The Presence of Biomarker Enzymes of Selected Scleractinian Corals of Palk Bay, Southeast Coast of India

The health and existence of coral reefs are in danger by an increasing range of environmental and anthropogenic impacts. The causes of coral reef decline include worldwide climate change, shoreline development, habitat destruction, pollution, sedimentation and overexploitation. These disasters have contributed to an estimated loss of 27% of the reefs. If the current pressure continues unabated, the estimated loss of coral reef will be about 60% by the year 2030. Therefore, the present study was aimed to analyze the enzymes involved in stress induced by coral pathogen and its resistance. We focused on the enzymes involved in melanin synthesis pathway (phenoloxidase (PO) and peroxidases (POD)) and free radical scavenging enzymes (super oxide dismutase (SOD), catalase (CAT)) and glutathione peroxidase (Gpx) in selected scleractinian corals such as Acropora formosa, Echinopora lamellosa, Favia favus, Favites halicora, Porites sp., and Anacropora forbesi. Overall, PO activity of coral was significantly lower than that of zooxanthellae except for Favia favus. Coral colonies with lower PO and POD activities are prone to disease. Maximum antioxidant defensive enzymes were observed in Favia favus followed by Echinopora lamellose. It is concluded that assay of these enzymes can be used as biomarkers for identifying the susceptibility of corals towards coral bleaching induced by pathogen.


Introduction
The coral holobiont is comprised not only of coral animal and endosymbiotic dinoflagellates (zooxanthellae) but also of microbial communities such as bacteria, archaea, and fungi as well as numerous viruses [1,2]. As a primitive organism, corals do have an innate immune system similar to that of other invertebrates [3][4][5][6] but do not possess the adaptive immune system of vertebrates. The defensive mechanism of the immune system plays a vital role in prevention of infection and in the maintenance of tissue integrity of corals [7,8]. The recognition of the coral defensive system involved in immune responses like other invertebrates includes Tolllike receptors [9] and the synthesis of melanin. The melanin synthesis is activated by phenoloxidase, immune cells and antioxidants, and peroxidase [10][11][12][13]. During PO pathways, the cytotoxic intermediates lead to inflammatory defense [5,14].
Prophenoloxidase is an important enzyme and a key component of innate immunity system of corals [15]. Pathogenic microbes trigger the synthesis of melanin through the cleavage of prophenoloxidase (PPO) into phenoloxidase (PO). Various phenoloxidases catalyse monophenol hydroxylation and diphenol oxidation as well as autocatalytic reactions which frequently lead to the formation of the pigment, melanin [14][15][16]. Oxidative burst is resulting in upregulation of PPO activity because PPO activity is the primary sources of oxidative stress during an invertebrate immune response [17,18]. This leads to the production of antioxidant enzymes such as SOD, CAT, POD, and GPx. Our study focused on the biomarker enzymes involved in defensive mechanism of selected corals of Palk Bay situated at southeast of India.  (AN). Coral fragments of about 2 cm 2 were collected using a hammer, stored in an ice pack, and immediately transported to the laboratory.

Materials and Methods
The coral samples were crushed using sterile sea water and the homogenate was obtained by centrifugation at 15,000 RPM for 15 minutes to separate the supernatant and pellets. The supernatant thus obtained was considered as substrate for the analysis of protein and enzyme activities of coral host [19] and the pellet was considered to be a substrate for analysis of protein and enzyme activities of zooxanthellae. The pellets containing the zooxanthellae were suspended in 2 mL of 100 mmol −1 phosphate buffer (pH-7.0) and were dissolved by sonication for 30 minutes in an ice bath. Triton X-100 (0.05%) solution was added to the sonicated suspension and kept for 10 minutes at room temperature and then the suspension was centrifuged at 14,000 rpm for 30 minutes and used as the zooxanthellae extract for protein and enzyme assays. The protein was estimated using standardized protocols prescribed by Lowry et al., 1951 [20].

Peroxidase and Phenoloxidase.
Peroxidase activity was assayed spectrophotometrically (SPECRTA max M2e) at 470 nm using guaiacol as a phenolic substrate with hydrogen peroxidase [21]. Five milliliters of the assay mixture for the peroxidase activity comprised 35 L of phosphate buffer (10 mmol −1 pH 6.0), 10 L of sample extract (coral tissue and zooxanthellae solution of six different coral samples), 40 L of guaiacol (25 mmol −1 ), and 25 L of H 2 O 2 (20 mmol −1 ). The reaction was stopped by adding 5% H 2 SO 4 • POD activity was expressed as U⋅mg −1 protein. Units were calculated using a molar absorptivity of 2.66 × 104 M −1 cm −1 for tetraguaiacol or 3,3 -dimethoxy-4,4 -biphenoquinone [22]. Phenoloxidase activity was calculated using the same procedure followed for the analysis of peroxidase but without peroxide.

