1 Introduction

The ornamental trade of marine organisms is a widespread and global component of international trade, fisheries, aquaculture and socio-economic development (Olivotto et al., 2006; Chambel et al., 2015).

A large variety of reef invertebrates, including soft corals, has been used as a potential source of biomolecules for diverse natural products with pharmaceutical or cosmetic value and as a source of organisms for the reef-aquarium trade (Forsman et al., 2006; Sella and Benayahu, 2010; Leal et al., 2013). Due to the intense demand for soft corals, it is essential to continue the studies that have been carried out in recent years with the objective of optimizing the production of corals in captivity (Schlacher et al., 2007; Sella and Benayahu, 2010). However successful coral culture is influenced by numerous factors, such as water movement, temperature, nutrients, heterotrophic feeding and light quality (Strychar et al., 2005; Khalesi et al., 2009; Schutter et al., 2010; Van Os et al., 2012; Mayfield et al., 2013; Rocha et al., 2013).

It is known that light is one of the main factors that influence coral well-being and that each species has specific lighting requirements that influence its growth (Titlyanov and Titlyanova, 2002; Osinga et al., 2011). Light variation is known to affect zooxanthellae density, photosynthetic pigment concentration and photosynthetic efficiency, as well as changes in the density of zooxanthellae that can affect coral physiology and its response to stress (Wang et al., 2008; Al-Hammady, 2013).

The process of reproduction by fragmentation is stressful for the coral and the maintenance of both the mother colony and the fragments generated under adequate lighting conditions certainly has a fundamental role on the post-fragmentation photophysiological processes and, therefore, on coral recovery (Rocha et al., 2013).

Although in the last years science has studied the effects of irradiance on coral and its algal symbionts, there is a gap regarding the study of the effect of spectral quality on the maintenance and reproduction of corals in captivity (Rocha et al., 2013).

It is well known each symbiotic coral have a specific spectral requisites, which is associated with ecophysiological differences among coral and symbiont species and with selective absorption of visible light by seawater (Iglesias-Prieto et al., 2004). The light released from a light-emitting diode has a specific wavelength/colour and each LED light colour is limited to a very narrow range of wavelength. The fact that certain wavelengths can be selected for means that useless wavelengths can be left out, increasing the growth and health of corals and minimizing the production costs with increasing the feasibility of ex situ production of ornamental corals.

The aim of the current study was to evaluate the effects of different light spectra, emitted by LEDs with different spectrum of light red, blue and white on the survival, cicatrisation recover photobiology and growth performance of one commercially important coral in the marine aquarium trade and bioprospecting on marine natural biocompounds, the leather coral Sarcophyton spp.

2 Materials and Methods

2.1 Coral husbandry and fragmentation

Two mothers colonies of the leather coral Sarcophyton spp. with 10 and 12 cm diameter were kept in a life support system and acclimatized during 3 weeks and were daily observed to detect any disease or parasite infection The life support system consisted of a recirculating system of four PVC tank (25-L water volume 60 • 40 • 25 cm, L • W • H) connected to a 100 L sump with protein skimmer, biological filter, UV sterilizer and sand filter. The recirculation was maintained with a submerged pump that provided of 300 L/h flow into each PVC tank. The system operates with natural seawater, filtered by UV sterilizer (V2ecton 18, TMC) and filter with 5 μm. The mother colonies were illuminated with T8 white lamp (4* T8 15 Watt) delivering a Photosynthetic Active Radiation (PAR) of 70 ± 10 μmol quantam⁻² s⁻¹ at the level of the colonies with a 12 h light: 12 h dark photoperiod. PAR value was measured with a Quantum Flux meter (ApogeeSQ-120, USA) with a submergible sensor. The mother colonies were weekly fed with artemianauplii (10 indml^-1). Salinity was maintained at 33 with daily addition of freshwater purified by a reverse osmosis at a volume equal to the evaporated water. Water quality, dissolved oxygen, temperature, salinity, pH, total ammonia, nitrite, nitrate and phosphate were measured weekly.

After acclimation period, each mother colonies were fragmented using a scalper producing 10 similar sized fragments per colony, with each one being individually attached with a rubber band to a labelled plastic coral stand (TMC Coral Cradle ®, UK).

