Lipid metabolism of sea urchin Paracentrotus lividus in two contrasting natural habitats
Results of our research showed that lipid accumulation in sea urchin gonads follows a periodic fluctuation, in agreement with previous observations22. The analysis of Fig. 1 suggests the key role of photoperiod in triggering and then modulating fat utilization and storage mechanisms in P. lividus gonads, while the effect of temperature in gametogenesis and spawning in echinoderms still remains uncertain5,22,28. In fact, a change in photoperiod anticipated the corresponding change in gonad total lipids content in both habitats, while the role of temperature was not very clear, since lipid changes seemed not to be associated with changes in temperature. Most likely, the combined effect of both parameters regulates reproductive cycle of sea urchins. Similar periodical trends in total lipid content were also observed in recent studies on P. lividus gonads19,20 collected in different geographical areas. For example, Rocha et al.20 reported that gonadal lipid content are likely influenced by the environmental conditions characterizing the harvest site in the Praia Norte (Portugal). In this work and in the abovementioned studies, total lipid content in gonads changed as a function of gametogenic cycle, i.e. increased until the recovery/growing stage (I–II) and then progressively decreased until the premature/mature stage (III–IV)27. In another detailed characterization of Arbacia dufresnii, Dìaz de Vivar et al.29 observed a marked dependence of the total lipid content with gonad maturation, with a significant decrease in lipid content in spawned compared to intact gonads, especially in female sea urchins.Assuming P. oceanica and H. scoparia were the main dietary sources of lipids in our study, gonad lipid content was relatively independent from dietary lipid intake, in agreement with data from other authors19,20,22. Indeed, total lipids in P. oceanica and H. scoparia were very low (approximately 1% D.W.) and seasonal variations of lipid levels in these main dietary substrates were definitely negligible. These results are further supported by the literature30,31. For a better understanding of the comparison between different scientific reports19,20,22, it should be recalled here that the displacement of periodical gametogenic cycles is strongly influenced by several environmental factors and is, therefore, dependent on the growing habitat16,32.As far as the commercial value of sea urchin gonads is considered, several reports20,33 suggest that the best harvesting period is when gonads are in the growing stage, when nutrient contents (i.e. proteins, lipids and carbohydrates) are at their highest levels, and when sensorial characteristics are optimal. In fact, gonad maturation decreases the overall quality of roe, and make them more bitter and less pleasant33,34. However, it is striking that very often the official regulation on the harvest of P. lividus in Sardinia allowed collection of sea urchins in the period from November to April, when products are nutrient-poor and in the late stages of gametogenesis (i.e. pre-mature, mature and spawning stages)35.Our observations suggest that total lipids from dietary sources concentrate in the gut. The amount of lipids in these latter samples is actually always much higher than in the sea grass and macroalgae analyzed. The concentration of lipids in the gut has been already observed in other echinoderms as well36,37. These evidences suggest that digestion phenomena occurring in the gut may include the concentration of nutrients. Moreover, our data show that lipid fatty acid composition in gut is considerably consistent, regardless dietary lipid. While further studies are needed, most recent findings strongly suggest that gut flora have a role in assisting digestion and absorption of nutrients in sea urchins38. De novo synthesis of fatty acids by microbiotes, an interesting hypothesis that would especially concern the modulation of short chain fatty acids levels, should be further and specifically investigated. Based on most recent findings, a possible role of bacteria in nutrient production and processing has been postulated39. However, it should be also reckoned that other lipids may come from other dietary sources beyond the main sea grass and macroalgae (“Supplementary Material”). This latter hypothesis, however, would not explain the substantial increase observed in gut lipids, since other possible sources do not have very high lipid contents and were taken in small percentage. For example, it was previously observed in adult Strongylocentrotus intermedius that algal pellets exceeded 80–90% (wet weight) of gut contents, complemented by detritus, small animals (e.g. small crustaceans and mollusks) and non-foods (e.g. sand, shell fragments)40. Moreover, in P. lividus sampled from natural conditions in Corsica (France), 95% of the total gut content was represented