Superoxide Dismutase
Activity. Quantification of SOD activity was based on the ability of SOD to inhibit the reduction of NBT by superoxide [23]. Two hundred microlitres of enzyme extract was added to a tube containing 0.2 mL of 0.1 M EDTA solution, 0.3 mM sodium cyanide, and 0.1 mL of 1.5 mM NBT. Then, 0.05 mL of 0.12 mM riboflavin was added at zero timed intervals and then all the tubes were incubated in a light box for 12 minutes and absorbance was recorded at 560 nm using SPECRTA max M2e. One unit of SOD was defined as the enzyme causing half the maximum inhibition of NBT reduction and SOD activity was expressed as U⋅mg −1 protein. The contents were incubated for 10 min at 37 ∘ C. In this mixture, 0.4 mL of 10% TCA was added to stop the reaction and the mixture was centrifuged at 3200 ×g for 20 min. The pellet was discarded and 0.5 mL of DTNB was added to the supernatant. The supernatant was assayed spectrophotometrically (SPECRTA max M2e) at 340 nm. One unit of glutathione peroxidase activity was defined as the amount of enzyme that oxidizes 1 mol of reduced glutathione and GPx activity was expressed as U⋅mg −1 protein.

Statistical Analysis.
Data were analyzed by one-way analysis of variance (ANOVA) with the Graph-Pad Prism 5 software and the least significant differences were compared at < 0.05.

Phenoloxidase Activity.
Phenoloxidase activity of both zooxanthellae and coral tissues of six healthy coral samples collected from the Palk Bay was presented in Figure 1.  Figure 1). Maximum PO activity of coral tissue was exhibited by F. favus. Except in F. favus, the PO activity of zooxanthellae was significantly higher than coral tissues in all species. The PO activity of zooxanthellae was significantly higher than that of their respective coral tissues except F. favus.  is presented in Figure 2. The peroxidase activity of zooxanthellae of Porites sp., A. forbesi, E. lamellosa, F. halicora, A. formosa, and F. favus was 5.65, 0.43, 7.1, 0.65, 1.22, and 6.91 U⋅mg −1 protein, respectively. Maximum POD activity was observed in zooxanthellae of E. lamellosa and the minimum was observed in zooxanthellae of A. forbesi (Figure 2). POD activity of A. forbesi was 17-fold higher than that of E. lamellosa.

Peroxidase
The peroxidase activity of coral tissues of six different coral species such as Porites sp., A. forbesi, E. lamellosa, F. halicora, A. formosa, and F. favus was 3.21, 0.34, 1.73, 1.13, 0.67, and 10.7 U⋅mg −1 protein, respectively. Maximum POD activity of coral tissues was found in F. favus and the minimum activity was found in A. forbesi (Figure 2). The POD activity of F. favus was 32-fold higher than that of A. forbesi (Figure 2). Significant increase in peroxidase activity was observed in zooxanthellae extracts when compared with coral tissue extracts of all coral samples except F. favus (Figure 2).

Antioxidant Defensive Factors of Corals.
The three major antioxidant enzymes are the superoxide dismutase (SOD) which produces H 2 O 2 by converting O 2 •− , then, the enzyme catalase (CAT) that splits H 2 O 2 to oxygen and water, and the third antioxidant enzyme, that is, glutathione peroxides (GPx), that converts H 2 O 2 and organic peroxides. Increased level of ROS activates these three antioxidant enzymes.

Superoxide Dismutase Activity.
Superoxide dismutase activity of both zooxanthellae and coral tissues of six different coral species is presented in Figure 3.  (Figure 3). SOD activity of E. lamellosa was 8-fold higher than that of A. formosa and 5-fold higher than that of Porites sp.
SOD activity of F. favus was found to be 11-fold higher than that of A. forbesi and F. halicora followed by 5-and 4-fold higher activity than E. lamellosa and Porites sp., respectively. Except A. forbesi and F. halicora, the SOD activity significantly varied between zooxanthellae and coral tissue in all species. Similarly, CAT activity of coral tissues of Porites sp., A. forbesi, E. lamellosa, F. halicora, A. formosa, and F. favus was 2.1, 0.6, 2.2, 1.1, 2.1, and 5.0 U⋅mg −1 protein, respectively ( Figure 4). The coral tissues of A. forbesi and F. favus exhibited minimum and maximum CAT activities, respectively (Figure 4). CAT activity of tissues of A. forbesi was 8.3-fold higher than that of F. favus and 3.7-fold higher than that of E. lamellosa.

