INTRODUCTION
The majority of studies concerning the biochemical composition of corals has been focused on the lipidic fraction and/or fatty acid composition (Rinkevich, Reference Rinkevich1989; Latyshev et al., Reference Latyshev, Naumenko, Svetashev and Latypov1991; Arai et al., Reference Arai, Kato, Heyward, Ikeda, Iizuka and Maruyama1993; Yamashiro et al., Reference Yamashiro, Oku, Higa, Chinen and Sakai1999; Oku et al., Reference Oku, Yamashiro, Onaga, Sakai and Iwasaki2003; Rodrigues et al., Reference Rodrigues, Grottoli and Pease2008; Imbs et al., Reference Imbs, Latyshev, Dautova and Latypov2010) and little attention has been given to the amino acid (AA) profile of these invertebrates. Some studies concerning these compounds were nevertheless performed on Hexacorallia, examining the composition of skeleton (Young; Reference Young1971; Goldberg et al., Reference Goldberg, Hopkins, Holl, Schaefer, Kramer and Morgan1994) and soluble organic matrix (Puverel et al., Reference Puverel, Tambutté, Pereira-Mouriès, Zoccola, Allemand and Tambutté2005), as well as the release (Tanaka et al., Reference Tanaka, Miyajima, Umezawa, Hayashibara, Ogawa and Koike2009) and uptake (Grover et al., Reference Grover, Maguer, Allemand and Ferrier-Pagès2008) of free amino acids (FAAs). Even less attention has been given to octocorals, with a few studies focusing on the FAA release by colonies (Schlichter & Liebezeit, Reference Schlichter and Liebezeit1991) and uptake by planula larvae (Ben-David-Zaslow & Benayahu, Reference Ben-David-Zaslow and Benayahu2000). Body proteins are responsible for growth, repair and maintenance of all cells, and protein-bound AAs are essential components of all life forms. Studying such AAs should therefore be imperative when pursuing a deeper understanding of any organism. On the other hand, FAAs are known osmolytes playing an important role in the osmoregulation of several invertebrates. Under hypoosmotic stress, the intracellular concentration of FAA decreases as a result of release, while under hyperosmotic stress, the intracellular concentration of FAA increases as a result of uptake (Zurburg & De Zwaan, Reference Zurburg and De Zwaan1981; Matsushima & Hayashi, Reference Matsushima and Hayashi1988). Some living groups (e.g. plants, bacteria and most fungi) are able to synthesize all of the 20 protein AAs. Yet, animals lack a number of AA synthetic pathways or have lower rates of synthesis of these AAs which are insufficient to meet metabolic needs. These AAs are designated as essential and must be obtained from the environment (Rosa & Nunes, Reference Rosa and Nunes2003; Rosa et al., Reference Rosa, Costa and Nunes2004). Regarding the AA biosynthesis in Cnidaria, only one study has been conducted so far, namely in scleractinian corals (Fitzgerald & Szmant, Reference Fitzgerald and Szmant1997). These authors showed that eight AAs normally considered essential for animals were made by the corals tested (valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, methionine and lysine) and this ability could be an indicator of a separate evolutionary history of the cnidarians from the rest of the Metazoa. Besides this study, only one other examined the AA composition of corals, also scleractinian hexacorals (Al-Lihaibi et al., Reference Al-Lihaibi, Al-Sofyani and Niaz1998). Yet, neither study contemplated seasonality, and the influence of biotic and abiotic factors on AA composition. Additionally, there is no information about AA composition of Octocorallia.
The finger-shaped sea pen, Veretillum cynomorium, is a colonial octocoral belonging to the order Pennatulacea that is widely distributed along the eastern Atlantic Ocean. It is found in shallow waters, inhabiting the soft sediment of beaches and sand plains (Kükenthal, Reference Kükenthal1915; Cornelius et al., Reference Cornelius, Manuel, Ryland, Hayward and Ryland1995), with its bathymetric distribution reported to range between 13 and 188 m (Williams, Reference Williams1990; López-González et al., Reference López-González, Gili and Williams2001). It is known that the presence and content of certain fatty acids allow a chemotaxonomic distinction of octocorals from other corals (Vysotskii & Svetashev, Reference Vysotskii and Svetashev1991; Svetashev & Vysotskii, Reference Svetashev and Vysotskii1998; Imbs et al., Reference Imbs, Latyshev, Dautova and Latypov2010). Yet, this biochemical differentiation has never been conducted with AAs. Thus, the objectives of this work were to investigate: (i) the seasonal variations in the total AA composition of the octocoral V. cynomorium; and (ii) interspecific differences between this octocoral and scleractinian hexacorals.
