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Effect of three diets on the growth and fatty acid profile of the common ragworm Hediste diversicolor (O.F. Müller, 1776)
Aquaculture 465 (2016) 37–42
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Effect of three diets on the growth and fatty acid profile of the common ragworm Hediste diversicolor (O.F. Müller, 1776) António Santos a, Luana Granada a, Teresa Baptista a, Catarina Anjos a, Tiago Simões a, Carla Tecelão a,b, Pedro Fidalgo e Costa c, José Lino Costa d, Ana Pombo a,⁎ a
MARE – Marine and Environmental Sciences Centre, ESTM, Polytechnic Institute of Leiria, 2520-641, Peniche, Portugal Linking Landscape, Environment, Agriculture and Food Research Unit (LEAF), Instituto Superior de Agronomia, University of Lisbon, Tapada da Ajuda, Lisbon, Portugal MARE - Marine and Environmental Sciences Centre, Laboratório Marítimo da Guia, Avenida Nossa Senhora do Cabo, 939, 2750-374 Cascais, Portugal d MARE – Marine and Environmental Sciences Centre, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal b c
a r t i c l e
i n f o
Article history: Received 12 February 2016 Received in revised form 11 August 2016 Accepted 17 August 2016 Available online 24 August 2016 Keywords: Aquaculture Polychaete Hediste diversicolor diets Specific growth rate Fatty acid profile
a b s t r a c t The polychaete Hediste diversicolor has a high physiological tolerance to extreme environmental factors, being easily farmed and reproduced in different types of conditions. Both in the field and under laboratory conditions, this worm can feed on different types of food. In order to highlight the potential of H. diversicolor for aquaculture, specific growth rate (SGR), daily growth rate (DGR), survival rate and fatty acid profile of juvenile worms, fed with three different diets, were assessed. The experiments were conducted using juvenile polychaete from a controlled reproduction with wild adults. H. diversicolor individuals were fed with two commercial diets, seabream dry feed (Aquagold) and semi-wet pellets for reared sole (Moist Sole), and with a non-processed diet consisting on mackerel's fillets (Trachurus trachurus). Juveniles fed with Aquagold had the highest final individual weight (0.89 ± 0.10 g). The SGR was higher in H. diversicolor fed with Aquagold and Moist Sole, (6.49 ± 0.30% d−1 and 6.54 ± 0.06% d−1, respectively. The highest DGR was observed for juveniles fed with Aquagold (0.146 ± 0.02 g d−1). The survival rate of ragworms under different treatments ranged from 96 to 100%. Regarding the protein content, the Moist Sole diet provided the highest percentage of protein in the reared worms (8.87%). Results showed that the total fat content of the diets was reflected in the fat content of the reared worms. The Moist Sole diet treatment had the highest fat content (2.25%) and individuals fed with seabream dry feed showed similar results (2.18%), while the lowest percentage was observed for the mackerel diet (0.85%). According to the fatty acid profile, the major fatty acids found in the juveniles fed with the three different diets were palmitic (C 16:0), with a higher value in the individuals fed with mackerel's fillets. Oleic (C 18:1 n9), eicosapentaenoic (C 20:5 n3), docosahexaenoic (C 22:6 n3) and stearic (C 18:0) acids presented high values in H. diversicolor fed with all the experimental diets. Statement of Relevance: The common ragworm Hediste diversicolor is a potential high quality fatty acids source for reared fish and shrimp. Previous studies suggested that diet could be a relevant factor affecting the fatty acid composition of this polychaete (Luis and Passos, 1995). This study aimed to assess the effect of different diets on growth and survival of common ragworms juveniles (H. diversicolor), as well as the fatty acid profile and protein content in their tissues, aiming to find an appropriate diet to be used in commercial aquaculture. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The common ragworm Hediste diversicolor is an Annelida Polychaeta, which inhabits the soft-bottoms of shallow marine and brackish waters in the temperate zone of the northern hemisphere, from North Africa to the North American Atlantic coast and throughout Europe (Breton et al., 2003; Scaps, 2002). Polychaetes play an important
⁎ Corresponding author. E-mail address: [email protected] (A. Pombo).
http://dx.doi.org/10.1016/j.aquaculture.2016.08.022 0044-8486/© 2016 Elsevier B.V. All rights reserved.
role in functioning ecosystems (Duport et al., 2006), once these individuals increase the flux of oxygen and nutrients over the sediment-water interface (Hedman et al., 2011). This is called bioturbation and leads to a modification of the physical, chemical and biological sediment properties. This species can grow and reproduce in different sediment's types, being able to tolerate extreme variations of temperature and salinity and to survive to drastic conditions of hypoxia. Moreover, it has relatively generalist feeding habits and a wide adaptation capacity to the food size, being able to behave as both deposit-feeder and suspension-feeder (Fidalgo e Costa et al., 2006). This high adaptability suggests that it is a suitable species for aquaculture.
