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Absorption of different macroalgae by sea cucumber Apostichopus japonicus (Selenka): Evidence from analyses of fatty acid profiles
Aquaculture 451 (2016) 421–428
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Absorption of different macroalgae by sea cucumber Apostichopus japonicus (Selenka): Evidence from analyses of fatty acid profiles Bin Wen a,b, Qin-Feng Gao a,b,⁎, Shuang-Lin Dong a,b, Yi-Ran Hou a,b, Hai-Bo Yu a,b, Xuan Xi c a b c
Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao, Shandong Province 266003, China Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, Shandong Province 266003, China College of Food Science and Engineering, Ocean University of China, Qingdao, Shandong Province 266003, China
a r t i c l e
i n f o
Article history: Received 23 August 2015 Received in revised form 5 October 2015 Accepted 6 October 2015 Available online 8 October 2015 Keywords: Apostichopus japonicus Macroalga Fatty acid Growth performance
a b s t r a c t Fatty acids (FAs) were used as trophic markers to examine the absorption of different algae species, including brown alga Sargassum muticum (S), red alga Gracilaria lemaneiformis (G) and green alga Ulva lactuca (U) by sea cucumber Apostichopus japonicus. A 56-day feeding experiment was carried out to investigate the FA profile of the A. japonicus feeding on 6 different types of diets with the ingredient of either pure powder of single algae species or mixtures of 3 algae species, i.e., the mixtures (1:1) of S. muticum and G. lemaneiformis (SG), S. muticum and U. lactuca (SU), and G. lemaneiformis and U. lactuca (GU). The FA profile of A. japonicus showed obvious changes over the experimental period and was remarkably affected by the different diets. Analyses of variations in the specific FAs and the multidimensional scaling (MDS) ordination of the overall FA profiles revealed that A. japonicus preferentially absorbed U. lactuca relative to S. muticum in the diet group SU while showed the discrimination against G. lemaneiformis in the diet groups SG and GU. Moreover, A. japonicus fed with U. lactuca displayed a similar or higher specific growth rate (SGR) than those fed on S. muticum or G. lemaneiformis, respectively. The results of the present study suggested that green alga U. lactuca might be used as a suitable substitute for replacing the traditional ingredient brown alga Sargassum thunbergii in the artificial feed for A. japonicus farming. Statement of relevance: The results of the present study could be used to further optimize the ingredient of A. japonicus artificial feeds. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Sea cucumber Apostichopus japonicus (Selenka) is one of the most commercially valuable species among 1100 species sea cucumber in the world (Okorie et al., 2008). Aquaculture of A. japonicus has been rapidly extended in China in the past 20 years (Chen, 2004; Xia et al., 2012). The total production of A. japonicus exceeded 201, 000 t in 2014 with a 3.8% increase compared to that in 2013 (MOAC, 2015). As the demand of A. japonicus increases, formulated feeds were widely used to improve the yield of this species. In littoral benthic ecosystems, deposit-feeding sea cucumber tend to take up the sedimentary organic matter containing abundant macroalgae detritus (Uthicke and Karez, 1999; Michio et al., 2003). A 1-yr in situ field study by Sun et al. (2013) indicated that A. japonicus consumed macroalgae as the principal food source throughout the investigation period. In aquaculture practice, algal powder is one of the main ingredients of formulated feeds in intensive and semi-intensive sea cucumber farming (Slater et al., 2009; Liu et al., 2010). ⁎ Corresponding author at: Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao, Shandong Province 266003, China. E-mail address: [email protected] (Q.-F. Gao).
http://dx.doi.org/10.1016/j.aquaculture.2015.10.008 0044-8486/© 2015 Elsevier B.V. All rights reserved.
Brown macroalgae, especially Sargassum thunbergii, is the most popular ingredient for the manufacture of A. japonicus feeds (Seo et al., 2011; Xia et al., 2012). With the rapid expansion of A. japonicus farming, over-harvest of Sargassum spp. has led to the sharp decline in the natural resource of this species and subsequent substantial rise in the price of Sargassum and elevated cost for A. japonicus culture (Gao et al., 2011). Accordingly, it is urgent to seek alternative macroalgae species as feed ingredient to relieve the shortage of S. thunbergii and to lower the cost of A. japonicus farming. Considerable studies have been carried out to examine the effects of various species of brown algae such as Sargassum polycystum, Laminaria japonica and Undaria pinnatifida and red algae such as Gracilaria lemaneiformis on the growth of A. japonicus (Liu et al., 2010; Seo et al., 2011; Gao et al., 2011; Xia et al., 2012). The high yield and easy availability of Ulva lactuca, one of the most popular seaweeds in the north of China, as well as its low price makes it potentially feasible to select this algae species as a new ingredient for the artificial feed of A. japonicus. However, studies on the uptake of green algae including U. lactuca by A. japonicus are scarce to the present (Xia et al., 2012). Feed utilization efficiency in terms of apparent digestibility coefficient (ADC) and feed conversion ratio (FCR) can give insights into digestive capacity and nutrient absorption in aquatic animals (Xia et al., 2012; Yu
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et al., 2015a). Accurate determinations of these parameters generally depend on the time-consuming collection of feed residue and feces. However, due to the nocturnal foraging behavior and the continuous feeding and defecation patterns of A. japonicus as well as the powder nature of the A. japonicus diet, such operations may be considerably difficult or even impossible (Dong et al., 2010; Yu et al., 2015a). Fatty acid (FA) trophic markers, which incorporate the dietary information of animals over longer periods of days and weeks, may be helpful to overcome such uncertainties (Dalsgaard et al., 2003; Budge et al., 2011). Most FAs synthesized by marine macroalgae are taxonomically specific, allowing the chemical discrimination between algae species using FA analyses. The sum of FAs 18:2n-6 and 18:3n-3, for example, is identified as the indicator for green algae, 20:4n-6 for brown algae, and 20:5n-3 for red algae (Meziane and Tsuchiya, 2000; Graeve et al., 2002; Kelly and Scheibling, 2011). FA profiles of animals can be distinctly changed due to the assimilation of different food items, and consequently specific FAs may provide evidence of food sources and dietary preferences of marine animals (Cook et al., 2000; Gao et al., 2006; Redmond et al., 2010; Ezgeta-Balić et al., 2012). The objectives of the present study were to identify the dietary preferences of A. japonicus for 3 different macroalgae using FA profiles and to examine the subsequent effects of food preferences on the growth performance of A. japonicus so as to provide scientific suggestions for optimizing the ingredients of artificial feed in the practice of sea cucumber farming. Three different algae species including brown alga Sargassum muticum, red alga G. lemaneiformis and green alga U. lactuca were used as feed ingredients. Temporal variations in FA compositions of A. japonicus fed on different diets were investigated to elucidate the accumulative effect of dietary FAs and the growth performance between different diet groups was compared. 2. Materials and methods 2.1. Diet preparation Six types of diets which were made of either a single or the mixture of 3 algae species were used in the experiment, i.e., single brown alga S. muticum (S), single red alga G. lemaneiformis (G), single green alga U. lactuca (U), mixture (1:1) of S. muticum and G. lemaneiformis (SG), mixture (1:1) of S. muticum and U. lactuca (SU) and mixture (1:1) of G. lemaneiformis and U. lactuca (GU). Each experimental diet was made of pure macroalgae to eliminate the influence of other ingredients. Dried algae were ground and sieved through a 150 mm mesh, then well mixed, slightly watered, stirred and extruded with a feed processing machine and the algal powder was thus pelletized under the mechanical pressure. Pelleted diets were dried at 60 °C for 36 h and stored at 4 °C for future use.
At the beginning of the experiment, 20 individuals were collected as day 0 samples and stored at − 80 °C for the initial FA analysis. During the acclimation and the following 56-d experiment, sea cucumbers were fed once a day at 15:00 with a daily ration of 5% of the body weight. Water temperature was maintained at 16.5 ± 0.5 °C, salinity ranged from 29 to 31, dissolved oxygen N 6.0 mg L−1 and a photoperiod 14:10 h (L/D) was used. Two-third volume of the water in each aquarium was exchanged with filtered seawater daily. Uneaten feed residue and feces were collected by siphoning at water exchange. 2.3. Sample collection To examine temporal changes in the FA profiles of the A. japonicus body walls over the experimental period, on days 0, 7, 14, 28, 42, and 56, 3 individuals from the same aquarium for each diet group were randomly sampled. Body walls of the collected A. japonicus were cleaned and kept in a freezer at − 80 °C. At the end of the experiment, A. japonicus individuals in the rest of the aquarium were starved for 48 h, then weighed, dissected and kept in a freezer at −80 °C for further analysis. 2.4. Fatty acid analysis Samples of freeze-dried body walls and algae ingredients were ground into fine powder using a tissue grinder. Total lipids were extracted with a chloroform/methanol mixture (2:1) according to the method of Bligh and Dyer (1959). Fatty acid methyl esters (FAMEs) were prepared by esterification using 2% sulphuric acid methanol as described in Gao et al. (2006). FAMEs were quantified with a gas chromatography (Shimadzu GC-2010 plus) equipped with an auto-sampler and a flame ionization detector (FID) instrument (GC-2010; Shimadzu, Kyoto, Japan) using an RTX-WAX fused silica capillary column (30 m × 0.25 mm × 0.25 μm), which was operated using following the temperature programs: 60°C for 1.0 min, rate of 10 °C/min to 190 °C, 2.0 °C/min to 236 °C and 236 °C for 2 min. The carrier gas was nitrogen and the linear velocity was 33.1 cm/s and the split ratio was 1:15. FAMEs were identified by comparing their retention time with those of the standards: 37-FAME Mix and 26-Bacterial Acid Methyl Esters Mix (Supelco, Bellefonte, PA, USA). 2.5. Calculation and statistical analysis The growth performance of A. japonicus in terms of specific growth rate (SGR) was calculated as: −1 SGR %d ¼ ð lnW f − ln Wi Þ=t 100
2.2. Feeding experiment The experiment was conducted at the laboratory of Qingdao National Ocean Scientific Research Center, Ocean University of China. The experimental A. japonicus juveniles were collected from a local commercial farm. Prior to the experiment, A. japonicus juveniles were cultured in 1000 L fiberglass tanks and were acclimatized for 14 days to the laboratory conditions. During the acclimatization period, sea cucumbers were fed with commercial feed which was used in the farm where the sea cucumber were collected so as to avoid the possible changes in the fatty acid (FA) profiles of A. japonicus before the beginning of the experiment. After 48 h starvation, a total of 216 acclimatized individuals with initial body weights of 5.57 ± 0.11 g were randomly selected and transferred into 36 glass aquaria (50 × 40 × 40 cm) in each of which 6 individuals were cultured. The 36 aquaria were further divided into 6 groups with 6 replicates for each group. Sea cucumbers in each of the 6 groups were fed with one of the 6 diets as described above, respectively.
where Wf and Wi are the final and initial weights of A. japonicus individuals in each aquarium, respectively and t is the duration of the experiment in days. To investigate the variations in overall FA profiles between different algae species and those between A. japonicus cultured with different diets, analyses of multidimensional scaling (MDS) were conducted based on the Euclidean distance resemblance matrix of the FA percentage after fourth root transformation. The differences in individual FAs and growth performance between the 6 diet groups were compared with one way analysis of variance (ANOVA) followed by a Tukey test for multiple comparisons at the significance level of 0.05 (p b 0.05). Prior to statistical analyses, raw data were diagnosed for normality of distribution and homogeneity of variance with the Kolmogorov– Smirnov test and Levene's test, respectively. The data were analyzed by the Primer 6.0 (Clarke and Gorley, 2006) and statistical software SPSS for Windows (Release 20.0).