MATERIALS AND METHODS
Biological sampling
Veretillum cynomorium colonies were hand collected in Caldeira de Tróia, a shallow water habitat near the mouth of Sado estuary, Portugal. Sampling was performed at low tide, in the intertidal zone, following a bimonthly periodicity, and comprising a one-year period (from April 2010 to February 2011). A total of 35 colonies were collected in each sampling.
Amino acid analysis
For the biochemical analysis, three independent pooled samples were taken from 35 colonies each month and homogenized with a grinder (Retsch Grindomix GM200, Düsseldorf, Germany; 5000 rpm; material: PP cup and stainless steel knifes), vacuum packed and frozen at –80°C. The frozen samples were freeze-dried for 48 hours at –50 °C under low pressure (approximately 10−1 atm) powdered and stored at –80°C. Moisture content was determined according to the AOAC (2005) methodologies, by drying the sample overnight at 105°C (laboratory heater, P-Selecta 207). To extract total AAs (protein bound + free), 14.8–15.8 mg of sample was placed in 10 ml ampoules with 3 ml of 6 M HCl (Merck) containing 0.1% phenol (Merck), according to the method described by AOAC (2005). Following the establishment of inert and anaerobic conditions, to prevent oxidative degradation of AAs, ampoules were sealed and samples hydrolysed at 110°C for 24 hours; hydrolysates were filtered (0.45 mm pore size) and dissolved with Milli-Q distilled water to 20 ml. Samples were then frozen at –80°C and freeze-dried for 48 hours at –50°C under low pressure (approximately 10−1 atm), after which they were dissolved in 5 ml of 0.1 M HCl (Merck) and stored at –80°C until AA separation. Finally, thawed samples were filtered (0.2 mm pore size) and separation was performed with high-performance liquid chromatography (Agilent 1100 HPLC) using precolumn o-phthalaldehyde and 3-mercaptopropionic acid in borate buffer (OPA, Agilent Technologies) and 9-fluorenylmethylchloroformate in acetonitrile (FMOC; Agilent Technologies) derivatization, a Phenomenex Gemini ODS C18 guard column (4 mm × 3 mm), and a Phenomenex Gemini ODS C18 110A column (4.6 mm × 150 mm, 5 µm). The solvents and gradient conditions were those described by Henderson et al. (Reference Henderson, Ricker, Bidlingmeyer and Woodward2000). Detection wavelengths were set at UV 338 and 262 nm and fluorescence 340/450 and 266/305 nm. The identity and quantity of the AAs were assessed by comparison with the retention times and peak areas of standard AAs (Sigma–Aldrich) using norvaline and sarcosine as internal standards. Tryptophan and cysteine were quantified in V. cynomorium; however, because these are partially lost during the acidic hydrolysis they were not considered for analysis.
Statistical analysis
Seasonal differences in the AA composition were tested with analysis of variance (ANOVA) followed by a multiple comparisons test (Tukey test). If necessary, data were transformed to satisfy normal distribution and homoscedasticity requirements. When data transformation still did not meet the assumptions of ANOVA, differences were analysed with the non-parametric ANOVA equivalent (Kruskal–Wallis). All statistical analyses were tested at 0.05 level of probability with the software STATISTICATM 6.1 (Statsoft, Inc., Tulsa, OK 74104, USA). Interspecific differences in AA profiles were compared using principal component analysis (PCA). PCA reduces the number of dimensions produced by the large number of variables and uses linear correlations (components) to identify those AAs that contributed most to the separation between octocorals (V. cynomorium) and scleractinian hexacorals (data from Fitzgerald & Szmant, Reference Fitzgerald and Szmant1997). Percentages of total AA values were used in order to remove the effect of concentration that would otherwise be the major controlling factor in the subsequent analysis. Prior to analysis, the proportion data were transformed with the formula: log %/(100%) to ensure homoscedasticity (see Rosa et al., Reference Rosa, Calado, Narciso and Nunes2007). It is worth noting that in this analysis we did not include data of scleractinian species obtained by Al-Lihaibi et al. (Reference Al-Lihaibi, Al-Sofyani and Niaz1998) because they did not present complete AA profiles, i.e. they lack information in some AAs, namely tyrosine and arginine.
