Fish Sci (2010) 76:375–380 DOI 10.1007/s12562-009-0213-y
ORIGINAL ARTICLE
Chemistry and Biochemistry
Distribution of lipids and fatty acids in the zooxanthellae and host of the soft coral Sinularia sp. Andrey B. Imbs • Irina M. Yakovleva Long Q. Pham
•
Received: 20 October 2009 / Accepted: 16 December 2009 / Published online: 4 February 2010 Ó The Japanese Society of Fisheries Science 2010
Abstract The lipid classes and the fatty acid (FA) compositions of the zooxanthellae, the host tissue, and intact coral were determined for the first time in a soft coral, Sinularia sp. The contents of monoalkyldiacylglycerol (MADAG), triacylglycerol, and polar lipids differed significantly between the zooxanthellae and the host fractions. The zooxanthellae were rich in polar lipids, whereas neutral lipids were concentrated in the host. MADAG comprised 35% of the host lipids and was practically absent in zooxanthellae. Hence, MADAG is only synthesized in animal tissues and serves as a biomarker for the host in the host–zooxanthellae association of these soft corals. Similar to the zooxanthellae of reef-building corals, the main FA in the zooxanthellae of Sinularia sp. were 18:4n-3, 20:5n-3, and 22:6n-3. In addition, 16:3n-4 and 16:4n-1 (8.9% in total) were found in these zooxanthellae. The ratios of 16:3n-4, 16:4n-1, 18:4n-3, 20:5n-3, and 22:6n-3 in the zooxanthellae to those in the host tissue were 4.2, 11.2, 10.1, 11.0, and 9.1, respectively. The proportions of some FA and lipid classes in the intact coral and its fractions showed that zooxanthellate lipids comprised 36 ± 15% of the total lipids in Sinularia sp. Two tetracosapolyenoic acids (24:5n-6 and 24:6n-3) are proposed as a biomarkers of the animal tissue and indicators of the purity of the zooxanthellae fractions from soft corals.
A. B. Imbs (&) I. M. Yakovleva A.V. Zhirmunsky Institute of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences, 690041 Vladivostok, Russian Federation e-mail:
[email protected] L. Q. Pham Institute of Natural Products Chemistry, Vietnamese Academy of Science and Technology, Hanoi, Vietnam
Keywords Biomarkers Host cells Lipid composition Soft corals Symbionts Tetracosapolyenoic acids Zooxanthellae
Introduction A wide variety of tropical hard and soft coral species harbor symbiotic dinoflagellates (Symbiodinium spp.), broadly known as zooxanthellae, in their endodermal cells. It is widely accepted that zooxanthellate corals can meet their energetic requirements via both heterotrophy (plankton and particulate organic matter) and autotrophy (primary production of zooxanthellae). The zooxanthellae transfer more than 90% of their photosynthetically fixed carbon to their host [1], contributing substantially to the host’s carbon and energy requirements [2, 3]. Autotrophic and heterotrophic feeding supplies corals with major compounds, such as glycerol and lipids [4, 5]. Lipids are mainly stored in the animal tissue as wax esters and triacylglycerols [6–8] or in the membranes as sterols and polar lipids [9]. The fatty acid (FA) composition of these lipids provides useful information on their autotrophic or heterotrophic origins [10]. The transfer (and possible accumulation) of FA synthesized by zooxanthellae to the coral host should significantly affect the host’s FA composition in view of the significant difference between the FA profiles of photoautotrophic organisms and those of the heterotrophic animal host [11, 12]. Several studies have described the FA and lipid compositions of the in hospite zooxanthellae and the animal fractions of hard coral species [12–17]. However, while the transfer of saturated FA in zooxanthellate reef-building corals has been demonstrated, and the transfer of some polyunsaturated FA (PUFA) between the zooxanthellae and the animal compartments in these corals
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has been hypothesized [2, 14, 15, 18, 19], the mechanism of this process is still under discussion [20]. In soft corals, the transfer of FA from the zooxanthellae to the animal partner has never been studied. Moreover, no study has ever attempted to quantify the lipid classes and FA compositions of the pure zooxanthellate and host fractions in any soft coral. At present, only one study has dealt with the presence of some polar lipid classes in the Okinawan soft coral Clavularia viridis and differences in the 20:4n-6 and 20:5n-3 contents of its algal and animal partners [21]. This paper reports the lipid classes and FA compositions of the total lipids of intact coral colonies, zooxanthellae and animal fractions freshly isolated from the soft coral Sinularia sp. The aims of this study were to obtain more information about the PUFA biomarkers of zooxanthellae, the production of lipids and PUFA in zooxanthellae, and their transfer to the host in soft corals.