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3. Results 3.1. Fatty acid profiles of feed ingredients Fatty acid (FA) compositions of the macroalgae used as feed ingredients including S. muticum, G. lemaneiformis and U. lactuca are presented in Table 1. FAs generally showed significant differences between the 3 algae species, particularly for the FAs which were used as trophic markers such as 18:1n-7, 18:2n-6, 20:4n-6 and 20:5n-3. For example, concentrations of 18:1n-7 and 18:2n-6 in U. lactuca were higher than those in S. muticum and G. lemaneiformis. Level of 20:4n-6 in S. muticum was higher compared to those in G. lemaneiformis and U. lactuca. G. lemaneiformis was characterized by higher levels of 20:5n-3 relative to S. muticum and U. lactuca. However, all 3 algae species contained trace amounts of 22:6n-3. Multidimensional scaling (MDS) analysis showed that the FA signatures of the macroalgae were classified into 3 separate groups in the MDS ordination plots (Fig. 1), indicating the substantial differences in the FA profiles between the 3 algae species. 3.2. Fatty acid compositions of A. japonicus Temporal changes in concentrations of the typical FA biomarkers including 18:1n-7, 18:2n-6, 20:4n-6 and 20:5n-3 in A. japonicus fed with different diets over the 56-d experimental period are shown in Fig. 2. During the experimental period, levels of 18:1n-7 for A. japonicus in the diet groups S and SG decreased while increased in the other diet groups (ANOVA, p b 0.05) (Fig. 2a). As shown in Fig. 2b, changes in the 18:2n-6 concentrations of A. japonicus in the diet group G were quite irregular and did not show any clear tendency over the experimental period while for other 5 diet groups, concentrations of 18:2n-6 showed continuous increases with the extension of the experimental period (ANOVA, p b
Table 1 Fatty acid profiles (% total fatty acids) of Sargassum muticum, Gracilaria lemaneiformis and Ulva lactuca which were used as feed ingredients in the experiment. Fatty acids in bold are those used as trophic markers for different algae. See text for the details. Data were presented as mean ± SD (n = 3). ND = not detected. Fatty acid
G. lemaneiformis
U. lactuca
Saturated fatty acids 14:0 6.07 ± 0.02 15:0 0.33 ± 0.00 i-15:0 0.93 ± 0.01 16:0 47.97 ± 0.09 17:0 0.46 ± 0.02 18:0 1.10 ± 0.01 20:0 0.66 ± 0.02 22:0 0.81 ± 0.01 24:0 0.49 ± 0.03 Subtotal 58.82 ± 0.08
S. muticum
4.99 ± 0.03 0.99 ± 0.02 0.42 ± 0.02 50.14 ± 0.17 1.48 ± 0.01 4.52 ± 0.05 0.59 ± 0.02 0.32 ± 0.00 0.33 ± 0.01 63.79 ± 0.15
3.75 ± 0.45 0.58 ± 0.02 0.69 ± 0.06 53.99 ± 0.35 0.72 ± 0.02 2.13 ± 0.12 0.78 ± 0.04 1.19 ± 0.39 0.75 ± 0.03 64.57 ± 0.86
Monounsaturated fatty acids 14:1n-5 0.27 ± 0.01 16:1n-7 7.71 ± 0.02 16:1n-9 1.27 ± 0.02 17:1n-9 0.35 ± 0.01 18:1n-7 1.16 ± 0.02 18:1n-9 9.80 ± 0.02 20:1n-9 3.32 ± 0.01 22:1n-9 0.34 ± 0.01 Subtotal 24.22 ± 0.06
0.33 ± 0.00 6.30 ± 0.03 0.76 ± 0.02 0.53 ± 0.01 4.33 ± 0.02 10.64 ± 0.08 2.03 ± 0.02 ND 24.93 ± 0.13
0.70 ± 0.02 4.57 ± 0.24 1.80 ± 0.10 0.97 ± 0.01 11.55 ± 0.04 2.69 ± 0.05 0.67 ± 0.11 ND 22.95 ± 0.38
Polyunsaturated fatty acids 18:2n-6 1.42 ± 0.03 18:3n-6 2.92 ± 0.01 18:3n-3 0.61 ± 0.00 20:2n-6 0.27 ± 0.02 20:3n-3 0.26 ± 0.01 20:4n-6 9.02 ± 0.02 20:5n-3 2.24 ± 0.04 22:6n-3 0.22 ± 0.01 Subtotal 16.96 ± 0.04
0.90 ± 0.01 0.80 ± 0.02 0.55 ± 0.01 0.39 ± 0.00 0.22 ± 0.01 0.89 ± 0.00 7.41 ± 0.05 0.13 ± 0.05 11.28 ± 0.02
6.57 ± 0.08 0.91 ± 0.03 3.04 ± 0.11 ND ND 0.72 ± 0.09 1.46 ± 0.12 ND 12.70 ± 0.09
Fig. 1. Multidimensional scaling (MDS) ordination with embedded Euclidean distance for the fatty acid profiles of the 3 algae species. S = Sargassum muticum, G = Gracilaria lemaneiformis and U = Ulva lactuca.
0.05). As for the level of 20:4n-6, it showed increase in A. japonicus in the diet group S and exhibited remarkable decreases in the diet groups G, U, SU and GU (ANOVA, p b 0.05). In the diet group SG, however, the concentration of 20:4n-6 kept relatively stable during the 56-d experimental period (ANOVA, p N 0.05) (Fig. 2c). For level of 20:5n-3 in A. japonicus, remarkable increase occurred in the diet group G but decreased in the other diet groups (ANOVA, p b 0.05) (Fig. 2d). As shown in Fig. 3a, ordinations of MDS analysis based on the overall FA profiles of A. japonicus revealed that the FA compositions of A. japonicus in the 3 diet groups of single macroalgae (diet S, G and U) tended to approach the FA profiles of corresponding diets from the beginning of the experiment (A0) to the end of the experiment (A-S56, A-G56 and AU56 for diets S, G and U, respectively), indicating the assimilation of FAs from S. muticum, G. lemaneiformis and U. lactuca by A. japonicus in the 3 diet groups, respectively. Fig. 3b illustrated the temporal changes in the FA compositions of the A. japonicus in the 3 mixture diet groups (diet groups SG, SU and GU) over the experimental period. In diet group SG, points in the MDS ordination plot representing the FA profiles of A. japonicus exhibited the tendency to approach those of S. muticum relative to G. lemaneiformis. While for diet groups SU and GU, MDS ordination suggested that A. japonicus assimilated FAs with the preference for U. lactuca vs S. muticum or G. lemaneiformis. FA compositions of the A. japonicus cultured with the 6 diets at the end of the experiment are shown in Table 2. Significant differences in the concentrations of 18:1n-7, 18:2n-6, 20:4n-6, 20:5n-3 and 22:6n-3 between the 6 diet groups were observed (ANOVA, p b 0.05). After 56 days of the feeding trial, the highest content of 18:1n-7 was detected in the diet group U while the lowest was found in the group SG. The highest proportion of 18:2n-6 was observed in the diet group SU while the lowest was found in the group G. A. japonicus in the diet groups S and G obtained the highest levels of 20:4n-6 and 20:5n-3, respectively. Concentrations of 22:6n-3 of A. japonicus in the diet group GU were significantly lower than those in other groups (ANOVA, p b 0.05) and no significant differences were observed between other groups (ANOVA, p N 0.05). Ordinations of MDS analysis based on the overall FA profiles of A. japonicus cultured with the 6 diets at the end of the experiment (A-S56, A-G56, A-U56, A-SG56, A-SU56 and A-GU56 for the 6 diet groups, respectively) are shown in Fig. 4. FA compositions of A. japonicus in all diet groups appeared obvious changes at the end of the experiment compared with the initial FA profiles of A. japonicus (A0). The positions of the points representing A. japonicus in different diet groups relative to those representing the 3 macroalgal species indicated the effects of dietary FAs on the FA profiles of A. japonicus. In the MDS ordination plot, the positions of the points representing the A. japonicus in diet groups S (A-S56), G (A-G56) and U (A-U56) were basically consistent with the points representing the 3 algae species S. muticum (S), G. lemaneiformis (G) and U. lactuca (U). For the 3 mixture diet groups, the sequence of
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Fig. 2. Temporal changes in fatty acid biomarkers of Apostichopus japonicus fed with different diets. (a) 18:1n-7, (b) 18:2n-6, (c) 20:4n-6 and (d) 20:5n-3. Data were presented as mean ± SD (n = 3). Different letters in each diet group mean significant differences (ANOVA with Tukey test for multiple comparisons, p b 0.05).
the points representing the FA profiles of A. japonicus in diet groups SG (A-SG56), SU (A-SU56) and GU (A-GU56) were also consistent with the sequence of those representing the 3 macroalgal species S. muticum (S), G. lemaneiformis (G) and U. lactuca (U). Moreover, the points representing the FA profiles of A. japonicus in the 3 mixture diet groups (A-SG56, A-SU56 and A-GU56) were all located within the points representing the FA profiles of A. japonicus in the 3 single algae diet groups (A-S56, A-U56 and A-U56), suggesting the simultaneous absorption of 2 macroalgal species by A. japonicus in the 3 mixture diet groups. 3.3. Growth performance Growth performance of A. japonicus in the 6 diet groups are shown in Table 3. At the beginning of the experiment, there were no significant differences in the initial body weights (IBW) of A. japonicus between the 6 diet groups (ANOVA, p N 0.05). In contrast, after the 56-d feeding trial, significant differences in the final body weight (FBW) and specific growth rate (SGR) were observed between the 6 diet groups (ANOVA, p b 0.05), with the highest FBW and SGR in the diet group SG and the lowest in the diet group SU. Moreover, the FBW and SGR of A. japonicus in the diet group G was significantly lower than those
in the diet groups S and U (ANOVA, p b 0.05) while no significant differences were observed between the diet groups S and U (ANOVA, p N 0.05). 4. Discussion Results of the present study showed the temporal changes in fatty acid (FA) compositions of A. japonicus feeding on 6 different diets, suggesting that the FA profiles of A. japonicus were remarkably affected by the FA components of the diets (Hasegawa et al., 2014; Yu et al., 2015b). Accordingly, analyses for the transfer and accumulation of dietary FAs into the tissues of A. japonicus might reveal dietary preferences between different algae species. Syntheses of FAs by aquatic macroalgae are generally taxonomically specific. Arachidonic acid (20:4n-6), for example, is generally selected as the indicator for brown algae, eicosapentaenoic acid (20:5n-3) for red algae and the sum of linoleic acid (18:2n-6) and linolenic acid (18:3n-3) for green algae (Meziane and Tsuchiya, 2000; Graeve et al., 2002; Kelly and Scheibling, 2011). In the present study, brown alga S. muticum, red alga G. lemaneiformis and green alga U. lactuca were characterized by high levels of 20:4n-6, 20:5n-3 and 18:2n-6,
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Fig. 3. Multidimensional scaling (MDS) ordination of the fatty acid profiles for Apostichopus japonicus and the dietary macroalgae. S = Sargassum muticum, G = Gracilaria lemaneiformis and U = Ulva lactuca and A0 = A. japonicus at the beginning of the experiment (day 0). A–S, A–G, A–U, A–SG, A–SU and A–GU = A. japonicus fed with S, G, U SG, SU and GU, respectively, and 7, 14, 28, 42 and 56 mean the experimental period in days on which the sample A. japonicus were collected.
