Comparison of chemical compounds associated with sclerites from healthy and diseased sea fan corals (Gorgonia ventalina)

Tissue collection and sclerite isolation

Sclerites used for this study were isolated from ten healthy and ten diseased Gorgonia ventalina colonies located at a depth of 1.5–2.0 m from El Escambrón beach, San Juan, Puerto Rico (18°28′00″N, 66°05′12″W). The tissue samples were collected under permit 2012-IC-086 issued to Claudia P. Ruiz Diaz, University of Puerto Rico (UPR) Rio Piedras campus, given by the Puerto Rico Department of Natural Resources, Commonwealth of Puerto Rico. Healthy colonies showed neither lesions nor tissue purpling, whereas diseased colonies showed at least one lesion usually overgrown by algae and surrounded by a ring of purple tissue. Previous studies have used the aforementioned pathology to diagnose algal tumor affliction, sea fan aspergillosis, as well as competitive interactions between inter and conspecific individuals (Morse, Morse & Duncan, 1997; Smith et al., 1996; Alker et al., 2004). However, since we performed no microbiological or histological analyses to determine the precise etiology of the injured tissue collected for this study, we avoided the use of any term that could be associated with the afflictions previously described for sea fans. One tissue fragment approximately 4 cm² was cut from the edge of each healthy fan (hereafter HH). In diseased fans, 2 cm² samples were cut from both healthy tissue at the edge of the fan (healthy-diseased or HD) as well as from the diseased area (diseased-diseased or DD). Fragments were placed individually in 50 mL centrifuge tubes and brought to the laboratory on ice. Once in the laboratory, each fragment was further cut into smaller fragments of 2–3 cm² in size and placed in 20.0 mL-sterilized leveled vials filled with 16.0 mL of distilled water. Then, to disassociate sclerites from the soft tissue matrix, vials were vortexed at room temperature, sonicated for 5 min, and then allowed to settle for approximately 4 hrs. This procedure was repeated over a period of 5-7 days to minimize impacts on the structure of the sclerites. After disassociated, sclerites were rinsed several times with distilled water, individually transferred to clean 55 mm Pyrex petri dishes, and dried at 35 °C for 72 hrs.

Gas chromatography-mass spectrometry analysis

From each dried sclerite sample, 0.10 g were grounded in a clean porcelain mortar and the homogenized powder was transferred to a 1.5 mL micro-test tube with 1 mL of acetone. Each tube was vortexed and centrifuged at 6,000 rpm for 5 min. Then, 1.5 mL of the resulting extract from each sample was filtered and transferred without introducing air bubbles to clean amber vials (32 × 12 mm). Afterward, samples were purged with nitrogen to remove dissolve oxygen and then septum caps were fitted to each vial and these were inverted to ensure no headspace was visible and finally, vials were hermetically sealed and not opened until analyzed to preserve the solution. Prior to injecting the solvent used for the extraction, reagents, glassware and other samples processing hardware were blank tested to ensure these materials will not add artifacts to the sclerite extracts analyses. Then, vials with sclerite extracts were placed in an Agilent 7890A gas chromatograph (GC) coupled to an Agilient 5,975 C mass-selective detector for splitless injection. Because removing a sample aliquot from the vials may compromise the integrity of the sample, multiple aliquots were injected to allow for the screening and re-analysis of the samples. The injection temperature was 250 °C. Separation of samples was performed using a fused silica capillary column (SLB^®-5 ms 30 m × 0.32 mm × 0.25 μm film thickness; Supelco, Bellefonte, PA, USA). The initial column temperature was held at 70 °C for 1 min, and then increased by 8 °C min⁻¹ until reaching 270 °C, where it was held for 3 min, for a total run time of 29 min per sample. Ultrapure helium at constant-flow of 1.2 mL min¹ was used as the carrier gas. Analysis of the generated chromatograms was performed using the AMDIS version 2.7/NIST version 2.0 computer program and the criteria for selection in the analysis for the screening compounds were: (1) R- match > 800; (2) S/N > 15; and (3) peak areas > 1,000. Compound identification was confirmed through the use of NIST library appropriated standards and retention time index (http://www.nist.gov/srd/nist1a.cfm). To determine the potential biological roles of each of the detected compounds, a scientific literature review using the IUPAC names of each compound was conducted.