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The increasing commercial importance of polychaetes as fishing bait and feed source in aquaculture and, consequently, their massive harvesting, are causing disturbance to the benthic community and the ecosystem (Fidalgo e Costa et al., 2000). For these reasons it is important the increase of polychaete production in aquaculture, to avoid the depletion of a natural resource and minimise the negative impacts in the environment (Nesto et al., 2012; Omena et al., 2012). Furthermore, H. diversicolor might be a suitable organism for integrated aquaculture since it can be a detritivorous feeder, producing valuable dietary compounds such as fatty acids from waste products from a primary fish aquaculture system. Bischoff et al. (2009) demonstrated that this species is able to recycle feed nutrients such as fatty acids when reared in an integrated recirculating aquaculture systems. The development and optimisation of new rearing techniques for H. diversicolor at industrial scale is crucial and one of the key steps for successful rearing of this species. In the last years, several studies have been conducted with polychaetes using different feeds, such as marine vascular and macroalgal plant sources (Nesto et al., 2012; Oliver et al., 1996), dried feed for ornamental fish Tetramin Mikromin (Tetra, Blacksburg, USA) (Fidalgo e Costa et al., 2000; Prevedelli and Vandini, 1998, 1999), phytoplankton (Riisgård et al., 1996), faeces of the carpet shell clam Ruditapes decussatus (Batista et al., 2003a), shrimp meat (Nielsen et al., 1995), decapsulated Artemia sp. cysts, sea bream dry feed and Lansy (diet for shrimp, INVE Aquaculture, Salt Lake City, USA) (Fidalgo e Costa et al., 2000). The best results were obtained with sea bream dry feed, shrimp meat and Tetramin. During the last decades, lipids and fatty acids in particular have been more studied as they are of great importance for the food webs, and are in high demand for both human and animal nutrition (Bischoff et al., 2009). It is clear that marine fish are not capable of converting shortchain PUFA to LC-PUFA (long-chain polyunsaturated fatty acids) (Tocher et al., 1989; Tocher and Sargent, 1990; Sargent et al., 2002; Glencross, 2009; Tocher, 2010; Monroig et al., 2013; Tocher, 2015) and, thereby, LC-PUFA are considered to be essential dietary nutrients. Modes of feeding, gametogenesis and environmental temperature are some of the factors influencing the fatty acid composition of marine animals (Luis and Passos, 1995). The polychaete H. diversicolor is a suitable source of high quality lipids, since the fatty acid content of marine polychaetes seems to play an important role in stimulating gonad development and spawning in several reared species, such as common sole (Solea vulgaris), Senegalense sole (Solea senegalensis) and penaeid shrimp (Penaeus kerathurus) (Bischoff et al., 2009; Fidalgo e Costa et al., 2000; Meunpol et al., 2005; Luis and Passos, 1995) and in wild caught broodstock of Penaeus monodon (Vijayan et al., 2005). The ragworm fatty acid content is important for reared fish, like sole, and for shrimp species as stated before and previous studies suggested that diet could be a relevant factor affecting the fatty acid composition of H. diversicolor (Luis and Passos, 1995). This study aimed to assess the effect of different diets on growth and survival of common ragworms (H. diversicolor), as well as the fatty acid profile and protein content in their tissues, aiming to find an appropriate feed to be used in commercial aquaculture. 2. Material and Methods 2.1. Broodstock conditioning, spawning and juvenile production Adult H. diversicolor were collected from a natural population on Óbidos Lagoon, Portugal (39°24′52.7″N 9°13′13.2″W). Thirty two ragworms were placed in rearing system. The rearing system consisted of two 80 L tanks (0.175 m2), filled with water and 15 cm of sand (250– 500 μm). This was maintained with constant aeration, while temperature and salinity were kept at 25 ± 1 °C and 15, respectively. A partial water exchange was made two times a week, in order to preserve the water quality. Ragworms were reared for one month, until they matured, and then they were induced to spawn by thermal shock,