RESULTS
The seasonal changes in the AA profile and in total AA content (% dry weight) of V. cynomorium are shown in Table 1 and Figure 1, respectively. Based on Fitzgerald & Szmant (Reference Fitzgerald and Szmant1997) findings in hexacorals, it is plausible to assume that this octocoral might also show non-essential AA that normally are considered essential for animals. Thus, valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, methionine and lysine were considered non-essential in the present study. The only essential AA was threonine.
Seasonal variation of total amino acid content in Veretillum cynomorium colonies, between April 2010 and February 2011. Values are means of triplicate samples (±SD). Different superscript letters represent significant differences between months (P < 0.05). Grey bars indicate highest frequency for Group I oocytes and presumed beginning of the development of Group I oocytes into Group II oocytes (October) and period of greater food availability (February). ΣTAA, total amino acid content.
Amino acid composition (% dry weight) of Veretillum cynomorium, between April 2010 and February 2011. Values are means of triplicate samples (±SD). Different superscript letters within rows represent significant differences between months (P < 0.05). Amino acid essentiality was defined based on Fitzgerald & Szmant (Reference Fitzgerald and Szmant1997).
ΣTAA, total amino acid content.
Quantitatively, the most important AAs in V. cynomorium colonies were: glutamic acid, varying from 3.92 to 5.94% dry weight (dw) (P < 0.05) and representing around 14–15% of total AA content (Table 2), aspartic acid (3.34–4.99% dw; P < 0.05; 11–12% of total AA) and glycine (2.87–4.57% dw; P < 0.05; 9–12% of total AA). On the other hand, the minor AAs were methionine (0.41–0.73% dw; P < 0.05; 1–2% of total AA) and histidine (0.54–0.76% dw; P < 0.05; 2% of total AA). Almost all AAs showed the same significant seasonal variations, with the highest values in February, second highest in October and the lowest in June. Concurringly, total AA content exhibited this seasonal trend (Figure 1). Lysine, phenylalanine and methionine did not follow this trend and showed the major peak in October.
Amino acid (AA) composition (% total AA) of Veretillum cynomorium, between April 2010 and February 2011. Values are means of triplicate samples (±SD). Amino acid essentiality was defined based on Fitzgerald & Szmant (Reference Fitzgerald and Szmant1997).
To discriminate potential AA markers from octocorals (V. cynomorium) and scleractinian hexacorals, a PCA with transformed percentage values was performed. The first principal component (PC) explained 66.61% of variance and the second PC explained 13.61% (Figure 2). The two coral groups were clearly separated along the PC1 (Figure 2A). The AAs that contributed most to the separation were arginine (Arg), tyrosine (Tyr), glycine (Gly) and glutamic acid (Glu) to the left (negative) side, and valine (Val), serine (Ser), histidine (His), isoleucine (Ile), alanine (Ala) and phenylalanine (Phe) to the right (positive) side of the PC1 (Figure 2B). Among the octocoral group, the month of October (V10) was clearly separated along the PC2, mainly due to the significant percentage increase of lysine (Table 2).
Principal component analysis based on the amino acid composition in Veretillum cynomorium colonies (present study) and scleractinian corals (Fitzgerald & Szmant, Reference Fitzgerald and Szmant1997). (A) Principal component plot; (B) loading plot of amino acids (AA) and their contribution to the spread along PC1 and PC2. Species abbreviations for Octocorallia: V, Veretillum cynomorium (numbers represent the different months); species abbreviations for Hexacorallia: Acer, Acropora cervicornis (zooxanthellate); Apoc, Astrangia poculata (non-zooxanthellate); Mfav, Montastraea faveolata (zooxanthellate); Pdiv, Porites divaricata (zooxanthellate), Tcoc, Tubastrea coccinea (non-zooxanthellate); AA abbreviations: Ala, alanine; Arg, arginine; Asp, aspartic acid; Glu, glutamic acid; Gly, glycine; His, histidine; Iso, isoleucine; Leu, leucine; Lys, lysine; Met, methionine; Phe, phenylalanine; Ser, serine; Thr, threonine; Tyr, tyrosine; Val, valine.