respectively, while U. lactuca was more enriched in the concentration of cis-vaccenic acid (18:1n-7) relative to other algae species. However, despite of relative low concentrations of 18:2n-6 and 18:1n-7 in the S. muticum and G. lemaneiformis, respectively, A. japonicus in the diet groups S and G showed increases in these two FAs, respectively. This might lead to misunderstanding the food sources in the mixed diet groups (groups SU and GU). In contrast, A. japonicus fed with S. muticum and G. lemaneiformis did not appear obvious increases in 18:1n-7 and 18:2n-6, respectively. Thus 18:1n-7 and 20:4n-6 could be used to distinguish U. lactuca and S. muticum while 18:2n-6 and 20:5n-3 could potentially be used to distinguish U. lactuca and G. lemaneiformis. Accordingly, 20:4n-6 was selected as the trophic marker for S. muticum, 20:5n-3 for G. lemaneiformis and 18:2n-6 and/or 18:1n-7 for U. lactuca. Multidimensional scaling (MDS) analysis showed the substantial differences in the FA patterns between the 3 algae species, indicating the feasibility of using FA profiles as trophic markers to trace the preferential absorption between multiple macroalgae species by A. japonicus.
After 56 days of the feeding trial, A. japonicus showed substantial weight gain, thus changes in FA profiles of A. japonicus might be attributable to the dilution process, rather than the result of FA turnover (Robin et al., 2003; Jobling, 2004; Budge et al., 2011). For the single diet groups, owing to the effects of FA compositions of S. muticum, G. lemaneiformis and U. lactuca, respectively, A. japonicus showed remarkable increases in 20:4n-6 and 18:2n-6 in the group S, 20:5n-3 and 18:1n-7 in the group G and 18:1n-7 and 18:2n-6 in the group U. Moreover, as illustrated in the ordinations of MDS analysis, the overall FA profiles of A. japonicus changed in the predictable manner that gradually resembled the FA profiles of the diets. As for A. japonicus cultured with mixed diets, analyses of variations in the specific FAs and the MDS ordination of the overall FA profiles revealed the dietary preferences between the 3 algae species. A. japonicus fed with the mixture of S. muticum and G. lemaneiformis showed remarkable increases in 20:4n-6 and 18:2n-6 while decreases in 20:5n-3 and 18:1n-7, indicating that A. japonicus accumulated more S. muticum against G. lemaneiformis. As for the A. japonicus cultured with the mixture of S. muticum and
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Table 2 Fatty acid compositions (mg g−1, dry weight basis) of Apostichopus japonicus fed with different diets at the end of the experiment (day 56). Fatty acids in bold are those used as trophic markers for different algae. See text for the details. Data were presented as mean ± SD (n = 3). Different letters in the same row mean significant differences (ANOVA with Tukey test for multiple comparisons, p b 0.05). ND = not detected. See text for detailed description of diets S, G, U, SG, SU, and GU. Fatty acids Saturated fatty acids 14:0 15:0 i-15:0 16:0 17:0 18:0 20:0 22:0 24:0 Subtotal
Diet S 0.34 ± 0.01 0.2 ± 0.01 ND 4.14 ± 0.06 0.23 ± 0.01 2.28 ± 0.09 0.80 ± 0.01 0.74 ± 0.01 0.13 ± 0.00 8.86 ± 0.14
Diet G
Diet U
Diet SG
Diet SU
Diet GU
0.76 ± 0.01 0.52 ± 0.01 0.21 ± 0.01 4.51 ± 0.10 0.54 ± 0.06 1.60 ± 0.05 0.45 ± 0.01 0.35 ± 0.02 0.14 ± 0.01 9.07 ± 0.06
0.42 ± 0.01 0.32 ± 0.01 0.16 ± 0.01 3.94 ± 0.12 0.83 ± 0.09 1.45 ± 0.04 0.67 ± 0.01 0.68 ± 0.02 0.16 ± 0.01 8.64 ± 0.21
0.41 ± 0.01 0.26 ± 0.01 0.11 ± 0.01 4.58 ± 0.01 0.36 ± 0.04 2.21 ± 0.04 0.66 ± 0.01 0.61 ± 0.02 0.14 ± 0.01 9.34 ± 0.07
0.41 ± 0.01 0.23 ± 0.01 0.11 ± 0.01 3.99 ± 0.04 0.41 ± 0.03 2.06 ± 0.09 0.69 ± 0.03 0.75 ± 0.04 0.15 ± 0.01 8.8 ± 0.21
0.56 ± 0.07 0.35 ± 0.04 0.19 ± 0.04 4.83 ± 0.32 0.54 ± 0.03 1.41 ± 0.08 0.63 ± 0.02 0.66 ± 0.04 0.17 ± 0.02 9.35 ± 0.41
Monounsaturated fatty acids 14:1n-5 0.29 ± 0.01 16:1n-7 2.1 ± 0.04 16:1n-9 0.11 ± 0.00 17:1n-9 0.11 ± 0.01 18:1n-7 1.65 ± 0.02a 18:1n-9 4.95 ± 0.03 20:1n-9 1.84 ± 0.01 22:1n-9 0.66 ± 0.01 Subtotal 11.72 ± 0.08
0.44 ± 0.01 3.62 ± 0.04 0.25 ± 0.01 0.27 ± 0.01 3.4 ± 0.01c 4.64 ± 0.01 1.65 ± 0.04 1.01 ± 0.14 15.3 ± 0.12
0.16 ± 0.00 3.51 ± 0.07 0.16 ± 0.01 0.43 ± 0.02 4.72 ± 0.16d 3.09 ± 0.03 1.68 ± 0.02 0.92 ± 0.19 14.67 ± 0.15
0.37 ± 0.01 2.83 ± 0.02 0.14 ± 0.01 0.17 ± 0.01 2.14 ± 0.01b 4.98 ± 0.05 1.84 ± 0.01 0.74 ± 0.02 13.2 ± 0.12
0.22 ± 0.01 2.79 ± 0.14 0.13 ± 0.01 0.24 ± 0.03 3.17 ± 0.15c 3.62 ± 0.10 1.51 ± 0.02 0.71 ± 0.03 12.39 ± 0.29
0.26 ± 0.04 3.10 ± 0.16 0.19 ± 0.03 0.27 ± 0.01 3.42 ± 0.05c 3.19 ± 0.06 1.30 ± 0.04 0.88 ± 0.21 12.61 ± 0.12
Polyunsaturated fatty acids 18:2n-6 0.58 ± 0.01b 18:3n-6 0.18 ± 0.01 18:3n-3 0.97 ± 0.01 20:2n-6 0.84 ± 0.06 20:3n-3 0.29 ± 0.01 20:4n-6 3.02 ± 0.08c 20:5n-3 1.39 ± 0.02a 22:6n-3 1.16 ± 0.01b Subtotal 8.42 ± 0.05
0.35 ± 0.03a 0.28 ± 0.01 0.82 ± 0.01 0.79 ± 0.03 0.27 ± 0.01 1.22 ± 0.02a 2.39 ± 0.08d 1.04 ± 0.04b 7.15 ± 0.17
0.71 ± 0.02c 0.44 ± 0.01 0.73 ± 0.01 0.59 ± 0.01 0.45 ± 0.01 1.70 ± 0.03b 1.68 ± 0.08c 1.00 ± 0.04b 7.29 ± 0.06
0.54 ± 0.01b 0.26 ± 0.01 0.88 ± 0.01 0.75 ± 0.01 0.27 ± 0.01 2.94 ± 0.05c 1.69 ± 0.07c 1.13 ± 0.03b 8.46 ± 0.14
U. lactuca and mixture of G. lemaneiformis and U. lactuca, the increases in the concentrations of 18:2n-6 and 18:1n-7 and the decreases in those of 20:4n-6 and 20:5n-3 suggested that A. japonicus preferentially absorbed U. lactuca relative to S. muticum and G. lemaneiformis. Furthermore, MDS ordination suggested that the points representing the FA profiles of A. japonicus showed the tendency to approach those of preferred algae species, confirming the preferential utilization of S. muticum by sea cucumber in the diet group SG and U. lactuca in the diet groups SU and GU. Such results are consistent with the previous findings that A. japonicus took up less G. lemaneiformis relative to S. thunbergii when offered the mixture of these two algal powder (Gao et al., 2011). The selective absorption between various algal sources by animals could be attributed to the intrinsic properties of different algae species,
Fig. 4. Multidimensional scaling (MDS) ordination with embedded Euclidean distance of the fatty acid profiles of algae species and Apostichopus japonicus at the end of the experiment (day 56). S = Sargassum muticum, G = Gracilaria lemaneiformis and U = Ulva lactuca and A0 = A. japonicus on day 0. A-S56, A-G56, A-U56, A-SG56, A-SU56 and A-GU56 = A. japonicus in the diet groups S, G, U SG, SU and GU on day 56, respectively.