decreasing temperature (5 ± 1 °C). They were fed with semi-wet pellets for cultured sole (Moist Sole, Sparos Lda, Olhão, Portugal) supplemented with Ergosan, an immunostimulant based on the macroalgae Laminaria digitata and Ascophyllum nodosum, with 0.65 g d−1 and 1 g d−1, before and after reproduction, respectively. 1.1. Growth experiment with H. diversicolor juveniles Juveniles of H diversicolor with similar size were selected from the reproduction assay. Ragworms were weighted (fresh weight 0.17 ± 0.03 g, mean ± SD) and placed into the tanks at a density of around 170 ind m− 2. The rearing system consisted of nine 80 L tanks (0.175 m2) (three replicates per treatment), filled with 15 cm of sand (250–500 μm) and water and 30 polychaetes were stocked per tank. This system was maintained with constant aeration, whereas temperature and salinity were kept at 25 ± 1 °C and 15, respectively (Fidalgo e Costa et al., 2000; Nielsen et al., 1995). Mortality was recorded daily and a partial water exchange was made every week. The ammonium concentration was monitored during the experiment with multiparameter photometer HANNA HI 83203. The ammonium concentration was the highest in the tanks of ragworms fed on mackerel's fillets and Moist Sole diet treatments (4.0 mg L−1). On the contrary, in tanks where Aquagold were applied, the ammonium concentration achieved was lower (2.0 mg L−1). Ragworms were fed with one of the three types of food for 60 days. The diets consisted of two commercial diets, seabream dry feed (Aquagold, Sorgal SA, Ovar, Portugal) and semi-wet pellets for cultured sole (Moist Sole, Sparos Lda, Olhão, Portugal), and a non-processed diet of mackerel's fillets (Trachurus trachurus). It was used a daily feeding rate of 3% total tank biomass, adjusted throughout the trial. The commercial diets, Aquagold (protein: 46.00%; Lipids: 18.00%) and Moist Sole (protein: 52.12%; Lipids: 20.03%), were developed by Sorgal SA and Sparos Lda, respectively. The biochemical composition for mackerel's fillets (Trachurus trachurus) (protein: 19.00%; Lipids: 2.50%) was similar to the results reported by Batista et al. (2008) (19.70%; Lipids: 2.90%). To further evaluate the nutritional value of dietary treatments used during experimental period, it was determined the fatty acid profile of the diets. At the end of the experiment, following a gentle sieving, all individuals were weighted. The specific growth rate (SGR): % d−1 = 100 [ln (final wet weight) − ln (initial wet weight)] / duration and the daily growth rate (DGR): g d−1 = (final wet weight − initial wet weight) / duration were calculated according to Batista et al. (2003b) and Fidalgo e Costa et al. (2000). The crude protein, total lipid content and fatty acid profile were estimate with sub-samples of juveniles fed the experimental diets at the end of the on-growing period (three replicates per dietary treatment). 2.2. Protein content analysis The Kjeldahl method (model Kjeltech 2006, Foss Tecator, Hillerod, Denmark) was used to determine the nitrogen content. The crude protein was estimated based on the equation: Protein content (%) = [(Va − Vb) × NHCl × 6.25 × 0.014] / m × 100, where Va is the titration volume of the sample, Vb is the titration volume of the blank, NHCl is the normality of HCl solution (0.1 N), 6.25 is the nitrogen conversion factor, 0.014 is the milliequivalent weight of nitrogen, and m stands for sample weight (g). 2.3. Lipid extraction and fatty acid profile Total lipid extraction method was adapted from Bligh and Dyer (1959) following a dry matter basis. Fatty acid methyl esters were prepared according to the methods of Lepage and Roy (1986) and Masood et al. (2005). 0.015 mg of crude fat was dissolved in 5 mL acetyl chloride:methanol (1:19 v/v) and heated in a water bath at 80 °C for 1 h. Then, 1 mL ultrapure water and 2 mL n-heptane were added and
A. Santos et al. / Aquaculture 465 (2016) 37–42
the solution was vortex-stirred for 1 min followed by centrifugation at 1500 × g for 5 min. The organic upper phase was recovered and analysed by gas chromatography (GC). A Finnigan Ultra Trace gas chromatograph equipped with a Thermo TR-FAME capillary column (30 m × 0.25 mm ID, 0.25 μm film thickness), an auto sampler AS 3000 from Thermo Electron Corp. (Boston, Mass., U.S.A.), and a flame ionization detector (FID) were used to quantify the fatty acid methyl esters. Fatty acid methyl esters were identified in comparison to an external standard, fatty acid methyl ester mix (PUFA No. 3 from Menhaden oil) was purchased from Supelco (Bellefonte, Pa., U.S.A.).
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Table 2 Initial and final individual weight (g), specific growth rate (% d−1), daily growth rate (g d−1) and survival (%) (n = 30). Results are presented as mean ± SD.
Initial weight (g) Final weight (g) SGR (% d−1) DGR (g d−1) Survival (%)
Aquagold
Moist Sole
T. trachurus
p-Values
0.18 ± 0.03 0.89 ± 0.10 a 6.49 ± 0.30 a 0.146 ± 0.02 a 96
0.16 ± 0.02 0.79 ± 0.03 b 6.54 ± 0.06 a 0.129 ± 0.01 b 100
0.18 ± 0.04 0.53 ± 0.02 c 5.62 ± 0.20 b 0.085 ± 0.005 c 99
0.23 b0.015 b0.0001 b0.019 0.07
a, b and c represent statistically significant differences between polychaetes fed with different feeds (one-way ANOVA, Tukey HSD).