DISCUSSION
Reproduction, environmental conditions and amino acids
The majority of the AAs revealed significant seasonal variations with the highest values being attained in February or, in the case of lysine, phenylalanine and methionine, in October (Figure 1; Table 1). These seasonal differences appear related to reproduction and environmental conditions (i.e. temperature and food availability). In fact, in our previous study (Baptista et al., Reference Baptista, Lopes, Pimentel, Bandarra, Marques and Rosa2012) we identified one spawning event in July. The mean oocyte size–frequency distributions indicated that all oocytes of the Group III (late-vitellogenic ones) were released in that month (i.e. during the aestival period), when water temperature was at its peak in Caldeira de Tróia. This was supported by the lack of these large oocytes in August. During the post-spawning period (August–October), there was a boost in early oogenesis observable by the increase in Group I oocytes (see figure 1 in Baptista et al., Reference Baptista, Lopes, Pimentel, Bandarra, Marques and Rosa2012). This may have been triggered by the increased water temperature (see figure 2 in Baptista et al., Reference Baptista, Lopes, Pimentel, Bandarra, Marques and Rosa2012). The metabolism of corals vary as a result of temperature (Oku et al., Reference Oku, Yamashiro, Onaga, Sakai and Iwasaki2003) and, therefore, warmer periods should imply higher metabolic rates with a consequent increase in feeding activity. Interestingly, a concomitant increase in AA values between June and October was observed. These results may indicate a special involvement of lysine, phenylalanine and methionine in the mid-stages of oogenesis in V. cynomorium. The specific functions of these AAs are unknown, but it is possible that they act in a similar way as glycine, which was reported to be involved in the gonadal development of another anthozoan (Kasschau & McCommas, Reference Kasschau and McCommas1982). By December, most of the smaller oocytes had already developed into median sized oocytes (Group II), and some of them started to develop into the larger oocytes by February (see figure 1 in Baptista et al., Reference Baptista, Lopes, Pimentel, Bandarra, Marques and Rosa2012). This phase of the oogenic cycle coincides with the period of greater food availability (prevernal: see figure 2 in Baptista et al., Reference Baptista, Lopes, Pimentel, Bandarra, Marques and Rosa2012) and greater AA contents. Spring blooms during the prevernal season, comprise high food availability. Moreover, because corals are polytrophic organisms, they are able to obtain nutrients from various food sources including phytoplankton (Migné & Davoult, Reference Migné and Davoult2002), zooplankton (Palardy et al., Reference Palardy, Grottoli and Matthews2005), bacteria (Sorokin, Reference Sorokin1973), particulate organic matter (Anthony, Reference Anthony2000; Anthony & Fabricius, Reference Anthony and Fabricius2000) and dissolved organic matter (Grover et al., Reference Grover, Maguer, Allemand and Ferrier-Pagès2008; Imbs et al., Reference Imbs, Latyshev, Dautova and Latypov2010), including free AAs (Grover et al., Reference Grover, Maguer, Allemand and Ferrier-Pagès2008). Thus, it is plausible to assume that the frequent sudden increase in available food observed in February acts as a trigger for the final stages of gametogenesis to commence. Finally, none of the observed variations in the content of AAs seem to have occurred as a result of salinity changes (see figure 2 in Baptista et al., Reference Baptista, Lopes, Pimentel, Bandarra, Marques and Rosa2012). For this reason, it appears that salinity did not have a medium/long-term effect in the yearly variations observed for AA content in V. cynomorium, meaning that even if there was an uptake or release of free AAs, it was not noticeable when analysing total AA content.