by plant material41. Similarly, animal taxa in our study represented a very low percentage of the gut content, and species populating the rocky bottom, other than H. scoparia, have low lipid content and likely had little relevance on sea urchins diet. Also Murillo-Navarro and Jimenez-Guirado25, in a yearlong investigation, found that H. scoparia was the most abundant brown alga in gut contents of P. lividus.Brown algae and leaves of P. oceanica are in fact generally considered among the primary components of adult P. lividus diets1,24,25. It has been also observed by other authors that sea urchins consume all parts of P. oceanica and preferentially green leaves colonised by epiphytes1,26,42,43,44. Epiphytes were not removed from our samples before analysis.The role of gut and stomach as nutrient storage organs is generally acknowledged41,45. This is demonstrated by the almost double lipid contents found in gut than in food sources in the present investigation and by other studies36,37. As a later digestion step, lipids are selectively stored in gonads, where almost three or even four times the lipids contents found in the gut were detected. This supports the hypothesis of lipid relocation from gut to gonads, thus confirming the role of gonads as an important storage tissue for P. lividus, as was previously established by other authors22,46 and correspondingly a role in lipid metabolism can be ascribed to the digestive tract. It also further proves that the amount of fat daily introduced with diet has only a limited influence on the seasonal evolution of total lipids in gonads. Of course, nutrients and especially lipids stored in gonads serve during gametogenesis, as an energy source for developing embryos and are mobilized during pre-feeding development of larvae5. In echinoderms, indeed, nutrients provided in the eggs are needed by developing embryos and larvae.In two recent investigations on P. lividus collected along the Atlantic coast of Portugal, Rocha et al.19,20 evidenced slightly different seasonal trends. They observed both a maximum lipid content and an increase in PUFA content in gonads during the fall season. In contrast, we observed a peak in total lipids during summer, and an increase in PUFA during winter. Likely, the different climatic and environmental conditions of the Atlantic coast with respect to the Mediterranean basin (especially seawater temperatures) induce different gametogenesis cycles16, which in turn modulate the lipid balance in gonads. Gametogenic stages are in fact differently distributed along the year in ours and the cited works by Rocha et al.19,20. In general, lipid content in gonads seem to increase during the recovery (stage I) and growing (stage II) gametogenic stages27, when gonads are packed with nutritive phagocytes and only few germ cells are present.Other studies suggested that specific fatty acids found in the gonads of sea urchins may be synthesized by other tissues such as the intestine and then mobilized to the gonads47.Regardless the different food availability in the two analyzed sites, our results show a remarkable robustness of the fatty acids profile of gut contents. This is particularly interesting since they show a regulation of physiologically essential C 20:5 n-3 and C 20:4 n-6 at gut level, which seem to quite finely level out according to season, regardless the dietary contents of these fatty acids.The increase in gonad PUFA observed in both habitats during winter did not seem to correlate with substantial changes in the main taxa isolated in the gut content of the sea urchin sampled in the P. oceanica meadow, nor to relevant changes in the specimens populating the rocky bottom habitat (“Supplementary Material”). This is consistent with our previous studies21,22, which linked the phenomenon to both the cold acclimatization effect and gametogenesis. Raise in PUFA in lower temperatures allows maintaining cell membrane fluidity and, consequently, supports its functionality.The questions arise whether the lipid species contained in the food sources can be directly and selectively absorbed by sea urchin gonads and how much food habits affect gonads composition. In order to answer these questions, discussion should be directed to each relevant fatty acid.The fatty acids of glycerolipids of higher-plants chloroplasts are highly unsaturated, and the most represented fatty acid is C 18:3 (n-3)48. Instead, brown algae, such as Phaeophyceae, contain a large amount of C 20:4 (n-6) and C 20:5 (n-3)49. During our studies, the most significant difference between the fatty acid profiles of P. oceanica and H. scoparia was related to C 18:3 (n-3). According to our data, P. oceanica contained, on average, more than ten times the amount of this FA in H. scoparia.The fatty acid profile of P. oceanica described in the present study is in agreement with previous reports50,51 and confirms that lipids of P. oceanica are mainly represented by