2.4. Statistical analysis Data were checked for normality and homoscedasticity. All data were subjected to one-way analysis of variance (ANOVA) to assess differences between diet treatments concerning growth rates, survival, total fat and protein content and fatty acid composition. When the conditions were not fulfilled, Kruskal-Wallis non-parametric test was performed. When differences were found, Tukey HSD post-hoc tests were employed. Where applicable, results are presented as mean ± SD. For all statistical tests, the significance level was set at p ≤ 0.05. All statistical tests were performed with IBM SPSS Statistics 20. 3. Results 3.1. Fatty acid profile of the diets The fatty acid profile of the three diets is present in Table 1. The most abundant saturated fatty acid in the experimental diets was palmitic (C 16:0). The stearic acid (C 18:0) presented a significantly higher value in T. trachurus diet (8.37 ± 0.35 g 100 g−1 FAME). The higher level of linoleic acid (18:2n-6) was obtained in Moist Sole diet. Regarding LCPUFAs, docosahexaenoic, DHA (C 22:6 n3), and arachidonic acid, ARA (C 20:4 n6), were significantly higher in T. trachurus diet (37.49 ± 2.32 and 2.11 ± 0.10 g 100 g−1 FAME, respectively). Also, significant statistical differences (p b 0.05) were found in the abundance of eicosapentaenoic acid, EPA (C 20:5 n3) between Aquagold diet and the other two experimental diets. This fatty acid was significantly higher in Aquagold diet (11.73 ± 1.00 g 100 g−1 FAME). 1.2. Growth experiment with H. diversicolor juveniles
Table 1 Dietary fatty acid composition (g 100 g−1 fatty acid methyl esters, FAME) (n = 9) of the experimental diets. Results are presented as mean ± SE. Fatty acid
Aquagold
Moist Sole
T. trachurus
C 14:0 C 16:0 C 16:1 C 16:3 n3 C 18:0 C 18:1 n7 C 18:1 n9 C 18:2 n6 C 18:3 n6 C 20:1 n9 C 20:2 n6 C 20:3 n9 C 20:4 n6 C 20:5 n3 C 22:5 n3 C 22:6 n3 ∑ SFA ∑ MUFA ∑ PUFA ∑ LC - PUFA
4.97 ± 0.80 19.61 ± 0.40 5.07 ± 0.54 0.32 ± 0.22 3.71 ± 0.23 1.67 ± 0.26 15.32 ± 1.54 6.78 ± 0.19 0.14 ± 0.03 1.53 ± 0.32 2.88 ± 0.49 0.19 ± 0.06 0.64 ± 0.22 11.73 ± 1.00 1.60 ± 0.35 12.22 ± 0.96 28.29 ± 1.43 23.59 ± 2.66 36.5 ± 4.62 26.19 ± 3.63
2.67 ± 0.29 15.41 ± 1.34 1.91 ± 0.39 0.12 ± 0.03 4.24 ± 0.44 2.37 ± 0.20 22.30 ± 1.62 10.22 ± 0.67 0.13 ± 0.04 0.50 ± 0.44 1.35 ± 0.26 0.13 ± 0.01 0.48 ± 0.41 8.10 ± 0.11 1.45 ± 0.20 15.75 ± 1.17 22.32 ± 2.07 27.08 ± 2.65 37.73 ± 2.9 25.78 ± 7.89
1.46 ± 0.11 19.05 ± 0.79 1.21 ± 0.61 0.31 ± 0.10 8.37 ± 0.35 2.53 ± 0.42 8.44 ± 0.18 1.19 ± 0.07 0.04 ± 0.07 0.22 ± 0.19 0.37 ± 0.46 0.16 ± 0.02 2.11 ± 0.10 8.22 ± 0.78 1.83 ± 0.45 37.49 ± 2.32 28.88 ± 1.25 12.4 ± 1.4 51.72 ± 4,37 49.65 ± 3.65
SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; and LC-PUFA, long-chain polyunsaturated fatty acid.