Amino acids in corals: a comparative approach
The main AAs in V. cynomorium colonies were, in descending order, glutamic acid, aspartic acid, glycine, arginine and leucine. Similar results were observed for scleractinian hexacorals, including both zooxanthellate and azooxanthellate species, although only protein-bound AAs were analysed in those studies (Fitzgerald & Szmant, Reference Fitzgerald and Szmant1997; Al-Lihaibi et al., Reference Al-Lihaibi, Al-Sofyani and Niaz1998). Fitzgerald & Szmant (1997) worked with zooxanthellate and azooxanthellate species, and for both observed that glutamic acid dominated, followed by aspartic acid and then by glycine and leucine which exhibited similar values. In that study, however, arginine was among the AAs exhibiting lowest concentration values (Fitzgerald & Szmant, Reference Fitzgerald and Szmant1997). In the work of Al-Lihaibi et al. (Reference Al-Lihaibi, Al-Sofyani and Niaz1998), only zooxanthellate species were studied, but main AAs differed substantially. Still, glutamic acid either dominated or was the second main AA, aspartic acid was always third, and glycine and leucine varied between second and fifth main AAs. Arginine was not detected, presumably as a result of this AA being masked by ammonia (Al-Lihaibi et al., Reference Al-Lihaibi, Al-Sofyani and Niaz1998). When considering such results, it becomes apparent that in corals, main AAs include glutamic acid, aspartic acid, glycine and leucine. Glutamic and aspartic acids are both indicated as major constituents in marine algae (Munda & Gubenesk, Reference Munda and Gubenesk1986). High content of these AAs may then partially occur as a result of zooxanthellae production (Al-Lihaibi et al., Reference Al-Lihaibi, Al-Sofyani and Niaz1998) or phytoplankton intake. Fatty acid composition of V. cynomorium is indicative of zooxanthellae absence (Baptista et al., Reference Baptista, Lopes, Pimentel, Bandarra, Marques and Rosa2012), leaving phytoplankton as the most probable source for these AAs. For all sampled months, arginine was the fourth main AA. In the study of Al-Lihaibi et al. (Reference Al-Lihaibi, Al-Sofyani and Niaz1998), as mentioned before, detection of this AA was not possible but in the study of Fitzgerald & Szmant (Reference Fitzgerald and Szmant1997), arginine occupied the 10th–13th position depending on studied species. No premature conclusion is intended, but there is the possibility of a high content of arginine being characteristic of octocorals when compared to hexacoralia. Either way, in respect to AA composition, a high content of arginine in V. cynomorium is the main characteristic distinguishing it from the above mentioned scleractinian species (Fitzgerald & Szmant, Reference Fitzgerald and Szmant1997; Al-Lihaibi et al., Reference Al-Lihaibi, Al-Sofyani and Niaz1998).
The octocoral V. cynomorium and the hexacorals studied by Fitzgerald & Szmant (Reference Fitzgerald and Szmant1997) were clearly distinguished in the PCA. This differentiation occurred along PC1, mainly due to higher percentages of arginine, tyrosine and glycine in V. cynomorium (irrespective of the month), and valine, serine, histidine, isoleucine and alanine in hexacorallia species (Figure 2). We speculate that such separation possibly derived from physiological differences related to phylogeny, and was not affected by reproductive or environmental seasonality. The differentiation of the octocoral group along PC2 (V10—October) was mainly due to the considerable percentage increase of lysine, together with the decrease of glycine and aspartic acid during that month (Table 2). These seasonal variations may be linked with the oogenic cycle of this octocoral species (discussed above). PCA findings for the Hexacorallia species also showed that the presence or absence of zooxanthellae had a negligible effect (Figure 2). Even still, minimal contributions of zooxanthellae and coral-associated bacteria should not be dismissed (Fitzgerald & Szmant, Reference Fitzgerald and Szmant1997). In the present study, there was no attempt to remove externally attached bacteria, and in fact, a high amount of bacterial fatty acids has been found in V. cynomorium indicating the presence of a bacterial community (Baptista et al., Reference Baptista, Lopes, Pimentel, Bandarra, Marques and Rosa2012). The existence of considerable amounts of bacterial fatty acids in azooxanthellate octocorals has been proposed to be an adaptation to the absence of symbiotic microalgae (Imbs et al., Reference Imbs, Latyshev, Zhukova and Dautova2007). A similar situation may occur regarding AA biosynthesis. Even if there is no incorporation of bacterial-produced AAs into coral tissues, such AAs could still be found in V. cynomorium as a result of bacteria/bacterial products ingestion or mere presence of bacteria on the corals' surface. So, one should keep in mind that bacterial AA contribution cannot be dismissed.
ACKNOWLEDGEMENT
The Portuguese Foundation for Science and Technology (FCT) supported this study through a Senior Research Position (Ciência 2007) to R.R.