Materials and methods Colonies of the soft coral Sinularia sp. (class Octocorallia, order Alcyonaria) were collected by scuba divers in Nha Trang Bay (South China Sea, Vietnam) at a depth of 4 m. Three different colonies were used for lipid and FA analysis. Isolation of the symbiotic dinoflagellates from the host tissue was performed according to Banaszak et al. [22] with some modifications. Briefly, coral fragments were first cleaned of debris by hand, chopped into small pieces, resuspended in extraction buffer (1.2 lm filtered seawater and 5 mM EDTA) and homogenized with a glass grinder. To separate the symbionts from the host tissue, the homogenate was centrifuged at 1,0009g for 5 min. The supernatant, which contained host tissue as determined by light microscopy (Nikon Optiphot-2, Japan) at 2009 magnification, was collected and centrifuged for 5 min at 9009g to completely remove symbionts. The resulting isolated host tissue was absorbed on a Whatman GF/C glass fiber filter (0.45 lm) under vacuum and processed for lipid analysis. The pellet, which mostly contained symbionts, was resuspended in extraction buffer, homogenized, 0.02% (v/v) Triton X-100 was added, and the mixture was gently mixed for 5 min. The slurry was centrifuged for 3 min at 9509g and the supernatant was discarded. The pellet was resuspended immediately in extraction buffer, mixed well, filtered through a 40 lm mesh and centrifuged at 1,5009g for 5 min. This step removed any remaining host cell debris as determined by observation with a light microscope (Nikon Optiphot-2) at 4009 magnification. The algal pellet was then washed twice more by centrifugation (1,5009g for 5 min, 2,0009g for 10 min). The final pellet was centrifuged at
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4,0009g for 4 min. The resulting algal pellet was processed for lipid analysis. Extraction of total lipids was conducted according to Bligh and Dyer [23]. Lipid classes were separated by onedimension silica gel TLC. The precoated Merck (Darmstadt, Germany) Kieselgel 60 G plates (10 cm 9 10 cm) were first developed to their full length with n-hexane/ Et2O/CH3COOH (70:30:1, by vol.) and finally to 25% length with CHCl3/CH3OH/C6H6/28% NH4OH (65:30:10: 6, by vol.). After drying in a stream of air, the plates were sprayed with 10% H2SO4/MeOH and heated at 180°C for 10 min. The chromatograms were scanned by an image scanner (Epson Perfection 2400 Photo, Nagano, Japan) in a grayscale mode. The software used for scanning was Adobe Photoshop (Adobe Systems). Percentages of lipid contents were determined basing on band intensity using an image analysis program (Sorbfil Densitometer, Krasnodar, Russia). The units were calibrated using known standards for each lipid class. Fatty acid methyl esters (FAME) were obtained by sequential treatment of the lipids with 1% MeONa/MeOH and 5% HCl/MeOH according to Carreau and Dubacq [24], and were purified by preparative TLC development in benzene. N-Acylpyrrolidide derivatives of FA were prepared by direct treatment of the FAME with pyrrolidine/ acetic acid (10:1, by vol.) in a capped vial (24 h, 25°C) followed by ethereal