Free radical scavenging assay

Free radical scavenging activity of sclerite extracts was estimated using 1,1,-diphenyl-2-picrylhydrazyl (DPPH) (MacDonald, Wood & Garg, 2006). Briefly, 0.10 g of homogenized powder not previously used from each of the sclerite samples was placed into labeled and clean 50 mL centrifuge tubes filled with 5.0 mL methanol. Then, each tube was vortexed for 10 s, placed in a water bath at 40 °C for 40 min, and sonicated afterward for 30 min. The samples were then centrifuged for 10 min (6,000 rpm) at 25 °C. Afterwards, 1.0 mL of the supernatant (“sclerite extract”) was transferred to a clean 50 mL centrifugal tube containing 5.0 mL methanol. Then, 1 µL of the sclerite extract and 39 µL of DPPH (0.09 mM) were added to a 96-well microplate and the absorbance of the each sample was recorded at 0, 1, 4 and 5 min and then every 5 min for a one-hour period at 560 nm in a Thermo Scientific Multiscan FC spectrophotometer. Only those samples with absorbances with absorbance <2 were used in the analysis. Scavenging activity in this study was expressed as IC₅₀, which represents the concentration of extracts (mg/mL) needed to inhibit 50% of the DPPH radicals. To calculate IC₅₀ for each sclerite sample, the percent inhibition of radical production (%I) was estimated using the following equation (Mishra, Ojha & Nabo Kumar Chaudhury, 2012): ${\displaystyle \text{\%}I=\left(A_i-A_f\right)\cdot 100∕A_i,}$

where A_i and A_f represent the initial and final absorbance, respectively. A linear regression analysis was performed with extract concentration as the independent variable and %I as the dependent variable. Finally, the resulting linear regression equations with a correlation coefficient R² >80% were used to estimate the IC₅₀.

Statistical analyses

Nonmetric Multi Dimensional Scaling (nMDS) along with an Analysis of Similarity (ANOSIM) with Bray-Curtis distances measurement were utilized to compare the composition of chemical compounds among the sclerites from different tissues. We used nMDS ordination, achieved by the metaMDS wrapper function from the package vegan in R version 3.2. The ordination was applied such that the data was scaled down to two dimensions.

For the analysis of free radical scavenging, a Kruskal-Wallis was performed to detect differences between IC₅₀ index from sclerites from different tissues. In this analysis, IC₅₀ index was assigned as the dependent variable and the origin of the sclerite was assigned as the independent variable. Finally, a Chi² test was conducted to determine statistical differences in the relative abundances of alcohols, alkanes and alkenes and sclerite origin. In all analyses, P-value was fixed at 0.05. Statistical analyses were performed using R version 3.2 (R Core Team, 2014). For the statistical analyses, HD and DD samples from the same colony where considered independent and analyzed accordingly. In modular organisms such as gorgonian corals, modules are semi-autonomous units. Consequently, any particular stressors (abiotic or biotic) may trigger or have a differential consequence in tissue subjected to different health states (Van Valen, 1978; Tuomi & Vuorisalo, 1989).

Semi-volatile organic compounds

A total of 70 SVOCs were found in the sampled sclerites; 37% of these were identified from DD sclerites, while 35% and 29% were identified from DH and HH sclerites, respectively. Yet the nMDS and ANOMIS analyses failed to group the chemical compounds according to their origin (e.g., HH, HD and DD sclerites). This holds true even when compounds found only once or twice across the samples were eliminated from the analysis.

In general, three major groups were identified: alcohols, alkanes and alkenes. When data was organized based on sclerite origin, alcohols and secondarily alkanes were the most frequently identified SVOCs in all samples, although the number of identified alkenes was higher in HD sclerites (Fig. 1). However, the Chi² test analysis revealed no statistical differences between alcohols, alkanes and alkenes and the sclerites origin. Furthermore, DD sclerites exhibited the highest number of exclusive compounds with a total of 17 unique SVOCs, followed by HH and HD sclerites with 15 and 8 unique SVOCs, respectively (Table S1 ; Fig. 2). The remaining SVOCs were either shared across all sclerites or between two distinct combinations of sclerites (Fig. 2).

Evidence from the scientific literature concerning the possible biological roles of the SVOCs identified in sclerites was obtained for 44% the compounds. Most were associated with immune related roles such as antimicrobials and antioxidants. Less represented roles were related to cytotoxic and apoptotic compounds, cell membrane components such as steroids and oxidized steroids derivatives, and intermediate signaling molecules.

Free radical scavenging assay

Thirteen out of the 30 sclerites sampled tested had R² >80% and thus were used for the free radical scavenging assays. Of these, six samples were HH sclerites, whereas four and three were DD and HD sclerite samples, respectively. The free radical scavenging IC₅₀ ranged from 0.21 mg dry extract/mL to 0.01 mg dry extract/mL DPPH. Observed differences in antioxidant activity among sclerites were not statistically different, however on average there was a trend for higher values in HH and lowest in DD sclerites (Fig. 3).