The initial and final weight, SGR, DGR and survival are reported in Table 2. There were no differences (p N 0.05) in the initial weight between treatments. The same was not observed for the final weight, which differed significantly (p b 0.05) between diets. Juveniles fed with Aquagold had the highest final weight (0.89 ± 0.10 g) and DGR (0.146 ± 0.02 g d−1). On the other hand, juveniles fed the diet consisting of mackerel's fillets had the lowest final individual weight (0.53 ± 0.02 g) and DGR (0.085 ± 0.005 g d−1). Regarding the SGR, the highest values were attained with the individuals fed with Aquagold (6.49 ± 0.30% d−1) and Moist Sole (6.54 ± 0.06% d−1). Individuals fed with mackerel's fillets had the lowest SGR (5.62 ± 0.20% d−1), which value differed significantly (p b 0.05) in relation to the other diets. Survival was high for all treatments and not significantly (p N 0.05) affected by the diets (96–100%). In particular, individuals fed with Moist Sole presented a survival of 100%. 3.1.1. Total fat and protein content Regarding the proportion of total fat content (Fig. 1), ragworms fed with Moist Sole and Aquagold presented higher values, 2.25 ± 0.11% and 2.18 ± 0.21%, respectively. On the contrary, 0.85 ± 0.05% was the lowest value corresponding to the individuals fed with mackerel's fillets. Concerning the concentration of crude protein (Fig. 1), ragworms fed with Moist Sole presented the highest value corresponding 8.87 ± 0.82%. The percentage of protein of ragworms fed with Aquagold was 8.65 ± 1.21% and the lowest value was observed for ragworms fed with mackerel's fillets 8.61 ± 1.18%. 3.1.2. Fatty acid profile The studied ragworms presented high values of important LC-PUFAs such eicosapentaenoic acid (EPA, C 20:5 n3), 17.38 ± 2.21%, 16.10 ± 3.97% and 14.80 ± 1.73% in individuals fed with Aquagold, mackerel's fillets and Moist Sole, respectively. Another key LC-PUFAs in aquafeeds, such DHA (C 22:6 n3), presented a higher value in H. diversicolor fed
#
#
*
Fig. 1. Values of total fat content and crude protein (%) of the ragworms fed the experimental diets at the end of the on-growing period. Results are presented as mean ± SD. Different symbols were significantly different (p b 0.05).
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A. Santos et al. / Aquaculture 465 (2016) 37–42
with mackerel's fillets (8.72 ± 1.52%), however, arachidonic acid (ARA, 20:4 n6) presented higher values with Aquagold and Moist Sole, 2.42 ± 1.09% and 2.50 ± 0.38%, respectively. The palmitic acid (C 16:0) was the most abundant fatty acid in the ragworms, independently of the diet they were fed; individuals fed with mackerel's fillets presented a value of 22.40 ± 0.11%, and 19.15 ± 0.33% and 16.63 ± 1.71% were the values observed for individuals fed with Aquagold and Moist Sole, respectively. In our study, significant statistical differences (p b 0.05) were found in the abundance of palmitic acid (C 16:0) between the ragworms fed with the three different diets. The abundance of eicosatrienoic acid (C 20:3) showed statistical significant differences between ragworms fed with Moist Sole and mackerel's fillets. It should also be noted that the accumulation of linoleic acid (LOA, 18:2 n6) in organisms fed with the Moist Sole is higher than with the other two dietary treatments, although no statistical significance was observed (Table 3). The mackerel's fillets diet afforded a higher DHA/EPA ratio in ragworms (0.54 ± 0.08). In individuals fed with Moist Sole and Aquagold, the DHA/EPA ratios were 0.45 ± 0.06 and 0.36 ± 0.03, respectively. On the other hand, the Moist Sole diet afforded a higher concentration of polyunsaturated fatty acids (PUFA) in ragworms (10.42 ± 0.02 mg g−1) and individuals fed with mackerel's fillets presented the lowest value (3.99 ± 0.01 mg g− 1). Aquagold and Moist Sole diets afforded higher concentrations of omega 3 (6.96 ± 0.01 mg g−1) and omega 6 (3.82 ± 0.00 mg g−1), respectively (Table 4). 4. Discussion In the present study a sea bream dry feed (Aquagold) was compared with semi-wet pellets for sole (Moist Sole) and mackerel's fillets (T. trachurus). The Aquagold feed led to promising results in previous studies (Fidalgo e Costa et al., 2000) and in experiments conducted by our research group at MARE-IPleiria (unpublished data). However, both Moist Sole and T. trachurus have not been studied before. The results showed that H. diversicolor was able to survive and grow with a single food source. The ragworms fed with mackerel's fillets presented a lower growth rate (Table 2); however, this diet can be used as a complement to other diets. Besides the fact that mackerel is a relatively expensive fish and, therefore, its use as aquaculture feed may be uneconomical, this experiment revealed that its use had a negative impact in water quality, resulting in high concentrations of ammonium. Moreira et al. (2006) reported that toxic concentrations of ammonium may lead to a reduction in feeding activity; that could explain the lower final weight of ragworms fed with mackerel's fillets (Table 2). Nevertheless, the obtained weight at the end of the experimental period