extraction from the acidified solution [25] and purification by preparative TLC developed in ethyl acetate. The gas–liquid chromatographic (GC) FAME analysis was carried out on a Shimadzu GC-2010 chromatograph (Kyoto, Japan) with a flame ionization detector on a Supelcowax 10 (Supelco, Bellefonte, PA, USA) capillary column (25 m 9 0.25 mm i.d.) at 210°C. Helium was used as the carrier gas. FAME were identified by a comparison with authentic standards and using a table of equivalent chain-lengths (ECL) [26]. The structures of fatty acids were confirmed by gas chromatography–mass spectrometry (GC–MS) of their methyl esters and N-acylpyrrolidide derivatives. The GC–MS analysis of the FAME was performed with a Shimadzu GCMS-QP5050A instrument (Kyoto, Japan). A Supelco MDN-5S capillary column (30 m 9 0.25 mm i.d.) was used at 160°C with a 2°C/min ramp to 240°C that was held for 20 min. Injector and detector temperatures were 250°C. GC–MS of N-acylpyrrolidides was performed on the same instrument; the injector and detector temperatures were 300°C, and the column temperature was 210°C with a 3°C/min ramp to 270°C that was held for 40 min. Homogeneity of data variance was verified using Levene’s test. All analyses were run with untransformed percentage values of lipid class and FA composition. Differences in lipid classes and FA between zooxanthellae
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and host tissue were tested using Student’s t test. Significance was set at P \ 0.01.
Table 2 Fatty acid compositions (% of total FA, mean ± SD, n = 3) of the total lipids in the intact colony, zooxanthellae, and host tissue of Sinularia sp. Fatty acids
Results Total lipids constituted 1.05 ± 0.15% of the wet weight of Sinularia sp. The percentages of individual lipid classes in the intact coral, zooxanthellae, and host tissue are shown in Table 1. The main lipid classes in the intact coral colony were polar lipids (PL), wax esters (WE), monoalkyldiacylglycerol (MADAG), and sterols (ST); the levels of triacylglycerol (TG) and free fatty acids (FFA) were lower than 10% of the total lipids. The same lipid classes were detected in both the zooxanthellae and the host fractions. The content of WE was significantly (P \ 0.01) higher in the host (31.7 ± 0.9%) than in the zooxanthellae (13.9 ± 1.6%). In contrast, the content of PL was significantly (P \ 0.01) higher in the zooxanthellae (56.2 ± 1.6%) than in the host (13.6 ± 1.0%). A substantial amount (35.0 ± 2.3%) of MADAG was detected in the host. A trace amount (less than 1% of the total lipids) of MADAG was found in the zooxanthellae fraction. There were no significant (P [ 0.01) differences in the contents of other lipid classes (TG, FFA, and ST) between the host and zooxanthellae fractions. In the total lipids of the intact coral colonies of Sinularia sp., the principal fatty acids (FA) were 16:0, 20:4n-6, and 18:0 (Table 2). The contents of 16:3n-4, 16:4n-1, 18:2n-7, 18:4n-3, 20:5n-3, 22:6n-3, and 24:5n-6 in the zooxanthellae and host fractions differed significantly (P \ 0.01) (Table 2). The zooxanthellae fraction was enriched with 16:3n-4, 16:4n-1, 18:4n-3, 