2.2 Experimental design

Five fragments (of the pool of twenty produced) were randomly distributed by one of the four PVC tanks of the culture system similar to the life support system of mother colonies. The experimental system consisted on a recirculating system of four PVC tank (25-L water volume 60 • 40 • 25 cm, L • W • H) connected to a 100 L sump with protein skimmer, biological filter, UV sterilizer, sand filter and submerged pump that provided of 300 L/h flow into each PVC tank. The system operates with natural seawater, filtered by UV sterilizer (V2ecton 18, TMC) and filter with 5 μm.

Each PVC tank was illuminated from above with same Photosynthetic Active Radiation (PAR) of 70 ± 10 μmol quantam⁻² s⁻¹, however light spectrum differs between different light sources, T8 white lamp (4* T8 15 Watt) (control group), white Led (3* 1 watt), red Led (3* 1 watt) and blue Led (3* 1 watt). The maintenance and monitoring of the culture system was identical to the described above for mother colonies.

2.3 Coral fragments growth

To determine the growth of coral fragments, buoyant weight measurements (Davies, 1989) were made at the start (day 0) and the end of the experiment (day 30). Each coral fragment was measured 5 times to guarantee reproducibility. The influence of the light spectrum was evaluated through the estimate of the specific growth rate (SGR% weight gain day^-1)¹.

¹ SGR = 100 (In Wt - In Wi)/t

(De Oliveira et al., 2012) Where Wi and Wt, are the initial and final coral weights and the t is time in days.

2.4 Zooxanthellae density

Zooxanthellae density was measured in the mother colonies and on the fragments at the end of the experiment. At each fragment, the zooxanthellae density was evaluated at two areas: fragmented and non-fragmented side. To determine zooxanthellae density a sample of coral tissue was removed with a scalpel and homogenized on falcon tubes containing 50 mL of filtred (0.2 μm) seawater. The solution was diluted to a known volume and homogenized before a zooxanthellae counting in a Burker chamber. To guarantee reproducibility, 3 samples of each homogenized was counting.

2.5 Statistical analysis

Growth performance parameters and zooxanthellae density are reported as means ± standard error of mean (SEM).

All data were checked for normality and homoscedasticity. A one-way analysis of variance (ANOVA) was used to determine significant differences of the different light spectra on coral growth and zooxanthellae density (Zar, 2009). Post hoc pairwise analysis (Tukey test) was conducted to determine significant differences among experimental combinations. When assumptions (that is, normality and homoscedasticity) were not met, Kruskal–Wallis, followed by Games-Howell test was employed as appropriate (Games and Howell, 1976; Kirk, 1982). For all statistical tests, the significance level was set at p≤0.05. All calculations were performed with IBM SPSS Statistics 22.

3 Results

During the experimental period the water quality was evaluated and maintained constant conditions with normal parameters values for the good maintenance of this specie: OD > 8.0 mg/L, pH between 7.8 and 8.4 temperature 24 to 26ºC, salinity 32 to 35 total ammonia and nitrite below 0.5 mg/L, nitrate < 10 mg/L and phosphates < 0.5 mg/L.

3.1 Coral fragments growth and survival

At the beginning of the experiment the average net weight of fragments was 0.720 ± 0.06 g for control group (T8 with light), 0.782 ± 0.06 g for white led light, 0.742 ± 0.03 g for blue led light and 0.690 ± 0.07 g for red led light. At the end of the period (30 days), the SGR (Figure 1) varied between 0.055 ± 0.09 %/day in control group and 0.091 ± 0.019%/day, 0.210 ± 0.031%/day and 0.380 ± 0.245%/day in, white, blue and red light, respectively. The highest SGR was obtained at blue and red led light , comparatively to the white light and control group used in this experiment (p<0.05). No mortality was recorded through the whole experiment period.

3.2 Zooxanthellae density

The density of zooxanthellae (individuals per gram of coral tissue dry weight) of mother colonies and coral fragments is shown on the Figure 2. At the start of the experiment (mother colonies) the zooxanthellae concentration was 1.067± 2.414. At end of the experiment the concentration was ±, ± and ± for white, blue and red light spectrum, respectively. No significance differences were noted between the concentration of zooxanthellae of mothers colonies and the corals exposed under different light spectrums (p>0.005) and between corals exposed under different light spectrums (p>0.05). Moreover no differences was obtained between the zooxanthellae density of the side initial fragmented and the opposite side, non-fragmented (p>0.005).