0.93 ± 0.04e 0.30 ± 0.01 0.86 ± 0.02 0.70 ± 0.02 0.35 ± 0.01 1.79 ± 0.03b 1.43 ± 0.03ab 1.14 ± 0.06b 7.51 ± 0.06
0.84 ± 0.03d 0.32 ± 0.01 0.62 ± 0.02 0.42 ± 0.04 0.38 ± 0.02 1.22 ± 0.12a 1.56 ± 0.06bc 0.80 ± 0.15a 6.15 ± 0.34
for instance, physical characteristics of algae such as toughness and calcification (Pennings et al., 1998; Gao et al., 2011), cell wall structure (Zemke-White et al., 2000; Shi et al., 2013) and nutritional content such as fiber (Zamora and Jeffs, 2011; Xia et al., 2012). A. japonicus tended to reject G. lemaneiformis, probably due to the harder texture and subsequent lower digestibility of this species relative to U. lactuca and S. muticum (Gao et al., 2011). Moreover, sea cucumber species generally have limited ability to produce endogenous cellulase and the digestion of cellulose mainly depends on beneficial microflora present in the digestive tract (Yingst, 1976; Ward-Rainey et al., 1996; Yuan et al., 2006). As a result, the digestion and absorption of G. lemaneiformis by A. japonicus is limited. As essential fatty acids (EFAs), n-3 and n-6 polyunsaturated fatty acids (PUFAs), e.g., ARA (20:4n-6), EPA (20:5n-3) and DHA (22:6n-3) are generally obtained from diets and play important roles in the physiological functions of marine animals such as egg and larval quality, immune function and membrane permeability (Cook et al., 2000; Bell and Sargent, 2003; Paulsen et al., 2014). However, certain marine invertebrates are capable of synthesizing long-chain FAs from shorter chain precursors, for example, converting 18:2n-6 to 20:4n-6 and 18:3n-3 to 20:5n-3 and/or 22:6n-3 (Bell et al., 2001; González-Durán et al., 2008). In spite of the fact that both U. lactuca and G. lemaneiformis contained trace amounts of 20:4n-6, A. japonicus fed with single U. lactuca accumulated more 20:4n-6 compared to those fed on single G. lemaneiformis. Similarly, although level of 20:5n-3 in U. lactuca was lower relative to that in S. muticum, A. japonicus in the diet group U showed a significantly higher proportion of 20:5n-3 than that in the diet group S. As U. lactuca was richer in 18:2n-6 and 18:3n-3 relative to S. muticum and G. lemaneiformis, these results might be explained by that A. japonicus was able to convert 18:2n-6 to 20:4n-6 and 18:3n3 to 20:5n-3. Hasegawa et al. (2014) and Yu et al. (2015b) also demonstrated that A. japonicus had the potential to synthesize 20:4n-6 and
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Table 3 Growth performance of Apostichopus japonicus fed with different diets. Data were presented as mean ± SD (n = 3). Different letters in the same row mean significant differences (ANOVA with Tukey test for multiple comparisons, p b 0.05). IBW = initial body weight, FBW = final body weight, SGR = specific growth rate (% d−1). See text for detailed description of diets S, G, U, SG, SU, and GU.
IBW FBW SGR
Diet S
Diet G
Diet U
Diet SG
Diet SU
Diet GU
5.57 ± 0.16 12.23 ± 1.06cd 1.40 ± 0.21b
5.62 ± 0.10 9.65 ± 1.26ab 0.96 ± 0.26a
5.52 ± 0.21 13.68 ± 1.12cd 1.61 ± 0.09b
5.60 ± 0.05 14.10 ± 1.48d 1.64 ± 0.18b
5.49 ± 0.02 9.07 ± 0.76a 0.89 ± 0.15a
5.61 ± 0.11 11.67 ± 1.04bc 1.30 ± 0.18b
20:5n-3 using the precursors with shorter carbon chains. As such, the requirements for EFAs may be met by a combination of dietary 18:2n6 and 18:3n-3. Although A. japonicus fed both single G. lemaneiformis and U. lactuca showed higher levels of 20:5n-3 compared with those fed with single S. muticum, comparable concentration of 22:6n-3 was recorded in the latter diet group, indicating that further elongation and desaturation of 20:5n-3 to 22:6n-3 might not happen in A. japonicus (Yu et al., 2015b). In general, monounsaturated fatty acids (MUFAs) are used as energy reserves in marine animals, and 18:1n-7 is selectively catabolized as the most dominant energy source during starvation (Smith et al., 2003; Mourente et al., 2005). Previous studies showed that large amounts of energy intake were used for metabolism in A. japonicus (Yuan et al., 2006; Liu et al., 2009). A. japonicus fed on single S. muticum showed decrease in 18:1n-7 due to the low level of this FA in S. muticum, suggesting that A. japonicus utilized 18:1n-7 for routine metabolism. However, if A. japonicus were fed with single U. lactuca or G. lemaneiformis which was richer in 18:1n-7 relative to S. muticum, they could use this FA as energy source in addition to maintaining the normal metabolism. Accordingly, elevated concentrations of 18:1n-7 were recorded in these two diet groups. As for the growth performance of the A. japonicus over the 70-d experimental period, A. japonicus in the diet group U showed a similar or higher specific growth rate (SGR) compared to specimens in the diet groups S or G, respectively. Such results are consistent with previous studies where A. japonicus feeding on U. lactuca showed a better growth performance than those fed with S. thunbergii (Xia et al., 2012). A. japonicus fed the mixture of S. muticum and G. lemaneiformis (diet SG) which was rich in both 20:4n-6 and 20:5n-3 showed the highest SGR, suggesting that these two FAs were vital for the growth of A. japonicus. The second highest SGR of A. japonicus in the diet group U might support the hypothesis that the requirements for 20:4n-6 and 20:5n-3 could be met by the combination of dietary 18:2n-6 and 18:3n-3. Thus, from the perspective of FAs, high concentrations of 18:1n-7, 18:2n-6 and 18:3n-3 in U. lactuca might account for the improved growth of A. japonicus feeding this alga. Despite of relative preferences for S. muticum and U. lactuca to G. lemaneiformis, A. japonicus fed the mixture of S. muticum and U. lactuca (diet SU) showed the lowest SGR. Moreover, A. japonicus in the diet group SU contained the lowest concentrations of 20:5n-3 indicating the insufficient supply of 20:5n-3 for the sea cucumber cultured with the mixture of S. muticum and U. lactuca, probably due to the low content of 20:5n-3 in both S. muticum and U. lactuca and due to the further dilution of 20:5n-3 when S. muticum and U. lactuca were mixed. These results might suggest that 20:5n-3 was more important to the growth of A. japonicus. Bell et al. (2001) reported that another echinoderm species sea urchin Psammechinus miliaris (Gmelin) was capable of biosynthesizing 20:5n3 from its precursor 18:3n-3. The conversion rate, which was equivalent to 0.09 μg/g tissue/mg 18:3n-3 eaten over 14 days, however, was quite low. Similarly, in the present study, even though A. japonicus had the potential to convert 18:3n-3 to 20:5n-3, it is possible that the capacity for endogenous production of 20:5n-3 may be inefficient to meet the requirements for the growth of sea cucumber. Moreover, S. muticum contained trace amounts of 18:3n-3 and the mixture of S. muticum with U. lactuca diluted the 18:3n-3 concentration of U. lactuca, leading to the reduction in the availability of 18:3n-3 for bioconversion of
18:3n-3 to 20:5n-3 by A. japonicus. Although G. lemaneiformis was rich in 20:5n-3, A. japonicus in the diet group G showed the second lowest SGR. In addition to the FAs, the growth of A. japonicus could be affected by other nutrients such as amino acids, carbohydrates and vitamins (Okorie et al., 2008; Zhao et al., 2011). The poor growth performance of A. japonicus in the diet group G might be explained by that A. japonicus could not utilize G. lemaneiformis effectively due to the low digestibility of this alga, affecting the absorption of other nutritional components by A. japonicus. In conclusion, results of the present study indicated that the FA profiles of A. japonicus were affected by the various FA components of different macroalgae species in the diets, and A. japonicus preferentially absorbed U. lactuca relative to S. muticum and tended to discriminate against G. lemaneiformis when cultured with the mixture of G. lemaneiformis and U. lactuca or and S. muticum. Moreover, A. japonicus fed with U. lactuca showed a similar or better growth performance than those fed on S. muticum or G. lemaneiformis, respectively. The green alga U. lactuca which is one of the most dominant seaweed species in northern China could therefore be utilized as a suitable substitute to replace the traditional ingredient S. thunbergii in the artificial feed for A. japonicus farming. Acknowledgements This study was funded by the National Natural Science Foundation of China (Grant Nos. 31172426 & 31372549) and the Ministry of Science and Technology of China (Grant No. 2011BAD13B03). References Bell, J.G., Sargent, J.R., 2003. Arachidonic acid in aquaculture feeds: current status and future opportunities. Aquaculture 218, 491–499. Bell, M.V., Dick, J.R., Kelly, M.S., 2001. Biosynthesis of eicosapentaenoic acid in sea urchin Psammechinus miliaris. Lipids 36, 79–92. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Budge, S.M., Penney, S.N., Lall, S.P., 2011. Response of tissue lipids to diet variation in Atlantic salmon (Salmo salar): implications for estimating diets with fatty acid analysis. J. Exp. Mar. Biol. Ecol. 409 (1), 267–274. Chen, J.X., 2004. Present status and prospects of sea cucumber industry in China. In: Lovatelli, A., Conand, C., Purcell, S., Uthicke, S., Hamel, J.F., Mercier, A. (Eds.), Advances in Sea Cucumber Aquaculture and Management 463. FAO, Rome, pp. 25–38. Clarke, K., Gorley, R.N., 2006. Primer v6: User Manual/Tutorial. PRIMER-E, Plymouth. Cook, E.J., Bell, M.V., Black, K.D., Kelly, M.S., 2000. Fatty acid composition of gonadal material and diets of the sea urchin, Psammechinus miliaris: trophic and nutritional implications. J. Exp. Mar. Biol. Ecol. 255, 261–274. Dalsgaard, J., St John, M., Kattner, G., Muller-Navarra, D., Hagen, W., 2003. Fatty acid trophic markers in the pelagic marine environment. Adv. Mar. Biol. 46, 225–340. Dong, G., Dong, S., Wang, F., Tian, X., 2010. Effects of light intensity on daily activity rhythm of juvenile sea cucumber, Apostichopus japonicus (Selenka). Aquac. Res. 41 (11), 1640–1647. Ezgeta-Balić, D., Najdek, M., Peharda, M., Blažina, M., 2012. Seasonal fatty acid profile analysis to trace origin of food sources of four commercially important bivalves. Aquaculture 334, 89–100. Gao, Q.F., Shin, P.K.S., Lin, G.H., Chen, S.P., Cheung, S.G., 2006. Stable isotope and fatty acid evidence for uptake of organic waste by green-lipped mussels Perna viridis in a polyculture fish farm system. Mar. Ecol. Prog. Ser. 317, 273–283. Gao, Q.F., Wang, Y., Dong, S., Sun, Z., Wang, F., 2011. Absorption of different food sources by sea cucumber Apostichopus japonicus (Selenka) (Echinodermata: Holothuroidea): evidence from carbon stable isotope. Aquaculture 319, 272–276. González-Durán, E., Castell, J.D., Robinson, S.M.C., Blair, T.J., 2008. Effects of dietary lipids on the fatty acid composition and lipid metabolism of the green sea urchin Strongylocentrotus droebachiensis. Aquaculture 276, 120–129.