20:5n-3, and 22:6n-3. The tetracosapolyenoic acids 24:5n-6 and 24:6n-3, as well as 18:2n-7, were concentrated in the host tissue. Comparable amounts of 16:2n-7 (4.1–6.0% of the total FA) were found in the intact coral and its freshly isolated zooxanthellae and animal fractions (Table 2). The level of 20:4n-6 was lower in the zooxanthellae than in the host. Table 1 Contents of individual lipid classes (% total lipids) in the intact colony, zooxanthellae, and host tissue of Sinularia sp. (mean ± SD, n = 3) Lipid class
Total
Zooxanthellae
Host
WE
19.8 ± 3.3
13.9 ± 1.6
31.7 ± 0.9
MADAGa
18.4 ± 1.1
0.8 ± 0.4
35.0 ± 2.3 10.7 ± 0.5
a
TG
7.3 ± 2.1
7.4 ± 2.2
FFA
4.8 ± 3.1
7.7 ± 3.2
3.5 ± 0.4
ST
13.9 ± 3.7
13.9 ± 5.1
5.4 ± 0.8
PLa
35.9 ± 2.9
56.2 ± 1.6
13.6 ± 1.0
a
The contents of this lipid class in the zooxanthellae and host tissue fractions are significantly different (P \ 0.01)
Total
Zooxanthellae
14:0*
2.5 ± 0.3
3.5 ± 0.3
14:1
0.2 ± 0.1
1.4 ± 0.3
15:0 16:0
0.3 ± 0.1 31.3 ± 4.6
0.3 ± 0.1 24.7 ± 1.0
16:1n-9
0.3 ± 0.1
0.8 ± 0.3
16:1n-7
2.1 ± 0.5
1.8 ± 0.0
Host 1.4 ± 0.3 – 0.2 ± 0.0 28.8 ± 4.1 – 2.0 ± 0.3
16:2n-7*
4.6 ± 0.6
6.0 ± 0.5
4.2 ± 2.5
16:3n-4**
1.6 ± 0.5
4.5 ± 0.3
1.1 ± 0.3
16:4n-1**
1.4 ± 0.6
4.4 ± 0.5
0.4 ± 0.2 14.3 ± 1.8
18:0*
9.1 ± 0.7
5.1 ± 0.6
18:1n-9
2.2 ± 0.3
2.9 ± 0.1
2.6 ± 0.3
18:1n-7*
0.3 ± 0.1
0.2 ± 0.1
0.7 ± 0.1
18:2n-7**
2.0 ± 0.4
0.8 ± 0.1
4.0 ± 0.7
18:2n-6
0.3 ± 0.1
0.4 ± 0.0
1.4 ± 0.6
18:3n-4*
1.0 ± 0.2
0.3 ± 0.1
1.7 ± 0.2
18:3n-3
0.4 ± 0.1
0.4 ± 0.1
0.4 ± 0.1
18:4n-3**
3.9 ± 0.3
13.3 ± 0.7
1.3 ± 0.2
20:0* 20:3n-6*
0.6 ± 0.0 0.3 ± 0.1
0.4 ± 0.1 0.1 ± 0.0
1.0 ± 0.3 1.0 ± 0.3 14.8 ± 1.7
20:4n-6**
18.1 ± 5.5
6.7 ± 0.8
20:4n-3**
0.9 ± 0.3
0.2 ± 0.1
1.3 ± 0.2
20:5n-3**
2.5 ± 0.3
6.3 ± 0.5
0.6 ± 0.1
22:4n-6
0.4 ± 0.1
0.1 ± 0.0
0.8 ± 0.5
22:6n-3**
2.1 ± 0.6
7.9 ± 0.8
0.9 ± 0.1
24:5n-6**
3.8 ± 1.0
0.8 ± 0.3
5.0 ± 0.9
24:6n-3*
1.2 ± 0.2
0.3 ± 0.2
1.6 ± 0.3
Other
6.0 ± 1.5
5.1 ± 0.6
8.1 ± 1.6
There are significantly different contents of the fatty acids marked by * and ** in the zooxanthellae and host tissue fractions at P \ 0.05 and P \ 0.01, respectively
The mass spectrum of the N-acylpyrrolidide derivative of 18:2n-7 exhibited a number of main peaks at m/z 113 (rel. intensity 100.0), 126 (73.1), 140 (6.5), 154 (4.9), 168 (12.9), 182 (3.9), 194 (2.1), 208 (12.9), 222 (5.5), 234 (2.7), 248 (1.9), 262 (2.1), 276 (1.8), 290 (1.5), 304 (1.2), 318 (0.4), and 333 (27.1). The molecular ion peak at m/z 333 (M?) and two pairs of fragments at m/z 182 and m/z 194, m/z 222 and m/z 234 indicated that the original FA contained 18 carbon atoms and that the double bonds were localized at the 8th and 11th carbon atoms [25]. A simple calculation based on the contents of some individual FA and lipid classes (Tables 1, 2, data with P \ 0.01) in the intact coral and its algal and animal fractions allowed us to define the proportion of the total lipids of the Sinularia sp. coral that came from the zooxanthellae. This estimation gave an unexpected large value
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(36 ± 15%), which confirmed the importance of zooxanthellate lipids to the total lipid budget of soft corals.
