the Ace, Chao1, and Shannon’s index values of S. trocheliophorum samples in the group M were significantly higher than the samples in the group L and H (P < 0.05), however, the Simpson’s index values in the group M were significantly lower than that in the group L and group H (P < 0.05). Combined with the Venn diagram, which can directly reflect the similarity and overlap of OTUs number composition of different light treatment coral samples (Fig. 4), we can see the quantity of OTUs in S. trocheliophorum group M was the largest, but the group H was the least.

Table 1

Microbial community diversities of *S. trocheliophorum* in different light intensities
Light treatments	OTU number	ACE index	Chao1 index	Shannon index	Simpson index
L	410.67 ± 12.47^a	381.00 ± 22.20^a	383.00 ± 22.99^a	3.36 ± 0.16^a	0.09 ± 0.02^a
M	497.33 ± 10.27^b	513.33 ± 11.88^b	524.00 ± 15.38^b	4.08 ± 0.07^b	0.07 ± 0.01^a
H	364.67 ± 22.29^a	433.00 ± 13.64^a	437.33 ± 12.71^a	3.00 ± 0.23^a	0.15 ± 0.04^b
Different superscript lower case (a and b) represent significant differences (P < 0.05) among different light treatments.

According to the classification results, the bacteria detected in 9 samples belonged to 35 phyla, 96 classes, 244 orders, 404 families, 756 genera, 1148 species and 1680 OTUs. The major phyla were Proteobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Acidobacteria, Verrucomicrobia, Firmicutes and Actinobacteria (Fig. 5). In group L, Proteobacteria (36.78%-67.50%) was the dominant group and Actinobacteria (12.05%-21.18%) was the secondary dominant group; Proteobacteria (31.02%-42.32%) and Actinobacteria (14.35%-30.97%) were also dominant in the group M; Proteobacteria (34.68%-78.13%) and Actinobacteria (7.15%-25.57%) were still the top two bacteria group in the group H. Although the composition of bacteria in different light groups was different, Proteobacteria and Actinobacteria were still the dominant bacteria at phylum level in S. trocheliophorum.

3.2. Effects of different photoperiods on S. trocheliophorum

3.2.1. Zooxanthellae density

At the beginning of the experiment (D₁), there were no significant differences (P > 0.05) in the density of zooxanthellae in S. trocheliophorum cultured under three different photoperiods (X, Y and Z) (Fig. 6). During the experiment, the density of zooxanthellae in group X decreased significantly (P < 0.05), but zooxanthellae density in groups Y and Z was not found significant differences (P > 0.05). As a result, at the end of the experiment (D₅), the zooxanthellae density of S. trocheliophorum in the group X (1.70×10⁸ ± 0.10×10⁸cells g^− 1 DW) was significantly lower than the group Y (2.40×10⁸ ± 0.07×10⁸cells g^− 1 DW) and Z (2.38×10⁸ ± 0.09×10⁸cells g^− 1 DW) (P < 0.05) (Fig. 6).

3.2.2. Chl a content

At D₁, no significantly differences in the content of Chl a were found in groups X, Y and Z (P > 0.05) (Fig. 7). Chl a content in group X decreased after D₁, but Chl a content in group Y and Z didn’t fluctuate significantly during the experiment (P > 0.05). At the end of the experiment (D₅), the Chl a content of S. trocheliophorum in group X (201.40 ± 9.77µg g^− 1 DW) was significantly lower than the group Y (287.78 ± 16.13µg g^− 1 DW) and Z (284.83 ± 16.22µg g^− 1 DW) (P < 0.05) (Fig. 7).

3.2.3. Enzyme activity

At the beginning of the experiment (D₁), the SOD, CAT and GST activities (Fig. 8a, b, c) in different groups (X, Y and Z) showed no significant differences (P > 0.05). And all the enzyme activity (Fig. 8a, b, c) in the group Y and Z didn’t fluctuate significantly (P > 0.05) either during the whole experiment. However, in group X, the SOD, CAT and GST activities (Fig. 8a, b, c) increased significantly after D₁ (P < 0.05). SOD and GST activities reached their peak values at D₄ (21.13 ± 0.27U/mg prot and 98.53 ± 3.20U/mg prot, respectively ) and CAT reached its maximum at D₃ (9.39 ± 0.28U/mg prot).

3.2.4. Diversity of microbial communities

Table 2

Microbial community diversities of *S. trocheliophorum* in different photoperiods
Light treatments	OTU number	ACE index	Chao1 index	Shannon index	Simpson index
X	350.00 ± 63.64^a	351.00 ± 59.70^a	357.67 ± 61.15^a	3.39 ± 0.28^a	0.10 ± 0.01^a
Y	550.33 ± 59.77^b	567.00 ± 53.75^b	577.33 ± 58.46^b	3.97 ± 0.08^b	0.06 ± 0.01^b
Z	465.00 ± 53.22^ab	474.67 ± 54.24^ab	446.00 ± 41.82^ab	3.94 ± 0.07^b	0.06 ± 0.01^b
Different superscript lower cases (a and b) represent significant differences (P < 0.05) among different light treatments.