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Absorption of different macroalgae by sea cucumber Apostichopus japonicus (Selenka): Evidence from analyses of fatty acid profiles Bin Wen a,b, Qin-Feng Gao a,b,⁎, Shuang-Lin Dong a,b, Yi-Ran Hou a,b, Hai-Bo Yu a,b, Xuan Xi c a b c
Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao, Shandong Province 266003, China Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, Shandong Province 266003, China College of Food Science and Engineering, Ocean University of China, Qingdao, Shandong Province 266003, China
a r t i c l e
i n f o
Article history: Received 23 August 2015 Received in revised form 5 October 2015 Accepted 6 October 2015 Available online 8 October 2015 Keywords: Apostichopus japonicus Macroalga Fatty acid Growth performance
a b s t r a c t Fatty acids (FAs) were used as trophic markers to examine the absorption of different algae species, including brown alga Sargassum muticum (S), red alga Gracilaria lemaneiformis (G) and green alga Ulva lactuca (U) by sea cucumber Apostichopus japonicus. A 56-day feeding experiment was carried out to investigate the FA profile of the A. japonicus feeding on 6 different types of diets with the ingredient of either pure powder of single algae species or mixtures of 3 algae species, i.e., the mixtures (1:1) of S. muticum and G. lemaneiformis (SG), S. muticum and U. lactuca (SU), and G. lemaneiformis and U. lactuca (GU). The FA profile of A. japonicus showed obvious changes over the experimental period and was remarkably affected by the different diets. Analyses of variations in the specific FAs and the multidimensional scaling (MDS) ordination of the overall FA profiles revealed that A. japonicus preferentially absorbed U. lactuca relative to S. muticum in the diet group SU while showed the discrimination against G. lemaneiformis in the diet groups SG and GU. Moreover, A. japonicus fed with U. lactuca displayed a similar or higher specific growth rate (SGR) than those fed on S. muticum or G. lemaneiformis, respectively. The results of the present study suggested that green alga U. lactuca might be used as a suitable substitute for replacing the traditional ingredient brown alga Sargassum thunbergii in the artificial feed for A. japonicus farming. Statement of relevance: The results of the present study could be used to further optimize the ingredient of A. japonicus artificial feeds. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Sea cucumber Apostichopus japonicus (Selenka) is one of the most commercially valuable species among 1100 species sea cucumber in the world (Okorie et al., 2008). Aquaculture of A. japonicus has been rapidly extended in China in the past 20 years (Chen, 2004; Xia et al., 2012). The total production of A. japonicus exceeded 201, 000 t in 2014 with a 3.8% increase compared to that in 2013 (MOAC, 2015). As the demand of A. japonicus increases, formulated feeds were widely used to improve the yield of this species. In littoral benthic ecosystems, deposit-feeding sea cucumber tend to take up the sedimentary organic matter containing abundant macroalgae detritus (Uthicke and Karez, 1999; Michio et al., 2003). A 1-yr in situ field study by Sun et al. (2013) indicated that A. japonicus consumed macroalgae as the principal food source throughout the investigation period. In aquaculture practice, algal powder is one of the main ingredients of formulated feeds in intensive and semi-intensive sea cucumber farming (Slater et al., 2009; Liu et al., 2010). ⁎ Corresponding author at: Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao, Shandong Province 266003, China. E-mail address: [email protected] (Q.-F. Gao).
http://dx.doi.org/10.1016/j.aquaculture.2015.10.008 0044-8486/© 2015 Elsevier B.V. All rights reserved.
Brown macroalgae, especially Sargassum thunbergii, is the most popular ingredient for the manufacture of A. japonicus feeds (Seo et al., 2011; Xia et al., 2012). With the rapid expansion of A. japonicus farming, over-harvest of Sargassum spp. has led to the sharp decline in the natural resource of this species and subsequent substantial rise in the price of Sargassum and elevated cost for A. japonicus culture (Gao et al., 2011). Accordingly, it is urgent to seek alternative macroalgae species as feed ingredient to relieve the shortage of S. thunbergii and to lower the cost of A. japonicus farming. Considerable studies have been carried out to examine the effects of various species of brown algae such as Sargassum polycystum, Laminaria japonica and Undaria pinnatifida and red algae such as Gracilaria lemaneiformis on the growth of A. japonicus (Liu et al., 2010; Seo et al., 2011; Gao et al., 2011; Xia et al., 2012). The high yield and easy availability of Ulva lactuca, one of the most popular seaweeds in the north of China, as well as its low price makes it potentially feasible to select this algae species as a new ingredient for the artificial feed of A. japonicus. However, studies on the uptake of green algae including U. lactuca by A. japonicus are scarce to the present (Xia et al., 2012). Feed utilization efficiency in terms of apparent digestibility coefficient (ADC) and feed conversion ratio (FCR) can give insights into digestive capacity and nutrient absorption in aquatic animals (Xia et al., 2012; Yu
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et al., 2015a). Accurate determinations of these parameters generally depend on the time-consuming collection of feed residue and feces. However, due to the nocturnal foraging behavior and the continuous feeding and defecation patterns of A. japonicus as well as the powder nature of the A. japonicus diet, such operations may be considerably difficult or even impossible (Dong et al., 2010; Yu et al., 2015a). Fatty acid (FA) trophic markers, which incorporate the dietary information of animals over longer periods of days and weeks, may be helpful to overcome such uncertainties (Dalsgaard et al., 2003; Budge et al., 2011). Most FAs synthesized by marine macroalgae are taxonomically specific, allowing the chemical discrimination between algae species using FA analyses. The sum of FAs 18:2n-6 and 18:3n-3, for example, is identified as the indicator for green algae, 20:4n-6 for brown algae, and 20:5n-3 for red algae (Meziane and Tsuchiya, 2000; Graeve et al., 2002; Kelly and Scheibling, 2011). FA profiles of animals can be distinctly changed due to the assimilation of different food items, and consequently specific FAs may provide evidence of food sources and dietary preferences of marine animals (Cook et al., 2000; Gao et al., 2006; Redmond et al., 2010; Ezgeta-Balić et al., 2012). The objectives of the present study were to identify the dietary preferences of A. japonicus for 3 different macroalgae using FA profiles and to examine the subsequent effects of food preferences on the growth performance of A. japonicus so as to provide scientific suggestions for optimizing the ingredients of artificial feed in the practice of sea cucumber farming. Three different algae species including brown alga Sargassum muticum, red alga G. lemaneiformis and green alga U. lactuca were used as feed ingredients. Temporal variations in FA compositions of A. japonicus fed on different diets were investigated to elucidate the accumulative effect of dietary FAs and the growth performance between different diet groups was compared. 2. Materials and methods 2.1. Diet preparation Six types of diets which were made of either a single or the mixture of 3 algae species were used in the experiment, i.e., single brown alga S. muticum (S), single red alga G. lemaneiformis (G), single green alga U. lactuca (U), mixture (1:1) of S. muticum and G. lemaneiformis (SG), mixture (1:1) of S. muticum and U. lactuca (SU) and mixture (1:1) of G. lemaneiformis and U. lactuca (GU). Each experimental diet was made of pure macroalgae to eliminate the influence of other ingredients. Dried algae were ground and sieved through a 150 mm mesh, then well mixed, slightly watered, stirred and extruded with a feed processing machine and the algal powder was thus pelletized under the mechanical pressure. Pelleted diets were dried at 60 °C for 36 h and stored at 4 °C for future use.
At the beginning of the experiment, 20 individuals were collected as day 0 samples and stored at − 80 °C for the initial FA analysis. During the acclimation and the following 56-d experiment, sea cucumbers were fed once a day at 15:00 with a daily ration of 5% of the body weight. Water temperature was maintained at 16.5 ± 0.5 °C, salinity ranged from 29 to 31, dissolved oxygen N 6.0 mg L−1 and a photoperiod 14:10 h (L/D) was used. Two-third volume of the water in each aquarium was exchanged with filtered seawater daily. Uneaten feed residue and feces were collected by siphoning at water exchange. 2.3. Sample collection To examine temporal changes in the FA profiles of the A. japonicus body walls over the experimental period, on days 0, 7, 14, 28, 42, and 56, 3 individuals from the same aquarium for each diet group were randomly sampled. Body walls of the collected A. japonicus were cleaned and kept in a freezer at − 80 °C. At the end of the experiment, A. japonicus individuals in the rest of the aquarium were starved for 48 h, then weighed, dissected and kept in a freezer at −80 °C for further analysis. 2.4. Fatty acid analysis Samples of freeze-dried body walls and algae ingredients were ground into fine powder using a tissue grinder. Total lipids were extracted with a chloroform/methanol mixture (2:1) according to the method of Bligh and Dyer (1959). Fatty acid methyl esters (FAMEs) were prepared by esterification using 2% sulphuric acid methanol as described in Gao et al. (2006). FAMEs were quantified with a gas chromatography (Shimadzu GC-2010 plus) equipped with an auto-sampler and a flame ionization detector (FID) instrument (GC-2010; Shimadzu, Kyoto, Japan) using an RTX-WAX fused silica capillary column (30 m × 0.25 mm × 0.25 μm), which was operated using following the temperature programs: 60°C for 1.0 min, rate of 10 °C/min to 190 °C, 2.0 °C/min to 236 °C and 236 °C for 2 min. The carrier gas was nitrogen and the linear velocity was 33.1 cm/s and the split ratio was 1:15. FAMEs were identified by comparing their retention time with those of the standards: 37-FAME Mix and 26-Bacterial Acid Methyl Esters Mix (Supelco, Bellefonte, PA, USA). 2.5. Calculation and statistical analysis The growth performance of A. japonicus in terms of specific growth rate (SGR) was calculated as: −1 SGR %d ¼ ð lnW f − ln Wi Þ=t 100
2.2. Feeding experiment The experiment was conducted at the laboratory of Qingdao National Ocean Scientific Research Center, Ocean University of China. The experimental A. japonicus juveniles were collected from a local commercial farm. Prior to the experiment, A. japonicus juveniles were cultured in 1000 L fiberglass tanks and were acclimatized for 14 days to the laboratory conditions. During the acclimatization period, sea cucumbers were fed with commercial feed which was used in the farm where the sea cucumber were collected so as to avoid the possible changes in the fatty acid (FA) profiles of A. japonicus before the beginning of the experiment. After 48 h starvation, a total of 216 acclimatized individuals with initial body weights of 5.57 ± 0.11 g were randomly selected and transferred into 36 glass aquaria (50 × 40 × 40 cm) in each of which 6 individuals were cultured. The 36 aquaria were further divided into 6 groups with 6 replicates for each group. Sea cucumbers in each of the 6 groups were fed with one of the 6 diets as described above, respectively.
where Wf and Wi are the final and initial weights of A. japonicus individuals in each aquarium, respectively and t is the duration of the experiment in days. To investigate the variations in overall FA profiles between different algae species and those between A. japonicus cultured with different diets, analyses of multidimensional scaling (MDS) were conducted based on the Euclidean distance resemblance matrix of the FA percentage after fourth root transformation. The differences in individual FAs and growth performance between the 6 diet groups were compared with one way analysis of variance (ANOVA) followed by a Tukey test for multiple comparisons at the significance level of 0.05 (p b 0.05). Prior to statistical analyses, raw data were diagnosed for normality of distribution and homogeneity of variance with the Kolmogorov– Smirnov test and Levene's test, respectively. The data were analyzed by the Primer 6.0 (Clarke and Gorley, 2006) and statistical software SPSS for Windows (Release 20.0).