Catalase
Comparing CAT activity between coral zooxanthellae and coral tissues of the six different species, it was observed that, except A. formosa and F. favus, CAT activity was higher in zooxanthellae than that of coral tissues in all species.

Glutathione Peroxidase.
Like SOD and CAT activities, glutathione peroxidase (GPx) activities of both zooxanthellae and coral tissues of six different corals were also estimated and presented in Figure 5. GPx activity of zooxanthellae of Porites sp., A. forbesi, E. lamellosa, F. halicora, A. formosa, and F. favus was 0.07, 0.05, 0.21, 0.04, 0.06, and 0.08 U⋅mg −1 protein, respectively. The zooxanthellae of E. lamellosa and F. halicora exhibited maximum and minimum GPx activities, respectively ( Figure 5). Comparatively, GPx activity of E. lamellose was 5.3-fold higher than that of F. halicora zooxanthellae. There was no significant difference between GPx activity of zooxanthellae of Porites sp. and F. favus while the remaining four species showed significant variations in activity.
Similar to GPx activity of zooxanthellae, the GPx activity of coral tissues has been presented in Figure 5  GPx activity and F. favus tissues exhibited maximum activity ( Figure 5). The GPx activities of both zooxanthellae and the coral tissues of Porites sp. were similar. But the GPx activity of coral tissues of A. forbesi and E. lamellosa was less than that of respective coral zooxanthellae activity. However, the activity was more in coral tissues of F. halicora, A. formosa, and F. favus than the respective coral zooxanthellae.

Discussion
As phenoloxidase plays a vital role in defensive mechanism of invertebrates, the presence of PO activity in all 6 coral species indicates the presence of baseline level of antimicrobial defense [13]. Generally, invertebrate animals with decreased phenoloxidase (PO) activity are more susceptible to disease [26][27][28]. In the present study, A. forbesi, F. halicora, and A. formosa showed lowest phenoloxidase activity. It reveals that these coralsare susceptible to bleaching, thermal stress, and diseases. At the same time, F. favus, E. lamellose, and Porites sp. indicated higher disease resistance towards pathogens [13]. The low level of PO activity in A. formosa and A. forbesi showed that branching corals are more susceptible to diseases. Similar results were observed in the study of susceptible ranking of 15 scleractinian corals by Palmer et al. [13].
Peroxidase activity induction includes oxidation of substrates and cytotoxic molecules [14,17] which act as antimicrobial agents [29]. In most cases, peroxidase provides resistance to the corals against fungal pathogens as in case of Aspergillosis [30]. In our findings, the maximum peroxidase activity was observed in the tissue of Favia favus and zooxanthellae of E. lamellosa and the lower activity was observed in A. forbesi. Pathogen recognition results in apoplastic generation of superoxide (O −1 ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals [31], thereby initiating the upregulation of antioxidant enzymes. ROS production is provided with an array of antioxidant enzymes, which either convert O 2 •− to H 2 O 2 (SOD), convert H 2 O 2 to water and oxygen (CAT), or use H 2 O 2 to oxidize substrates (glutathione peroxidase) [31]. The principal findings reveal that corals such as F. favus and E. lamellosa have strong resistance towards bleaching and disease and, at the same time, Anacropora forbesi are more susceptible to bleaching and diseases [13]. The SOD activities of zooxanthellae were too high when compared with the SOD activities of coral tissue of all coral samples analyzed in this study. The findings suggested the production of SOD by coral host in response to O 2 production during photosynthesis of its particular host.
The maximum catalase activity was observed in zooxanthellae compared to coral tissue. This may be due to the conversion of superoxide ion produced during photosynthesis into hydroxyl ion which was denoted by the increase in GPx and SOD activity, which in turn resulted in hydrogen peroxide free radical in the zooxanthellae. Hence, the increase in CAT activities was observed as a result of increased H 2 O 2 concentrations in the zooxanthellae [32].
Glutathione peroxidase (GPx) plays a vital role in cleaving H 2 O 2 as a compensatory mechanism under severe oxidative challenge where the catalase activity may be inhibited [33,34]. The antioxidant enzymes such as SOD, CAT, and GPx are reported to be increased during thermal and salinity stress in response to ROS generated during these stresses [35].

Conclusion
The present study focused on the enzymes involved in the coral resistance among the selected coral species of the Palk Bay region, southeast coast of India. The results of this study described that the corals E. lamellosa, followed by F. favus and Porites sp., were exhibited maximum activities of defensive enzymes. Accordingly, it was concluded that these corals were highly resistant towards the coral pathogens. While the corals of A. formosa, F. halicora, and A. forbesi exhibited minimum activities of the defensive enzymes indicating susceptibility to coral pathogens, these susceptible corals are prone to disease and should be prioritized to prevent transmittance of disease.