the C 18:3 (n-3), C 18:2 (n-6), and C 16:051. On the contrary, the fatty acid composition of H. scoparia seems to be quite variable considering previously published reports, although literature generally agrees on the most abundant fatty acids (i.e. C 16:0, C 18:2 n-6, C 20:5 n-3 and C 20:4 n-6)31,52.Both in rocky bottom and in P. oceanica meadows, gonadal C 18:3 (n-3) decreased when sea urchins metabolism is mainly influenced by production of gametes (from November), i.e. when gonads reached premature/mature stages, as previously observed15,22. Our data showed a decrease of C 18:3 (n-3) in gut roughly corresponding to an increase of the same FA in gonads (Fig. 4), suggesting that dietary C 18:3 (n-3) was not selectively and directly retained in gonads from the diet, but likely took active part to metabolic processes of bioconversion or is catabolized during β-oxidation of lipids.Also C 18:2 (n-6) showed a similar behaviour in our study and in other previous investigations15,20.Remarkably, C 20:5 (n-3) and C 20:4 (n-6) were the most abundant LC-PUFA in both gut and gonads, in contrast with the composition of the main dietary sources of lipids in the two habitats. In fact, while high percentages of these fatty acids were found in the brown algae H. scoparia, they were present only in very low percentages in the P. oceanica samples. In sea urchins, the fatty acid profile of diet is often scarcely reflected in gut contents and gonads53. From July to March we detected higher percentages of C 20:5 (n-3) in gonad samples collected from P. oceanica meadow than in the corresponding samples from rocky bottom. Moreover, our data clearly show that the C 20:5 (n-3) contained in either gonads and gut does not reflect seasonal variations of this FA in the main sea grass and macroalgae populating the two sites. This result supports earlier observations5,21.Beyond P. oceanica, green algae, especially C. cylindracea, represented additional dietary sources of C 20:5 (n-3) in the P. oceanica meadow. P. lividus usually feeds on brown algae and only less frequently on green algae1,15. In fact, green algae represented less than 5% of the gut content in P. oceanica meadow all year long but from October to December, when they increased from 10 to 25%. In this period, C 20:4 (n-6) and C 20:5 (n-3) in gonads reached their lowest values, but the C 20:5 (n-3) content in gut noticeably increased. After January, when sea urchin reduced feeding in green algae and again less than 5% of green algae was found in the gut content, C 20:5 (n-3) and C 20:4 (n-6) content in sea urchins gut started increasing. To explain this observation, we recall that it was found in S. droebachiensis that dietary FA were not incorporated in sea urchin tissues after short feeding experiments54, but longer experiments allowed to observe diet-related modifications in tissues36. Therefore, it is reasonable to think that nutrients are transferred from gut to gonads. Among other dietary sources of lipids, brown algae in P. oceanica meadow likely did not significantly contribute to increase LC PUFA in gut contents and gonads prior to gametogenesis, being brown algae intake almost always low in the present study.The observed increase of C 20:4 (n-6) in gonads in December was less correlated to the dietary availability of this FA, but was likely associated to cold adaptation and to the growth and maturation of gametes21. In fact, even when the main dietary source of lipids, P. oceanica, was almost completely devoid of this FA, the percentage of C 20:4 (n-6) in gonads was 10–15% and not significant increase of this FA was observed in gut contents from October to December.As for most aquatic consumers, C 20:5 (n-3) and C 20:4 (n-6) can be selectively retained in gonads from dietary sources or accumulated through the conversion of other essential 18-carbon FA.Since we found similar amount of C 20:5 (n-3) C 20:5 (n-3) and C 20:4 (n-6) in P. lividus gonads and gut contents and these values were much higher than in dietary sources, retention or biosynthesis should have occurred already at intestinal level, as previously suggested for other echinoderms36,37,47. As previously hypothesized for Strongylocentrotus intermedius, likely these FA were transferred to gonads after being processed and stored in the digestive tract47.Recently, Kabeya et al.55 found that P. lividus possesses desaturases that are able to convert C 18:3 (n-3) and C 18:2 (n-6) into C 20:5 (n-3) and C 20:4 (n-6), respectively. Han et al.47 characterized the expression of fatty acid desaturases (SiFad1) in different tissues of S. intermedius and concluded that the highest expression is in the intestine, while gonads have lower expression level. Therefore, while retention from diet and biosynthesis from C18 precursors of essential lipid species such as C 20:5 (n-3) and C 20:4 (n-6) might occur already in the gut36,37,41,45, also gonads might possess some, likely lower, biosynthetic