In the study, 332236 sequences were obtained totally. As shown in Table 2, the Ace, Chao1 and Shannon’s index values in the group Y were significantly higher than the samples in the group X and Z (P < 0.05), but the Simpson’s index values of samples in the group Y were significantly lower than that in the group X and group Z (P < 0.05). From the Venn diagram (Fig. 9), we can get that the quantity of OTUs in S. trocheliophorum of group Y was the largest, but group X was the least.

According to the classification results, the bacteria detected in 9 samples belong to 34 phyla, 95 classes, 253 orders, 418 families, 822 genera, 1276 species and 1931 OTUs. The main phylumwere Proteobacteria, Actinobacteria, Firmicutes, Chloroflexi, Cyanobacteria, Acidobacteria, Verrucomicrobia and Bacteroidetes (Fig. 10). In the group X, the top three bacteria were Proteobacteria (32.2%-50.1%), Actinobacteria (16.4%-40.4%) and Firmicutes (24.7%-41.3%). In group Y, the main bacteria were Proteobacteria (36.7%-45.9%), Actinobacteria (36.1%-37.7%) and Firmicutes (8.05%-16.8%). In the group Z, Proteobacteria (49.6%-54.3%), Actinobacteria (22.3%-34.6%) and Firmicutes (8.15%-21.5%) were still the dominant bacteria. Thus, Proteobacteria, Actinobacteria and Firmicutes were the dominant bacteria at the phylum level of corals in different photoperiod groups.

4.1. Effects of different light intensity treatments on S. trocheliophorum

4.1.1. Zooxanthellae density and Chl a content

As the most important primary producer of the coral reef ecosystem, symbiotic zooxanthellae provides sufficient oxygen and energy for the growth of host coral through photosynthesis. Many studies have stated that the health and growth of corals are closely tied to the number of zooxanthellae (Jahajeeah et al. 2020). In our study, soft coral in group M possessed relatively higher zooxanthellae density than group L and H. We could accept that some zooxanthellae could leave the host coral under light intensity conditions. This seems to be in line with Rocha et al (2013b), who claimed that leather coral Sarcophyton cf. glaucum exposed to the 120µmol m^− 2 s^− 1 light intensity possessed fewer zooxanthellae than under 80µmol m^− 2 s^− 1. And Rocha et al (2013a) also found the same pattern in the Sinularia flexibilis, which exposed to the 80µmol m^− 2 s^− 1 light intensity possessed the highest zooxanthellae density.

Additionally, as one of the major pigments in zooxanthellae, Chl a content could be easily influenced by environmental situations, such as light and temperature, and may be impacted by the change of zooxanthellae density or pigment concentration per zooxanthellae (Myers et al. 1999; Kuguru et al. 2010). Yu et al (2020) reported that, after acute heat stress, the zooxanthellae density of coral Acropora pruinosa declined following the decrease of Chl a content. In our study, the decreased Chl a content in S. trocheliophorum cultured under the 30 and 130µmol m^− 2 s^− 1 light intensities may account for the change of zooxanthellae density.

4.1.2. Enzyme activity

Inappropriate light intensities may cause light stress on corals, leading to the production of ROS, and the increased content of ROS is frequently accompanied by the increase in the activity of the antioxidant enzyme (Yu et al. 2020). As defense free radical scavengers, SOD provided the first line of defense against the toxic effects of elevated levels of ROS (Gill et al. 2010); CAT catalyzes the dismutation of H₂O₂ to O₂ (Anila et al. 2021); GST is a detoxifying enzyme and as a co-factor of antioxidant enzyme (Lushchak 2011). In this study, the antioxidant enzyme activities (SOD, CAT and GST) in group M showed no change, which indicated that 80µmol m^− 2 s^− 1 light intensity did not cause ROS damage to soft coral. Conversely, we also found the increased activity of the antioxidant enzyme in group L and H, indicating that 30 and 130µmol m^− 2 s^− 1 light intensities can result in the increased production of ROS in coral, and the increased antioxidant enzymes could be considered as a protective response to oxidative stress. This was in line with Tomihiko et al (2015), who proved that antioxidant enzyme activity increased in coral Montipora digitata under high light intensity conditions indicating that ROS levels also increased. Correspondingly, there is also a decrease in the zooxanthellae density of corals in the 30 and 130µmol m^− 2 s^− 1 light intensities. Furthermore, oxidative stress has been demonstrated to be one of the main causes of the decrease of the zooxanthellae amount and its photosynthetic efficiency (Lesser 1996; Tomihiko et al. 2015). Therefore, the following conclusions can be drawn: 30 and 130µmol m^− 2 s^− 1 light intensities may enhance oxidative stress, resulting in a faster decline in zooxanthellae density in coral compared with 80µmol m^− 2 s^− 1 light intensity.