Table 3 Fatty acid composition (% total FAME) of H. diversicolor fed the experimental diets at the end of the on-growing period (n = 9). Results are presented as mean ± SE. Fatty acid
Aquagold
Moist Sole
T. trachurus
p-Values
C 14:0 C 16:0 C 16:1 C 16:3 C 18:0 C 18:1 n7 C 18:1 n9 C 18:2 n6 C 18:3 n6 C 20:1 n9 C 20:2 C 20:3 C 20:4 n6 C 20:5 n3 C 22:4 n6 C 22:5 n3 C 22:6 n3
2.02 ± 0.55 19.15 ± 0.33 b 2.46 ± 0.06 4.45 ± 1.81 7.21 ± 1.14 2.54 ± 0.26 10.71 ± 3.16 4.50 ± 0.58 0.88 ± 0.34 3.20 ± 1.42 1.57 ± 0.48 2.70 ± 0.42 a,b 2.42 ± 1.09 17.38 ± 2.21 1.08 ± 0.82 2.66 ± 0.22 6.72 ± 1.41
1.56 ± 0.31 16.63 ± 1.71 c 1.09 ± 0.05 5.80 ± 1.14 6.04 ± 0.72 2.45 ± 0.45 13.45 ± 3.88 8.03 ± 1.45 0.57 ± 0.25 2.90 ± 0.67 1.97 ± 0.25 3.44 ± 0.21 a 2.50 ± 0.38 14.80 ± 1.73 1.04 ± 0.21 1.87 ± 0.28 6.72 ± 0.81
0.97 ± 0.49 22.40 ± 0.11 a 2.23 ± 1.03 4.07 ± 1.62 8.35 ± 0.90 3.31 ± 0.85 5.89 ± 4.72 5.21 ± 4.72 1.00 ± 0.48 2.17 ± 1.05 1.16 ± 0.05 2.23 ± 0.49 b 1.25 ± 0.83 16.10 ± 3.97 2.14 ± 0.33 3.15 ± 0.83 8.72 ± 1.52
0.081 b0.001 0.61 0.409 0.062 0.214 0.299 0.346 0.387 0.535 0.051 b0.024 0.196 0.561 0.071 0.061 0.169
a, b and c represent statistically significant differences between polychaetes fed with different diets, for each fatty acid (one-way ANOVA, Tukey HSD).
Table 4 Abundance of fatty acids (mg g−1 wet weight) of H. diversicolor fed the experimental diets at the end of the on-growing period (n = 9). Results are presented as mean ± SE. Means values with different superscripts were significantly different (p b 0.001). Fatty acids ∑ Saturated ∑ Unsaturated ∑ Polyunsaturated ∑ n3 ∑ n6 n3/n6 DHA/EPA
Aquagold
Moist Sole a
6.91 ± 0.03 3.41 ± 0.01 b 9.29 ± 0.02 a 6.96 ± 0.01 2.19 ± 0.01 3.18 ± 0.66 0.36 ± 0.03
T. trachurus a
5.75 ± 0.02 5.03 ± 0.05 a 10.42 ± 0.02 a 6.43 ± 0.01 3.83 ± 0.00 1.68 ± 0.91 0.45 ± 0.06
3.04 ± 0.01 b 0.89 ± 0.02 c 3.99 ± 0.01 b 3.05 ± 0.01 0.87 ± 0.00 3.49 ± 0.51 0.54 ± 0.08
(approximately 0.5 g per individual) corresponded to H. diversicolor commercial minimum weight that Nesto et al. (2012) observed with a high protein food after 6 weeks. This could be an advantage since, within the same period, the ragworms are available to commercialization and less mature and, subsequently, with less probability to reproduce. Both Aquagold and Moist Sole were efficiently assimilated by the ragworms. By observation during the experimental period, it is known that the most part of individuals had a deposit feeding behaviour, collecting the food near its burrow opening (Scaps, 2002). It is likely that there was also a suspension feeding behaviour by the presence of mucous inside galleries (Fidalgo e Costa et al., 2006). Ragworms fed with Aquagold showed the highest final weight (Table 2). This is in accordance with the studies reported by Fidalgo e Costa et al. (2000) and by Batista et al. (2003b), in which the sea bream dry feed afforded one of the highest growth rates. Moist Sole, with high protein content, afforded high growth rates as well, being a good alternative as polychaete aquaculture's feed. Nesto et al. (2012) suggested that high protein commercial diet determines higher growth rates, which was observed in this experiment (Table 2). However, besides the fact that Moist Soil was the diet with higher protein content, ragworms fed with Aquagold presented higher values of final weight and daily growth rate (Table 2). Regarding the specific growth rate (Table 2), the obtained values were higher than 6% d− 1, being in accordance with several authors that had similar results with commercial feeds (Batista et al., 2003b; Fidalgo e Costa et al., 2000; Nesto et al., 2012; Nielsen et al., 1995). Concerning the survival rates (Table 2), there were no significant differences between treatments, however, it is worth noting that Moist Sole afforded 100% survival, probably due to the immunostimulant Ergosan included in this feed that contained red algae extracts. This immunostimulant was already used with other species, such as trout. Heidarieh et al. (2012) reported that diet supplementation with Ergosan improved growth parameters, feed intake and digestive enzymes, and decreased pathogenic bacteria of rainbow trout (Oncorhynchus mykiss). The overall results of survival are also in accordance with Fidalgo e Costa et al. (2000) and Batista et al. (2003b). Moreover, these results may have been favoured by the low stocking density used during the experimental period, since high density is associated with high polychaete mortality (Nesto et al., 2012; Safarik et al., 2006). The results of protein content of H. diversicolor (Fig. 1) suggested that the protein assimilation by ragworms reflected the protein content of diet. Individuals fed diet with the highest content of protein (Moist Sole) presented the highest content in their tissues and, contrariwise, ragworms fed with mackerel's fillets, the diet with the lowest content of protein had the lowest content in their tissues. The results of other authors for other worm's species are not similar with those obtained in this experiment. Lemieux et al. (1997) obtained a maximum value of 41.4 mg g−1 for Nereis virens; Neuhoff (1979) observed a mean value of 458 mg g−1 for Nereis succinea but in natural conditions. As observed for the protein content, the results of fat content also suggested that the nutritional composition of ragworms reflected the dietary composition. Ragworms fed with Moist Sole had a higher fat content, followed by the ragworms fed with Aquagold. Ragworms fed with mackerel's