Discussion Patton et al. [27] have shown that approximately 19% of the intact coral lipid resides with the symbiotic algae in the host– zooxanthellae association of the reef-building coral Pocillopora capitata. The two neutral lipids (WE and TG) comprised 75% of the intact coral lipid, with only 8% of these two lipids occurring in the zooxanthellae. Structural lipids (ST, phospholipids, galactolipids, etc.) made up approximately 67% of the zooxanthellate lipids and 16% of the host lipids in P. capitata. Monogalactosyldiacylglycerol and digalactosyldiacylglycerol have been isolated from the total lipids of the zooxanthellate fractions of eight reef-building coral species [13]. A qualitative TLC analysis has indicated that polar lipids of pure zooxanthellae from the Okinawan soft coral Clavularia viridis contain mainly glycolipids and few phospholipids [21]. In this study, the lipid compositions of the intact coral and the algal and animal fractions of the same coral colony were determined for the first time for a soft coral, Sinularia sp. Similar to the hard coral P. capitata, structural lipids (PL) prevailed in the zooxanthellae of this soft coral, but its host fraction was rich in neutral lipids (WE, MADAG, TG, and ST) (Table 1). MADAG was the main lipid class (35%) found in the host lipids of Sinularia sp. MADAG has highly stable chemical bounds and its hydrolysis by lipases is hindered. We agree with Joseph [28] that MADAG may stabilize coral membranes, which are exposed to highly active hydrolytic enzymes. The presence of MADAG (up to 17% of total lipids) in scleractinian, soft, and hydroid corals has been previously shown [11, 28, 29]. Taking into consideration the low content (0.8%) of MADAG in the zooxanthellae, we can postulate that MADAG is only synthesized in animal tissues, and that it can serve as a biomarker of the host in the host–zooxanthellae association of the soft coral. Bishop and Kenrick [13] have demonstrated that 18:3n6, 18:4n-3, 18:5n-3, 20:5n-3, and 22:6n-3 are the major FA in the total lipids of zooxanthellae isolated from eight species of hard corals. Among the total FA of galactolipids from these zooxanthellae, depending on the species, 18:3n6, 18:4n-3, 18:5n-3, and 20:5n-3 comprised up to 26.0, 66.8, 24.0, and 40.5%, respectively. It has been confirmed that the zooxanthellae from several reef-building corals show high proportions of the PUFA mentioned above in comparison to their proportions in the intact corals [14, 16, 29]. However, only Papina et al. [15] and Treignier et al. [17] determined the FA compositions for each fraction (algae, animal) of the same hard coral colony. The ratios of
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18:3n-6, 18:4n-3, 20:5n-3, and 22:6n-3 of zooxanthellae to those of the host tissue were 2.04, 3.92, 6.48, 6.59 in Montipora digitata and 2.13, 6.04, 5.30, 4.35 in Turbinaria reniformis, respectively. Neither of these studies [15, 17] recorded 18:5n-3. Dinoflagellates vary in FA composition according to their taxonomic positions and environments, but some PUFA, such as 18:4n-3 and 22:6n-3, are regarded as biomarkers of these algae [30, 31]. Based on restriction fragment length polymorphism (RFLP) analysis or the sequence of the rRNA-encoding gene, similar phylogenetic clades of zooxanthellae are shown to form associations with both hard and soft corals [32]. Thus, it is very likely that the distinctive PUFA are the same in the zooxanthellae of both scleractinian and soft corals. In fact, a significant predominance of 18:4n-3, 20:5n-3, and 22:6n-3, which were characteristic of zooxanthellae in hard corals, was also detected in zooxanthellae of the soft coral Sinularia sp. (Table 2). We found a substantial content of 16:3n-4 and 16:4n-1 (8.9% in total) in zooxanthellae of Sinularia sp. C16 PUFA have never been previously detected in zooxanthellae of hard corals. The ratios of 16:3n-4, 16:4n1, 18:4n-3, 20:5n-3, and 22:6n-3 in zooxanthellae of Sinularia sp. to those in the host tissue were 4.2, 11.2, 10.1, 11.0, 9.1, respectively, which were twice those seen in hard corals [15, 17]. In the case of soft corals, the biosynthesis of C16 PUFA may be presented as the pathway 16:1n-7 ? 