4.1.3. The diversity of microbial communities

Microbial symbionts play an irreplaceable role in the health and adaptive response of coral to environmental changes (Bourne et al. 2013; Van et al. 2018). Because the stability of the symbiotic structure is an important guarantee for maintaining host health and the change of microbial community structure will affect the function of the whole micro-habitat, so the species and quantity of bacteria could be an index to judge the health status of corals (Chen et al. 2020).

The diversity indices commonly used in microbial community diversity analysis are Ace, Chao1, Shannon and Simpson indices. Among them, the Ace index represents abundance-based coverage estimator of species richness; Chao1 index represents abundance-based coverage estimator of species richness; Shannon and Simpson's indices represent estimator of species richness and species evenness (Yang et al. 2015; Kim et al. 2017). In our study, the results demonstrated that the Ace, Chao1 and Shannon indices in the light intensity group of 80µmol m^− 2 s^− 1 were the biggest and the Simpson index was the smallest, on the contrary, the coral in the group of 130µmol m^− 2 s^− 1 light intensity possesses the smallest Ace, Chao1, Shannon indices and biggest Simpson index. Consequently, we could consider that the microbial community diversity of coral in group M was the highest, and group H was the lowest. Combined with the results of zooxanthellae and enzyme activity, we guessed that, compared to the coral in group M, the decrease of the microbial community diversity in groups L and H might be related to the light stress on coral and the decrease of the number of zooxanthellae. Taken together, the coral cultured under the light intensity of 80µmol m^− 2 s^− 1 performed better than the coral in 30 and 130µmol m^− 2 s^− 1 light intensities.

At present, the identified symbiotic bacteria in coral at the phylum level mainly include Proteobacteria, Firmicutes, Cyanobacteria, Chlorobia, Actinobacteria, Bacteroidetes and so on (Lin et al. 2020). Proteobacteria and Cyanobacteria play a crucial role in the photosynthesis of zooxanthellae for corals (Wu et al. 2018). In the present study, the main bacterial composition of corals in different light intensity groups was the same at the phylum level; for example, the dominant phylum in S. trocheliophorum were Proteobacteria, Actinobacteria and Cyanobacteria.

4.2. Effects of different photoperiod treatments on S. trocheliophorum

4.2.1. Zooxanthellae density and Chl a content

This study showed that the number of zooxanthellae and the Chl a content were relatively stable in the Y and Z, while group X showed a significant downward trend. Unsuitable photoperiod might cause zooxanthellae to escape from the polyps to avoid the adverse environment, resulting in a decline in the number of zooxanthellae and the content of Chl a (Nakamura et al. 2017; Wells et al. 2021). In addition, the photosynthetic efficiency of the coral-symbiotic zooxanthellae would decrease when illumination time was insufficient, thus affecting the growth of corals at the end.

4.2.2. Enzyme activity

Under the photoperiod of 18D:6L, the activities of CAT, SOD and GST, showed a trend of significant increase at first and then decreased. These results indicated that a large amount of ROS was produced in S. trocheliophorum at first under the 18D:6L photoperiod, which promoted the related enzyme activities. At a later stage, the decrease of ROS quantity was accompanied by the decrease of enzyme activity level. Fan et al (2020) have also reported that prolonged darkness could lead to increased oxidative stress and oxidative damage, consistent with our study. Accordingly, 6D:18L and 12D:12L photoperiods didn't cause ROS damage to soft coral.

Nevertheless, corals under the 18D:6L photoperiod suffered severe oxidative damage. Correspondingly, there was also a decrease in the zooxanthellae density under the18D:6L photoperiod. Therefore, we could conclude that 18D:6L photoperiod may enhance oxidative stress on coral, resulting in a faster decline in zooxanthellae density compared with 6D:18L and 12D:12L photoperiods.