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3. Results 3.1. Fatty acid profiles of feed ingredients Fatty acid (FA) compositions of the macroalgae used as feed ingredients including S. muticum, G. lemaneiformis and U. lactuca are presented in Table 1. FAs generally showed significant differences between the 3 algae species, particularly for the FAs which were used as trophic markers such as 18:1n-7, 18:2n-6, 20:4n-6 and 20:5n-3. For example, concentrations of 18:1n-7 and 18:2n-6 in U. lactuca were higher than those in S. muticum and G. lemaneiformis. Level of 20:4n-6 in S. muticum was higher compared to those in G. lemaneiformis and U. lactuca. G. lemaneiformis was characterized by higher levels of 20:5n-3 relative to S. muticum and U. lactuca. However, all 3 algae species contained trace amounts of 22:6n-3. Multidimensional scaling (MDS) analysis showed that the FA signatures of the macroalgae were classified into 3 separate groups in the MDS ordination plots (Fig. 1), indicating the substantial differences in the FA profiles between the 3 algae species. 3.2. Fatty acid compositions of A. japonicus Temporal changes in concentrations of the typical FA biomarkers including 18:1n-7, 18:2n-6, 20:4n-6 and 20:5n-3 in A. japonicus fed with different diets over the 56-d experimental period are shown in Fig. 2. During the experimental period, levels of 18:1n-7 for A. japonicus in the diet groups S and SG decreased while increased in the other diet groups (ANOVA, p b 0.05) (Fig. 2a). As shown in Fig. 2b, changes in the 18:2n-6 concentrations of A. japonicus in the diet group G were quite irregular and did not show any clear tendency over the experimental period while for other 5 diet groups, concentrations of 18:2n-6 showed continuous increases with the extension of the experimental period (ANOVA, p b
Table 1 Fatty acid profiles (% total fatty acids) of Sargassum muticum, Gracilaria lemaneiformis and Ulva lactuca which were used as feed ingredients in the experiment. Fatty acids in bold are those used as trophic markers for different algae. See text for the details. Data were presented as mean ± SD (n = 3). ND = not detected. Fatty acid
G. lemaneiformis
U. lactuca
Saturated fatty acids 14:0 6.07 ± 0.02 15:0 0.33 ± 0.00 i-15:0 0.93 ± 0.01 16:0 47.97 ± 0.09 17:0 0.46 ± 0.02 18:0 1.10 ± 0.01 20:0 0.66 ± 0.02 22:0 0.81 ± 0.01 24:0 0.49 ± 0.03 Subtotal 58.82 ± 0.08
S. muticum
4.99 ± 0.03 0.99 ± 0.02 0.42 ± 0.02 50.14 ± 0.17 1.48 ± 0.01 4.52 ± 0.05 0.59 ± 0.02 0.32 ± 0.00 0.33 ± 0.01 63.79 ± 0.15
3.75 ± 0.45 0.58 ± 0.02 0.69 ± 0.06 53.99 ± 0.35 0.72 ± 0.02 2.13 ± 0.12 0.78 ± 0.04 1.19 ± 0.39 0.75 ± 0.03 64.57 ± 0.86
Monounsaturated fatty acids 14:1n-5 0.27 ± 0.01 16:1n-7 7.71 ± 0.02 16:1n-9 1.27 ± 0.02 17:1n-9 0.35 ± 0.01 18:1n-7 1.16 ± 0.02 18:1n-9 9.80 ± 0.02 20:1n-9 3.32 ± 0.01 22:1n-9 0.34 ± 0.01 Subtotal 24.22 ± 0.06
0.33 ± 0.00 6.30 ± 0.03 0.76 ± 0.02 0.53 ± 0.01 4.33 ± 0.02 10.64 ± 0.08 2.03 ± 0.02 ND 24.93 ± 0.13
0.70 ± 0.02 4.57 ± 0.24 1.80 ± 0.10 0.97 ± 0.01 11.55 ± 0.04 2.69 ± 0.05 0.67 ± 0.11 ND 22.95 ± 0.38
Polyunsaturated fatty acids 18:2n-6 1.42 ± 0.03 18:3n-6 2.92 ± 0.01 18:3n-3 0.61 ± 0.00 20:2n-6 0.27 ± 0.02 20:3n-3 0.26 ± 0.01 20:4n-6 9.02 ± 0.02 20:5n-3 2.24 ± 0.04 22:6n-3 0.22 ± 0.01 Subtotal 16.96 ± 0.04
0.90 ± 0.01 0.80 ± 0.02 0.55 ± 0.01 0.39 ± 0.00 0.22 ± 0.01 0.89 ± 0.00 7.41 ± 0.05 0.13 ± 0.05 11.28 ± 0.02
6.57 ± 0.08 0.91 ± 0.03 3.04 ± 0.11 ND ND 0.72 ± 0.09 1.46 ± 0.12 ND 12.70 ± 0.09
Fig. 1. Multidimensional scaling (MDS) ordination with embedded Euclidean distance for the fatty acid profiles of the 3 algae species. S = Sargassum muticum, G = Gracilaria lemaneiformis and U = Ulva lactuca.
0.05). As for the level of 20:4n-6, it showed increase in A. japonicus in the diet group S and exhibited remarkable decreases in the diet groups G, U, SU and GU (ANOVA, p b 0.05). In the diet group SG, however, the concentration of 20:4n-6 kept relatively stable during the 56-d experimental period (ANOVA, p N 0.05) (Fig. 2c). For level of 20:5n-3 in A. japonicus, remarkable increase occurred in the diet group G but decreased in the other diet groups (ANOVA, p b 0.05) (Fig. 2d). As shown in Fig. 3a, ordinations of MDS analysis based on the overall FA profiles of A. japonicus revealed that the FA compositions of A. japonicus in the 3 diet groups of single macroalgae (diet S, G and U) tended to approach the FA profiles of corresponding diets from the beginning of the experiment (A0) to the end of the experiment (A-S56, A-G56 and AU56 for diets S, G and U, respectively), indicating the assimilation of FAs from S. muticum, G. lemaneiformis and U. lactuca by A. japonicus in the 3 diet groups, respectively. Fig. 3b illustrated the temporal changes in the FA compositions of the A. japonicus in the 3 mixture diet groups (diet groups SG, SU and GU) over the experimental period. In diet group SG, points in the MDS ordination plot representing the FA profiles of A. japonicus exhibited the tendency to approach those of S. muticum relative to G. lemaneiformis. While for diet groups SU and GU, MDS ordination suggested that A. japonicus assimilated FAs with the preference for U. lactuca vs S. muticum or G. lemaneiformis. FA compositions of the A. japonicus cultured with the 6 diets at the end of the experiment are shown in Table 2. Significant differences in the concentrations of 18:1n-7, 18:2n-6, 20:4n-6, 20:5n-3 and 22:6n-3 between the 6 diet groups were observed (ANOVA, p b 0.05). After 56 days of the feeding trial, the highest content of 18:1n-7 was detected in the diet group U while the lowest was found in the group SG. The highest proportion of 18:2n-6 was observed in the diet group SU while the lowest was found in the group G. A. japonicus in the diet groups S and G obtained the highest levels of 20:4n-6 and 20:5n-3, respectively. Concentrations of 22:6n-3 of A. japonicus in the diet group GU were significantly lower than those in other groups (ANOVA, p b 0.05) and no significant differences were observed between other groups (ANOVA, p N 0.05). Ordinations of MDS analysis based on the overall FA profiles of A. japonicus cultured with the 6 diets at the end of the experiment (A-S56, A-G56, A-U56, A-SG56, A-SU56 and A-GU56 for the 6 diet groups, respectively) are shown in Fig. 4. FA compositions of A. japonicus in all diet groups appeared obvious changes at the end of the experiment compared with the initial FA profiles of A. japonicus (A0). The positions of the points representing A. japonicus in different diet groups relative to those representing the 3 macroalgal species indicated the effects of dietary FAs on the FA profiles of A. japonicus. In the MDS ordination plot, the positions of the points representing the A. japonicus in diet groups S (A-S56), G (A-G56) and U (A-U56) were basically consistent with the points representing the 3 algae species S. muticum (S), G. lemaneiformis (G) and U. lactuca (U). For the 3 mixture diet groups, the sequence of
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Fig. 2. Temporal changes in fatty acid biomarkers of Apostichopus japonicus fed with different diets. (a) 18:1n-7, (b) 18:2n-6, (c) 20:4n-6 and (d) 20:5n-3. Data were presented as mean ± SD (n = 3). Different letters in each diet group mean significant differences (ANOVA with Tukey test for multiple comparisons, p b 0.05).
the points representing the FA profiles of A. japonicus in diet groups SG (A-SG56), SU (A-SU56) and GU (A-GU56) were also consistent with the sequence of those representing the 3 macroalgal species S. muticum (S), G. lemaneiformis (G) and U. lactuca (U). Moreover, the points representing the FA profiles of A. japonicus in the 3 mixture diet groups (A-SG56, A-SU56 and A-GU56) were all located within the points representing the FA profiles of A. japonicus in the 3 single algae diet groups (A-S56, A-U56 and A-U56), suggesting the simultaneous absorption of 2 macroalgal species by A. japonicus in the 3 mixture diet groups. 3.3. Growth performance Growth performance of A. japonicus in the 6 diet groups are shown in Table 3. At the beginning of the experiment, there were no significant differences in the initial body weights (IBW) of A. japonicus between the 6 diet groups (ANOVA, p N 0.05). In contrast, after the 56-d feeding trial, significant differences in the final body weight (FBW) and specific growth rate (SGR) were observed between the 6 diet groups (ANOVA, p b 0.05), with the highest FBW and SGR in the diet group SG and the lowest in the diet group SU. Moreover, the FBW and SGR of A. japonicus in the diet group G was significantly lower than those
in the diet groups S and U (ANOVA, p b 0.05) while no significant differences were observed between the diet groups S and U (ANOVA, p N 0.05). 4. Discussion Results of the present study showed the temporal changes in fatty acid (FA) compositions of A. japonicus feeding on 6 different diets, suggesting that the FA profiles of A. japonicus were remarkably affected by the FA components of the diets (Hasegawa et al., 2014; Yu et al., 2015b). Accordingly, analyses for the transfer and accumulation of dietary FAs into the tissues of A. japonicus might reveal dietary preferences between different algae species. Syntheses of FAs by aquatic macroalgae are generally taxonomically specific. Arachidonic acid (20:4n-6), for example, is generally selected as the indicator for brown algae, eicosapentaenoic acid (20:5n-3) for red algae and the sum of linoleic acid (18:2n-6) and linolenic acid (18:3n-3) for green algae (Meziane and Tsuchiya, 2000; Graeve et al., 2002; Kelly and Scheibling, 2011). In the present study, brown alga S. muticum, red alga G. lemaneiformis and green alga U. lactuca were characterized by high levels of 20:4n-6, 20:5n-3 and 18:2n-6,
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Fig. 3. Multidimensional scaling (MDS) ordination of the fatty acid profiles for Apostichopus japonicus and the dietary macroalgae. S = Sargassum muticum, G = Gracilaria lemaneiformis and U = Ulva lactuca and A0 = A. japonicus at the beginning of the experiment (day 0). A–S, A–G, A–U, A–SG, A–SU and A–GU = A. japonicus fed with S, G, U SG, SU and GU, respectively, and 7, 14, 28, 42 and 56 mean the experimental period in days on which the sample A. japonicus were collected.