16:2n-7 ? 16:3n-4 ? 16:4n-1, which is performed by sequential enzymatic actions: D6, D12, and D15 desaturations. D12 and D15 desaturases are specific for plants, but D6 desaturase is also present in animals. Thus, a transfer of 16:2n-7 between the zooxanthellae and the host or a parallel action of D6 desaturase in these symbionts may lead to the comparable levels of 16:2n-7 in the fractions of Sinularia sp. Most likely, C2 elongation of 16:2n-7 to 18:2n-7 occurred in the host tissue, where the amount of 18:2n-7 was the highest (Table 2). The absence of 20:4n-6 in pure zooxanthellae from the soft coral Clavularia viridis has been reported [21]. In contrast, in zooxanthellae of the soft coral Sinularia sp., we found a noticeable amount of 20:4n-6 (6.7%), which correlated well with the amount of this acid found in zooxanthellae of hard corals [15–17]. Similar to C. viridis [21], the FA composition of Sinularia host tissue was distinguished by its very low content of 20:5n-3 (0.6%) and its high content of 20:4n-6 (14.8%). The tetracosapolyenoic acids (TPA) 24:5n-6 and 24:6n-3 were also concentrated in the host tissue (Table 2). It is noteworthy that hard corals (Hexacorallia), irrespective of the presence of zooxanthellae, have no TPA; this is their main chemotaxonomic distinction from soft corals (Octocorallia) [33]. It was also shown that the level of TPA
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was several times higher in zooxanthellae-free soft corals compared to zooxanthellate ones [34]. Since both azooxanthellate and zooxanthellate soft corals contain TPA, it is obvious that TPA are synthesized from C22 PUFA in the coral host, and that the enzymes of zooxanthellae do not take part in this stage. Earlier, newly revised pathways for the biosynthesis of n3 and n-6 PUFA in animal cells were proposed [35]. This report concludes that 24:5n-6 is formed by the elongation of 20:4n-6 to 22:4n-6 and then to 24:5n-6, followed by further desaturation (20:4n-6 ? 22:4n-6 ? 24:4n-6 ? 24:5n-6); a similar formation route for 24:6n-3 from 20:5n-3 has been proposed. C2 degradation via b-oxidation of 24:5n-6 and 24:6n-3 acids is postulated as a pathway to 22:5n-6 and 22:6n-3 acids, respectively. It is likely that soft corals utilize a similar biosynthetic route, thus providing another interesting system for studying this type of biogenesis. We propose these TPA as biomarkers of animal host tissue and indicators of the purity of zooxanthellae fractions of soft corals. The total FA of the host and zooxanthellae in Sinularia sp. contained 6.0 and 1.1% TPA, respectively. If we reject the hypothesis about the transfer of FA from the host to zooxanthellae, the total FA obtained from zooxanthellae, which appear to be absolutely pure under light microscopy, actually contain about 20% host FA. The method applied to isolate pure zooxanthellae is commonly used for reef-building corals, but TPA are absent in hermatypic corals, and we cannot apply these FA markers to check the purity of hexacoral zooxanthellae. Acknowledgments This work was supported by the Russian Foundation of Basic Research (09-04-01040, 09-04-90304, and 0904-98542) and the project ‘‘The study of biochemical resistance factors of the bleaching of Vietnamese Coral reefs’’ (NAFOSTED 2009-2011).
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