4.2.3. The diversity of microbial communities

In our study, the results demonstrate that the Ace, Chao1 and Shannon indices of S. trocheliophorum in the group Y were the biggest and the Simpson index was the smallest; on the contrary, the coral in the group X possesses the smallest Ace, Chao1 and Shannon indices and biggest Simpson index. Consequently, we could consider that the microbial community diversity of coral in group X was lower than the group Y and Z. Combined with the results of zooxanthellae and enzyme activities, we found that compared the coral in group Y and Z, the decrease of the microbial community diversity in group X might be related to the light stress on coral and the decrease of the number of zooxanthellae. Taken together, the coral cultured under the photoperiod of 12D:12L and 6D:18L performed better than the coral in the photoperiod of 12D:6L. Environmental stress might affect corals' growth by reducing the ability to regulate symbiotic microorganisms (Zaneveld et al. 2016; Mcdevitt-Irwin et al. 2017). Microorganisms were sensitive to changes in the environment, and even if corals appeared healthy on the surface, their microbial composition would change to some extent (Ziegler et al., 2016; Rice et al., 2019). But we found the dominant bacteria composition in all three different photoperiod groups at the phylum level were Proteobacteria, Actinobacteria and Firmicutes, which was similar to the results of many related studies (Matthews et al. 2020; Patel et al. 2021). In summary, insufficient light, like 18D:6L photoperiod, could decrease the microbial community diversity of soft coral, but it doesn’t change the main bacteria composition at the phylum level.

In this study, the effect of light intensity and photoperiod on the cultivation of the S. trocheliophorum was reported for the first time. The present study allowed us to point out 80µmol m^− 2 s^− 1 and 12D:12L as the suitable light intensity and photoperiod to apply in S. trocheliophorum cultivation, as higher zooxanthellae density, Chl a content and microbial diversity were found in these light conditions. 130µmol m^− 2 s^− 1 light intensity and 18D:6L photoperiod could cause oxidative damage to coral and led to the decline of zooxanthellae density and microbial diversity. Consequently, symbiotic zooxanthellae, enzyme activity and microbial diversity could be the indicators to evaluate the effects of environmental stresses on corals, which have reference value for the conservation and cultivation of other corals in the future.

Declarations

Authors’ information

College of Fisheries, Ocean University of China, Qingdao, China

Ruiguang Dong, Xiefa Song, Xian Li and Dengpan Dong

College of Ecology and Environment, Hainan University, Haikou, China

Weidong Li

Key laboratory of utilization and protection of tropical marine living resources, Ministry of Education, Hainan Tropical Ocean University, Sanya, China

Peizheng Wang

Declarations

Conflict of interest

The authors declare no conflict of interest.

Consent to participate

All authors consented to participate in all aspects of this research and publication.

Consent for publication

All authors agree to the publication of this manuscript in Aquaculture International.

Ethics approval

All the trials were performed in accordance with the Regulations of the Laboratory Animal Ethics Committee of Ocean University of China and were approved by the Laboratory Animal Ethics Committee of Ocean University of China. All handling of S. trocheliophorum specimens was conducted in strict accordance with Animal Care Quality Assurance in China.

Funding

This work was supported by the National Key Research and Development Program of China (No. 2019YFD0900505) and Hainan Province Science and Technology Department Key Research and Development Programme (No. ZDYF2021SHFZ104).

Authors’ contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Ruiguang Dong, Weidong Li, Peizheng Wang, Dengpan Dong, Xiefa Song and Xian Li. The first draft of the manuscript was written by Ruiguang Dong and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors would like to thank Xvtao Sun, Hao Lu Yuexin Wu and Wenxue Che for the assistance in the breeding of experimental coral.

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Cultivation and conservation of the soft coral Sarcophyton trocheliophorum: Light intensity and photoperiod regulation

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Abstract

Figures

1. Introduction

2. Materials and method

2.1. Experimental coral and culture system

2.2. Effect of light intensities and photoperiods on S. trocheliophorum

2.3. Sample analysis

2.3.1. Density determination of zooxanthellae

2.3.2. Determination of chlorophyll a content

2.3.3. Determination of enzyme activity

2.3.4. Analysis of microbial diversity

2.3.4.1. Sample collection and DNA extraction

2.3.4.2. PCR amplification and high throughput sequencing

2.4. Data analysis

3. Results

3.1. Effects of different light intensity treatments on S. trocheliophorum

3.1.1. Zooxanthellae density

3.1.2. Chl a content

3.1.3. Enzyme activity

3.1.4. Diversity of microbial communities

3.2. Effects of different photoperiods on S. trocheliophorum

3.2.1. Zooxanthellae density

3.2.2. Chl a content

3.2.3. Enzyme activity

3.2.4. Diversity of microbial communities

4. Discussion

4.1. Effects of different light intensity treatments on S. trocheliophorum

4.1.1. Zooxanthellae density and Chl a content

4.1.2. Enzyme activity

4.1.3. The diversity of microbial communities

4.2. Effects of different photoperiod treatments on S. trocheliophorum

4.2.1. Zooxanthellae density and Chl a content

4.2.2. Enzyme activity

4.2.3. The diversity of microbial communities

5. Conclusion

Declarations

Declarations

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Funding

Authors’ contributions

Acknowledgements

References

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