respectively, while U. lactuca was more enriched in the concentration of cis-vaccenic acid (18:1n-7) relative to other algae species. However, despite of relative low concentrations of 18:2n-6 and 18:1n-7 in the S. muticum and G. lemaneiformis, respectively, A. japonicus in the diet groups S and G showed increases in these two FAs, respectively. This might lead to misunderstanding the food sources in the mixed diet groups (groups SU and GU). In contrast, A. japonicus fed with S. muticum and G. lemaneiformis did not appear obvious increases in 18:1n-7 and 18:2n-6, respectively. Thus 18:1n-7 and 20:4n-6 could be used to distinguish U. lactuca and S. muticum while 18:2n-6 and 20:5n-3 could potentially be used to distinguish U. lactuca and G. lemaneiformis. Accordingly, 20:4n-6 was selected as the trophic marker for S. muticum, 20:5n-3 for G. lemaneiformis and 18:2n-6 and/or 18:1n-7 for U. lactuca. Multidimensional scaling (MDS) analysis showed the substantial differences in the FA patterns between the 3 algae species, indicating the feasibility of using FA profiles as trophic markers to trace the preferential absorption between multiple macroalgae species by A. japonicus.
After 56 days of the feeding trial, A. japonicus showed substantial weight gain, thus changes in FA profiles of A. japonicus might be attributable to the dilution process, rather than the result of FA turnover (Robin et al., 2003; Jobling, 2004; Budge et al., 2011). For the single diet groups, owing to the effects of FA compositions of S. muticum, G. lemaneiformis and U. lactuca, respectively, A. japonicus showed remarkable increases in 20:4n-6 and 18:2n-6 in the group S, 20:5n-3 and 18:1n-7 in the group G and 18:1n-7 and 18:2n-6 in the group U. Moreover, as illustrated in the ordinations of MDS analysis, the overall FA profiles of A. japonicus changed in the predictable manner that gradually resembled the FA profiles of the diets. As for A. japonicus cultured with mixed diets, analyses of variations in the specific FAs and the MDS ordination of the overall FA profiles revealed the dietary preferences between the 3 algae species. A. japonicus fed with the mixture of S. muticum and G. lemaneiformis showed remarkable increases in 20:4n-6 and 18:2n-6 while decreases in 20:5n-3 and 18:1n-7, indicating that A. japonicus accumulated more S. muticum against G. lemaneiformis. As for the A. japonicus cultured with the mixture of S. muticum and
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Table 2 Fatty acid compositions (mg g−1, dry weight basis) of Apostichopus japonicus fed with different diets at the end of the experiment (day 56). Fatty acids in bold are those used as trophic markers for different algae. See text for the details. Data were presented as mean ± SD (n = 3). Different letters in the same row mean significant differences (ANOVA with Tukey test for multiple comparisons, p b 0.05). ND = not detected. See text for detailed description of diets S, G, U, SG, SU, and GU. Fatty acids Saturated fatty acids 14:0 15:0 i-15:0 16:0 17:0 18:0 20:0 22:0 24:0 Subtotal
Diet S 0.34 ± 0.01 0.2 ± 0.01 ND 4.14 ± 0.06 0.23 ± 0.01 2.28 ± 0.09 0.80 ± 0.01 0.74 ± 0.01 0.13 ± 0.00 8.86 ± 0.14
Diet G
Diet U
Diet SG
Diet SU
Diet GU
0.76 ± 0.01 0.52 ± 0.01 0.21 ± 0.01 4.51 ± 0.10 0.54 ± 0.06 1.60 ± 0.05 0.45 ± 0.01 0.35 ± 0.02 0.14 ± 0.01 9.07 ± 0.06
0.42 ± 0.01 0.32 ± 0.01 0.16 ± 0.01 3.94 ± 0.12 0.83 ± 0.09 1.45 ± 0.04 0.67 ± 0.01 0.68 ± 0.02 0.16 ± 0.01 8.64 ± 0.21
0.41 ± 0.01 0.26 ± 0.01 0.11 ± 0.01 4.58 ± 0.01 0.36 ± 0.04 2.21 ± 0.04 0.66 ± 0.01 0.61 ± 0.02 0.14 ± 0.01 9.34 ± 0.07
0.41 ± 0.01 0.23 ± 0.01 0.11 ± 0.01 3.99 ± 0.04 0.41 ± 0.03 2.06 ± 0.09 0.69 ± 0.03 0.75 ± 0.04 0.15 ± 0.01 8.8 ± 0.21
0.56 ± 0.07 0.35 ± 0.04 0.19 ± 0.04 4.83 ± 0.32 0.54 ± 0.03 1.41 ± 0.08 0.63 ± 0.02 0.66 ± 0.04 0.17 ± 0.02 9.35 ± 0.41
Monounsaturated fatty acids 14:1n-5 0.29 ± 0.01 16:1n-7 2.1 ± 0.04 16:1n-9 0.11 ± 0.00 17:1n-9 0.11 ± 0.01 18:1n-7 1.65 ± 0.02a 18:1n-9 4.95 ± 0.03 20:1n-9 1.84 ± 0.01 22:1n-9 0.66 ± 0.01 Subtotal 11.72 ± 0.08
0.44 ± 0.01 3.62 ± 0.04 0.25 ± 0.01 0.27 ± 0.01 3.4 ± 0.01c 4.64 ± 0.01 1.65 ± 0.04 1.01 ± 0.14 15.3 ± 0.12
0.16 ± 0.00 3.51 ± 0.07 0.16 ± 0.01 0.43 ± 0.02 4.72 ± 0.16d 3.09 ± 0.03 1.68 ± 0.02 0.92 ± 0.19 14.67 ± 0.15
0.37 ± 0.01 2.83 ± 0.02 0.14 ± 0.01 0.17 ± 0.01 2.14 ± 0.01b 4.98 ± 0.05 1.84 ± 0.01 0.74 ± 0.02 13.2 ± 0.12
0.22 ± 0.01 2.79 ± 0.14 0.13 ± 0.01 0.24 ± 0.03 3.17 ± 0.15c 3.62 ± 0.10 1.51 ± 0.02 0.71 ± 0.03 12.39 ± 0.29
0.26 ± 0.04 3.10 ± 0.16 0.19 ± 0.03 0.27 ± 0.01 3.42 ± 0.05c 3.19 ± 0.06 1.30 ± 0.04 0.88 ± 0.21 12.61 ± 0.12
Polyunsaturated fatty acids 18:2n-6 0.58 ± 0.01b 18:3n-6 0.18 ± 0.01 18:3n-3 0.97 ± 0.01 20:2n-6 0.84 ± 0.06 20:3n-3 0.29 ± 0.01 20:4n-6 3.02 ± 0.08c 20:5n-3 1.39 ± 0.02a 22:6n-3 1.16 ± 0.01b Subtotal 8.42 ± 0.05
0.35 ± 0.03a 0.28 ± 0.01 0.82 ± 0.01 0.79 ± 0.03 0.27 ± 0.01 1.22 ± 0.02a 2.39 ± 0.08d 1.04 ± 0.04b 7.15 ± 0.17
0.71 ± 0.02c 0.44 ± 0.01 0.73 ± 0.01 0.59 ± 0.01 0.45 ± 0.01 1.70 ± 0.03b 1.68 ± 0.08c 1.00 ± 0.04b 7.29 ± 0.06
0.54 ± 0.01b 0.26 ± 0.01 0.88 ± 0.01 0.75 ± 0.01 0.27 ± 0.01 2.94 ± 0.05c 1.69 ± 0.07c 1.13 ± 0.03b 8.46 ± 0.14
U. lactuca and mixture of G. lemaneiformis and U. lactuca, the increases in the concentrations of 18:2n-6 and 18:1n-7 and the decreases in those of 20:4n-6 and 20:5n-3 suggested that A. japonicus preferentially absorbed U. lactuca relative to S. muticum and G. lemaneiformis. Furthermore, MDS ordination suggested that the points representing the FA profiles of A. japonicus showed the tendency to approach those of preferred algae species, confirming the preferential utilization of S. muticum by sea cucumber in the diet group SG and U. lactuca in the diet groups SU and GU. Such results are consistent with the previous findings that A. japonicus took up less G. lemaneiformis relative to S. thunbergii when offered the mixture of these two algal powder (Gao et al., 2011). The selective absorption between various algal sources by animals could be attributed to the intrinsic properties of different algae species,
Fig. 4. Multidimensional scaling (MDS) ordination with embedded Euclidean distance of the fatty acid profiles of algae species and Apostichopus japonicus at the end of the experiment (day 56). S = Sargassum muticum, G = Gracilaria lemaneiformis and U = Ulva lactuca and A0 = A. japonicus on day 0. A-S56, A-G56, A-U56, A-SG56, A-SU56 and A-GU56 = A. japonicus in the diet groups S, G, U SG, SU and GU on day 56, respectively.