functions. Kabeya et al.55 did not specifically quantify the expression of desaturases in different tissues of P. lividus, therefore further research in this sense would be beneficial.It should be mentioned that sex-induced difference of fatty acid profiles of sea urchin gonads were not studied in the present work, but males and females specimens were pooled together. Some previous reports have evidenced differences in lipid classes and fatty acids profiles between sexes15,29, while other studies did not spot statistically significant gender-related discrepancies5. Fatty acids profiles of gonads are likely to be related by sea urchin gender, but it is reasonable to believe that such differences would not disprove the aforementioned considerations on lipid storage and metabolism at gut and at gonad level. In particular, the differences in C 18:3 n-3, C 18:2 n-6, C 20:4 n-6 and C 20:5 n-3 found in previous studies between male and female gonads were quite low (maximum 2–4% of total FAME). Gender differences are ascribable to the increasing presence of lipid-rich gametes (oocytes or sperm) during the gonad maturation period. Also differences in lipid classes are expected in this period, being triglycerides mainly present in female gametes29,56. According to previous reports, during the reproductive period females of both P. lividus and Arbacia lixula showed lower proportions of 20:4n-6, while 20:5n-3 was higher in males of P. lividus and in females of A. lixula56. In P. lividus, such differences were found to be very limited for 20:4n-6 and 20:5n-3 (0.1% and 1.3%, respectively, between mean values of total FAME percentage)56. Also in Arbacia dufresnii the differences between male and female intact gonads for 20:4n-6, while 20:5n-3 were found to be not very important, but both fatty acids seemed to be slightly more concentrated in male tissues29.In any case, the present study confirms that during maturation stages of gonads, when their nutritive content decreases20,22, C 20:5 (n-3) and C 20:4 (n-6) levels increase, and so does their nutritional quality. C 20:5 (n-3) consumption is in fact associated to reduced risk of several chronic diseases57. At the same time, previous reports showed that the best commercial value of sea urchin gonads is before the onset of gametogenesis20,33. These results are quite relevant not only because they allow to deepen the knowledge of the metabolic response of sea urchin P. lividus to season and diet, but also for both improving echinoculture practices and guiding relevant policies directed to regulate the harvest of wild populations. Changes in the concentration of biochemical components in the gonads of sea urchins impact their sensory quality20,33,34. In particular, gonads in their mature stages were described as more bitter34 and of lower quality overall33 than when they are in the growing stage. On the other hand, gonads in the growing stage reach the highest contents of nutrients (protein, fat, carbohydrates)20. Harvest of wild sea urchin during the reproductive time should be avoided, and this is particularly important for an endangered species such as P. lividus. Echinoculture could provide sea urchin roe for which the harvest time should be carefully scheduled as a function of analytical quality parameters and based on expected use.In conclusion, P. oceanica and H. scoparia, primarily constituted P. lividus diet in two contrasting sites within the same geographical area. Green algae, especially C. cylindracea, supplemented sea urchin diet in the P. oceanica meadow prior to gametogenesis, demonstrating the ability of P. lividus to select their diet according to requirements. Total lipid content in gonads changed periodically as a function of gametogenic cycle, being relatively independent from dietary lipid intake and showing a maximum during the growing stage and a minimum in mature gonads. Fatty acid profiles of P. oceanica and H. scoparia were significantly different from each other throughout the year. C 18:3 (n-3) was the main differential dietary marker in P. lividus gonads and gut contents. The main PUFA of P. lividus gonads, C 20:5 (n-3) and C 20:4 (n-6) were associated to increased consumption of green algae in P. oceanica meadow. LC-PUFA were selectively allocated in gonads as a function of reproductive cycle. Conversion of C 18:3 (n-3) to C 20:5 (n-3) and of C 18:2 (n-6) to C 20:4 (n-6) at gut level cannot be excluded, although further research in this sense is desirable. It is worth to note that harvest is generally allowed in Sardinia during gonad maturation, when main nutrients (lipids, carbohydrates, proteins) are at lowest level and also the sensory quality of roe is low, but gonads are rich in healthy LC-PUFA. Our results suggest that rearing of P. lividus would be possible with diets very poor in LC-PUFA given a supplement of this nutrients is provided prior to gametogenesis, when gonads are in the growing/premature stages. More