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fillets, the diet with the lowest fat content, also had the lowest fat content in their tissues. However, Techaprempreecha et al. (2011), reported the highest values of fat content for the sandworm P. nuntia, farmed with a commercial shrimp diet, that presented a fat content of 340 mg g−1 and wild individuals presented a fat content of 273 mg g−1. There are still few studies regarding the fatty acids abundance and profile in ragworms. The present study, where diets with different profiles were administered, may give a better comprehension about the subject. Also, according to Fidalgo e Costa et al. (2000), the fatty acid profile of the diets seems to play a significant role in the survival and growth rates of H. diversicolor under laboratorial conditions. Regarding the saturated fatty acids, the palmitic acid (C 16:0) was the most abundant, being the highest value observed for ragworms fed with mackerel's fillets, followed by ragworms fed with Aquagold and Moist Sole (Table 3). This possibly reflected the diets composition, since this fatty acid was abundant in all diets (Table 1). Palmitic acid may be important, since it is a precursor of many types of molecules with physiological relevance (membrane lipids, fats and waxes) (García-Alonso et al., 2008). In their studies, Bischoff et al. (2009); García-Alonso et al. (2008) and Luis and Passos (1995) also observed that the palmitic acid was one of the most abundant fatty acids in H. diversicolor. The ragworms also presented high concentrations of the stereatic acid (C 18:0), especially those who were fed with mackerel's fillet, with a value of 8.37 ± 0.35 %. The abundance of this fatty acid in ragworms reflected its abundance in the diet. However, the obtained value was higher than the maximum values reported by other authors, 6%, 6.1% and 7.6%, respectively (Bischoff et al., 2009; García-Alonso et al., 2008; Luis and Passos, 1995). It was observed that the cis-vaccenic (C 18:1 n7) and eicosenoic (C 20:1 n9) acids were found in more abundance in ragworms than in the diets. Regarding the PUFA, the highlight was gamma-linolenic acid (C 18:3 n6) in ragworms fed with mackerel's fillets. In relation to LC-PUFAs, the highlights were DHA (C 22:6 n3) in ragworms fed with mackerel's fillets, the arachidonic acid (C 20:4 n6) in ragworms fed with Moist Sole, and EPA (C 20:5 n3) in ragworms fed with Aquagold. In fact, aquaculture industry value Nereid worms because they are an excellent source of PUFA and, thus, they have potential to supplement fish oil for feeds (Brown et al., 2011). The majority of PUFA was present in higher concentrations in ragworms tissues than in diets. This may be linked with the important role that these fatty acids play in ragworms metabolism and with the ability of these organisms to synthesise PUFAs de novo from acetyl-coenzyme A, using several fatty acid desaturase and elongase enzymes (Vásquez et al., 2014). Also, ragworms fed with mackerel's fillets presented the highest DHA/EPA ratio (Table 4), even though the other diets afforded similar results, wherein all the DHA/EPA ratios were all less than one. This may be related to the omnivorous and opportunistic feeding behaviour of H. diversicolor, since usually carnivorous marine animals have higher ratios. It is known that if the amount of EPA or DHA is low in the diet, the worms can synthesise it de novo. However, when abundant in diet, the worms can retain nearly all the EPA from their diet, while they just can metabolise about 50% of DHA (Fidalgo e Costa et al., 2000). In the overall, the most abundant fatty acids were the palmitic and oleic acids and EPA, in ragworms fed with mackerel's fillets, Moist Sole and Aquagold, respectively. These results are in accordance with Bischoff et al. (2009); García-Alonso et al. (2008) and Luis and Passos (1995), who also observed higher concentrations for these fatty acids. 5. Future prospects In further experiments, the photoperiod should be monitored, which did not happen in the present study. According to several authors, this is a crucial parameter for the control of maturation and reproduction of reared worms (Last and Olive, 1999; Olive et al., 1998; Rees and Olive, 1999).