0.93 ± 0.04e 0.30 ± 0.01 0.86 ± 0.02 0.70 ± 0.02 0.35 ± 0.01 1.79 ± 0.03b 1.43 ± 0.03ab 1.14 ± 0.06b 7.51 ± 0.06
0.84 ± 0.03d 0.32 ± 0.01 0.62 ± 0.02 0.42 ± 0.04 0.38 ± 0.02 1.22 ± 0.12a 1.56 ± 0.06bc 0.80 ± 0.15a 6.15 ± 0.34
for instance, physical characteristics of algae such as toughness and calcification (Pennings et al., 1998; Gao et al., 2011), cell wall structure (Zemke-White et al., 2000; Shi et al., 2013) and nutritional content such as fiber (Zamora and Jeffs, 2011; Xia et al., 2012). A. japonicus tended to reject G. lemaneiformis, probably due to the harder texture and subsequent lower digestibility of this species relative to U. lactuca and S. muticum (Gao et al., 2011). Moreover, sea cucumber species generally have limited ability to produce endogenous cellulase and the digestion of cellulose mainly depends on beneficial microflora present in the digestive tract (Yingst, 1976; Ward-Rainey et al., 1996; Yuan et al., 2006). As a result, the digestion and absorption of G. lemaneiformis by A. japonicus is limited. As essential fatty acids (EFAs), n-3 and n-6 polyunsaturated fatty acids (PUFAs), e.g., ARA (20:4n-6), EPA (20:5n-3) and DHA (22:6n-3) are generally obtained from diets and play important roles in the physiological functions of marine animals such as egg and larval quality, immune function and membrane permeability (Cook et al., 2000; Bell and Sargent, 2003; Paulsen et al., 2014). However, certain marine invertebrates are capable of synthesizing long-chain FAs from shorter chain precursors, for example, converting 18:2n-6 to 20:4n-6 and 18:3n-3 to 20:5n-3 and/or 22:6n-3 (Bell et al., 2001; González-Durán et al., 2008). In spite of the fact that both U. lactuca and G. lemaneiformis contained trace amounts of 20:4n-6, A. japonicus fed with single U. lactuca accumulated more 20:4n-6 compared to those fed on single G. lemaneiformis. Similarly, although level of 20:5n-3 in U. lactuca was lower relative to that in S. muticum, A. japonicus in the diet group U showed a significantly higher proportion of 20:5n-3 than that in the diet group S. As U. lactuca was richer in 18:2n-6 and 18:3n-3 relative to S. muticum and G. lemaneiformis, these results might be explained by that A. japonicus was able to convert 18:2n-6 to 20:4n-6 and 18:3n3 to 20:5n-3. Hasegawa et al. (2014) and Yu et al. (2015b) also demonstrated that A. japonicus had the potential to synthesize 20:4n-6 and
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Table 3 Growth performance of Apostichopus japonicus fed with different diets. Data were presented as mean ± SD (n = 3). Different letters in the same row mean significant differences (ANOVA with Tukey test for multiple comparisons, p b 0.05). IBW = initial body weight, FBW = final body weight, SGR = specific growth rate (% d−1). See text for detailed description of diets S, G, U, SG, SU, and GU.
IBW FBW SGR
Diet S
Diet G
Diet U
Diet SG
Diet SU
Diet GU
5.57 ± 0.16 12.23 ± 1.06cd 1.40 ± 0.21b
5.62 ± 0.10 9.65 ± 1.26ab 0.96 ± 0.26a
5.52 ± 0.21 13.68 ± 1.12cd 1.61 ± 0.09b
5.60 ± 0.05 14.10 ± 1.48d 1.64 ± 0.18b
5.49 ± 0.02 9.07 ± 0.76a 0.89 ± 0.15a
5.61 ± 0.11 11.67 ± 1.04bc 1.30 ± 0.18b
20:5n-3 using the precursors with shorter carbon chains. As such, the requirements for EFAs may be met by a combination of dietary 18:2n6 and 18:3n-3. Although A. japonicus fed both single G. lemaneiformis and U. lactuca showed higher levels of 20:5n-3 compared with those fed with single S. muticum, comparable concentration of 22:6n-3 was recorded in the latter diet group, indicating that further elongation and desaturation of 20:5n-3 to 22:6n-3 might not happen in A. japonicus (Yu et al., 2015b). In general, monounsaturated fatty acids (MUFAs) are used as energy reserves in marine animals, and 18:1n-7 is selectively catabolized as the most dominant energy source during starvation (Smith et al., 2003; Mourente et al., 2005). Previous studies showed that large amounts of energy intake were used for metabolism in A. japonicus (Yuan et al., 2006; Liu et al., 2009). A. japonicus fed on single S. muticum showed decrease in 18:1n-7 due to the low level of this FA in S. muticum, suggesting that A. japonicus utilized 18:1n-7 for routine metabolism. However, if A. japonicus were fed with single U. lactuca or G. lemaneiformis which was richer in 18:1n-7 relative to S. muticum, they could use this FA as energy source in addition to maintaining the normal metabolism. Accordingly, elevated concentrations of 18:1n-7 were recorded in these two diet groups. As for the growth performance of the A. japonicus over the 70-d experimental period, A. japonicus in the diet group U showed a similar or higher specific growth rate (SGR) compared to specimens in the diet groups S or G, respectively. Such results are consistent with previous studies where A. japonicus feeding on U. lactuca showed a better growth performance than those fed with S. thunbergii (Xia et al., 2012). A. japonicus fed the mixture of S. muticum and G. lemaneiformis (diet SG) which was rich in both 20:4n-6 and 20:5n-3 showed the highest SGR, suggesting that these two FAs were vital for the growth of A. japonicus. The second highest SGR of A. japonicus in the diet group U might support the hypothesis that the requirements for 20:4n-6 and 20:5n-3 could be met by the combination of dietary 18:2n-6 and 18:3n-3. Thus, from the perspective of FAs, high concentrations of 18:1n-7, 18:2n-6 and 18:3n-3 in U. lactuca might account for the improved growth of A. japonicus feeding this alga. Despite of relative preferences for S. muticum and U. lactuca to G. lemaneiformis, A. japonicus fed the mixture of S. muticum and U. lactuca (diet SU) showed the lowest SGR. Moreover, A. japonicus in the diet group SU contained the lowest concentrations of 20:5n-3 indicating the insufficient supply of 20:5n-3 for the sea cucumber cultured with the mixture of S. muticum and U. lactuca, probably due to the low content of 20:5n-3 in both S. muticum and U. lactuca and due to the further dilution of 20:5n-3 when S. muticum and U. lactuca were mixed. These results might suggest that 20:5n-3 was more important to the growth of A. japonicus. Bell et al. (2001) reported that another echinoderm species sea urchin Psammechinus miliaris (Gmelin) was capable of biosynthesizing 20:5n3 from its precursor 18:3n-3. The conversion rate, which was equivalent to 0.09 μg/g tissue/mg 18:3n-3 eaten over 14 days, however, was quite low. Similarly, in the present study, even though A. japonicus had the potential to convert 18:3n-3 to 20:5n-3, it is possible that the capacity for endogenous production of 20:5n-3 may be inefficient to meet the requirements for the growth of sea cucumber. Moreover, S. muticum contained trace amounts of 18:3n-3 and the mixture of S. muticum with U. lactuca diluted the 18:3n-3 concentration of U. lactuca, leading to the reduction in the availability of 18:3n-3 for bioconversion of
18:3n-3 to 20:5n-3 by A. japonicus. Although G. lemaneiformis was rich in 20:5n-3, A. japonicus in the diet group G showed the second lowest SGR. In addition to the FAs, the growth of A. japonicus could be affected by other nutrients such as amino acids, carbohydrates and vitamins (Okorie et al., 2008; Zhao et al., 2011). The poor growth performance of A. japonicus in the diet group G might be explained by that A. japonicus could not utilize G. lemaneiformis effectively due to the low digestibility of this alga, affecting the absorption of other nutritional components by A. japonicus. In conclusion, results of the present study indicated that the FA profiles of A. japonicus were affected by the various FA components of different macroalgae species in the diets, and A. japonicus preferentially absorbed U. lactuca relative to S. muticum and tended to discriminate against G. lemaneiformis when cultured with the mixture of G. lemaneiformis and U. lactuca or and S. muticum. Moreover, A. japonicus fed with U. lactuca showed a similar or better growth performance than those fed on S. muticum or G. lemaneiformis, respectively. The green alga U. lactuca which is one of the most dominant seaweed species in northern China could therefore be utilized as a suitable substitute to replace the traditional ingredient S. thunbergii in the artificial feed for A. japonicus farming. Acknowledgements This study was funded by the National Natural Science Foundation of China (Grant Nos. 31172426 & 31372549) and the Ministry of Science and Technology of China (Grant No. 2011BAD13B03). References Bell, J.G., Sargent, J.R., 2003. Arachidonic acid in aquaculture feeds: current status and future opportunities. Aquaculture 218, 491–499. Bell, M.V., Dick, J.R., Kelly, M.S., 2001. Biosynthesis of eicosapentaenoic acid in sea urchin Psammechinus miliaris. Lipids 36, 79–92. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Budge, S.M., Penney, S.N., Lall, S.P., 2011. Response of tissue lipids to diet variation in Atlantic salmon (Salmo salar): implications for estimating diets with fatty acid analysis. J. Exp. Mar. Biol. Ecol. 409 (1), 267–274. Chen, J.X., 2004. Present status and prospects of sea cucumber industry in China. In: Lovatelli, A., Conand, C., Purcell, S., Uthicke, S., Hamel, J.F., Mercier, A. (Eds.), Advances in Sea Cucumber Aquaculture and Management 463. FAO, Rome, pp. 25–38. Clarke, K., Gorley, R.N., 2006. Primer v6: User Manual/Tutorial. PRIMER-E, Plymouth. Cook, E.J., Bell, M.V., Black, K.D., Kelly, M.S., 2000. Fatty acid composition of gonadal material and diets of the sea urchin, Psammechinus miliaris: trophic and nutritional implications. J. Exp. Mar. Biol. Ecol. 255, 261–274. Dalsgaard, J., St John, M., Kattner, G., Muller-Navarra, D., Hagen, W., 2003. Fatty acid trophic markers in the pelagic marine environment. Adv. Mar. Biol. 46, 225–340. Dong, G., Dong, S., Wang, F., Tian, X., 2010. Effects of light intensity on daily activity rhythm of juvenile sea cucumber, Apostichopus japonicus (Selenka). Aquac. Res. 41 (11), 1640–1647. Ezgeta-Balić, D., Najdek, M., Peharda, M., Blažina, M., 2012. Seasonal fatty acid profile analysis to trace origin of food sources of four commercially important bivalves. Aquaculture 334, 89–100. Gao, Q.F., Shin, P.K.S., Lin, G.H., Chen, S.P., Cheung, S.G., 2006. Stable isotope and fatty acid evidence for uptake of organic waste by green-lipped mussels Perna viridis in a polyculture fish farm system. Mar. Ecol. Prog. Ser. 317, 273–283. Gao, Q.F., Wang, Y., Dong, S., Sun, Z., Wang, F., 2011. Absorption of different food sources by sea cucumber Apostichopus japonicus (Selenka) (Echinodermata: Holothuroidea): evidence from carbon stable isotope. Aquaculture 319, 272–276. González-Durán, E., Castell, J.D., Robinson, S.M.C., Blair, T.J., 2008. Effects of dietary lipids on the fatty acid composition and lipid metabolism of the green sea urchin Strongylocentrotus droebachiensis. Aquaculture 276, 120–129.
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