4 Discussions

The development of reliable and sustainable hatchery procedures for the captive breeding of reef fishes is now becoming essential to reduce pressure on wild populations. In addition, the rearing organisms in closed systems is likely to lead to the production of hardier specimens that are far better in captivity and survive longer (Pomeroy et al., 2006; Olivotto et al., 2011). The aquaculture of ornamentals organisms can only be achieved through combination of science and technology in order to develop current techniques for ornamental aquaculture, in fact despite the exponential growth of marine ornamental market very few species are presently cultured (Olivotto et al., 2011).

This study showed a positive role of use a specific light spectrum in coral growth, namely blue and red spectrum with a specific growth rate of 0.210 to 0.380%/day. The results on control and with led are similar to the results obtained on previous studies (Rocha et al., 2013), where the authors obtained a specific growth rate of 0.040 ± 0.010%/day, 0.038 ± 0.007%/day and 0.035 ± 0.009%/day when exposed fragments Sarcophytum spp. at an intensity of 50, 80 and PAR 120 respectively, using a metal halide illumination (HQI) of 150 Watt.

As mentioned on results section, there was an influence of different illumination spectra in the growth rate of Sarcophyton spp. fragments. The utilization of light with specific wavelengths spectra, is to date poorly studded, however the few studies existing are contradictory Wijgerde et al. (2014), when using illuminations of white, blue and red spectrum found a negative response on the coral Stylophorapistillata, showed an negative response of coral fragments exposed to red light and a positive response in blue light. However, Kinzie and Hunter (1987) found no differences in Montiporaverrucosa response when fragments were exposed to different lighting, white and blue. These different responses of different species, show the need to increase knowledge about specific growing conditions ex situ for each species in order to guarantee a supply of cultivation conditions to guarantee maximum profitability.

In the presented study there was no influence of the lighting spectrum in zooxanthellae density. However the density obtained in the different treatments is in the order of magnitude between and. This data is in agreement with the results obtained by Rocha et al. (2013) that the exposed coral fragments Sarcophytonglaucum species for 60 days under different intensities of PAR (50, 80, 120) obtained zooxanthellae densities on the order of 107. In contrast, Wijgerde et al. (2014) who obtained average values in the order of 106 when submitted coral Stylophorapistillata species for 6 weeks to PAR intensities of 128 and 256 in different LED lights (blue LED, red LED, blue LED mix and red and white lighting control T5), in these conditions the author found that a pair of the different LED 128 does not affect the amount of zooxanthellae, but there influences using a PAR 256 to give over zooxanthellae the blue LED lighting.

Also, it was found that the fragmented area of each of coral doesn’t differs in zooxanthellae density with non-fragmented area of the same coral. This result indicates that there was a regeneration of the tissue after healing since, after fragmentation and an expected to decrease of zooxanthellae in the affected area. Whereas the zooxanthellae in all treatments were within acceptable values according to the literature and knowing the density increases very zooxanthellae when present the optimal light conditions (Rocha et al. 2013) this study showed that the four types of lighting provided good conditions for the growth of zooxanthellae.

In this study, we also evaluated the effect on survival rate, growth, healing and zooxanthellae density of Sarcophytumspp fragments using LED lights with different spectrum (white, blue and red) comparatively to a traditional white illumination T8. It is concluded from this study that fragments of this species of soft coral, have a best response to when exposed to Red and Blue LED, moreover differences weren't observed between the use of  white LED and T8 Fluorescent light.

The LEDs used in this study have energy consumption of 3 watt and the white traditional lighting T8 a 72 watt. Accordingly beyond the LED lights can increasing the growth of corals, they can do it with less energy cost. Thus, knowing that energy costs are a major cost to consider in aquaculture a substantial reduction in energy costs of lighting can be synonymous of economic viability of ex situ coral aquaculture.

In order to confirm the potential of aquaculture profitability ex situ coral through the use of LED lighting, more studies are needed, including the extension of this study to other species of coral, as well as the study of the combination of different LED spectrums, to optimize growth rates.

Although this study will contribute to increased knowledge and for the feasibility of soft corals aquaculture there is still a long way to go, with many studies that can be done to optimize the growth of coral for ornamental and biotechnological purposes.

Acknowledgments

This study had the support of Fundação para a Ciência e Tecnologia (FCT), through the strategic project UID/MAR/04292/2013 granted to MARE.

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