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Moreover, several studies demonstrated that H. diversicolor is able to reduce the production of waste in integrated multitrophic recirculation systems, while its reproductive fitness is enhanced (Granada et al., 2015). Also, according to the environmental conditions, this species is able to switch its feeding behaviour (Scaps, 2002) and, thus, in these systems fish faeces, uneaten fish feed and sediment borne bacteria are possible sources of different dietary fatty acids to the worms (Bischoff et al., 2009). In order to make, not just the ragworm use as bait, but also its own aquaculture more sustainable it is necessary to develop integrated systems using these organisms as suggested by Carvalho et al. (2013) and “waste” resources should be tested. Additionally, in order to improve this species aquaculture, we consider that is crucial to study how to promote the growth of ragworms and, at the same time, delay the maturation, since the reproduction is followed by the death of the mature worms.
Acknowledgements This study had the support of Fundação para a Ciência e Tecnologia (FCT), through the strategic project UID/MAR/04292/2013 granted to MARE, and the PROMAR Program through the project 31-03-05-FEP42: LIVE BAIT - Annelid polychaetes as live bait in Portugal: harvesting, import and rearing management.
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Effect of three diets on the growth and fatty acid profile of the common ragworm Hediste diversicolor (O.F. Müller, 1776) António Santos a, Luana Granada a, Teresa Baptista a, Catarina Anjos a, Tiago Simões a, Carla Tecelão a,b, Pedro Fidalgo e Costa c, José Lino Costa d, Ana Pombo a,⁎ a
MARE – Marine and Environmental Sciences Centre, ESTM, Polytechnic Institute of Leiria, 2520-641, Peniche, Portugal Linking Landscape, Environment, Agriculture and Food Research Unit (LEAF), Instituto Superior de Agronomia, University of Lisbon, Tapada da Ajuda, Lisbon, Portugal MARE - Marine and Environmental Sciences Centre, Laboratório Marítimo da Guia, Avenida Nossa Senhora do Cabo, 939, 2750-374 Cascais, Portugal d MARE – Marine and Environmental Sciences Centre, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal b c
a r t i c l e
i n f o
Article history: Received 12 February 2016 Received in revised form 11 August 2016 Accepted 17 August 2016 Available online 24 August 2016 Keywords: Aquaculture Polychaete Hediste diversicolor diets Specific growth rate Fatty acid profile
a b s t r a c t The polychaete Hediste diversicolor has a high physiological tolerance to extreme environmental factors, being easily farmed and reproduced in different types of conditions. Both in the field and under laboratory conditions, this worm can feed on different types of food. In order to highlight the potential of H. diversicolor for aquaculture, specific growth rate (SGR), daily growth rate (DGR), survival rate and fatty acid profile of juvenile worms, fed with three different diets, were assessed. The experiments were conducted using juvenile polychaete from a controlled reproduction with wild adults. H. diversicolor individuals were fed with two commercial diets, seabream dry feed (Aquagold) and semi-wet pellets for reared sole (Moist Sole), and with a non-processed diet consisting on mackerel's fillets (Trachurus trachurus). Juveniles fed with Aquagold had the highest final individual weight (0.89 ± 0.10 g). The SGR was higher in H. diversicolor fed with Aquagold and Moist Sole, (6.49 ± 0.30% d−1 and 6.54 ± 0.06% d−1, respectively. The highest DGR was observed for juveniles fed with Aquagold (0.146 ± 0.02 g d−1). The survival rate of ragworms under different treatments ranged from 96 to 100%. Regarding the protein content, the Moist Sole diet provided the highest percentage of protein in the reared worms (8.87%). Results showed that the total fat content of the diets was reflected in the fat content of the reared worms. The Moist Sole diet treatment had the highest fat content (2.25%) and individuals fed with seabream dry feed showed similar results (2.18%), while the lowest percentage was observed for the mackerel diet (0.85%). According to the fatty acid profile, the major fatty acids found in the juveniles fed with the three different diets were palmitic (C 16:0), with a higher value in the individuals fed with mackerel's fillets. Oleic (C 18:1 n9), eicosapentaenoic (C 20:5 n3), docosahexaenoic (C 22:6 n3) and stearic (C 18:0) acids presented high values in H. diversicolor fed with all the experimental diets. Statement of Relevance: The common ragworm Hediste diversicolor is a potential high quality fatty acids source for reared fish and shrimp. Previous studies suggested that diet could be a relevant factor affecting the fatty acid composition of this polychaete (Luis and Passos, 1995). This study aimed to assess the effect of different diets on growth and survival of common ragworms juveniles (H. diversicolor), as well as the fatty acid profile and protein content in their tissues, aiming to find an appropriate diet to be used in commercial aquaculture. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The common ragworm Hediste diversicolor is an Annelida Polychaeta, which inhabits the soft-bottoms of shallow marine and brackish waters in the temperate zone of the northern hemisphere, from North Africa to the North American Atlantic coast and throughout Europe (Breton et al., 2003; Scaps, 2002). Polychaetes play an important
⁎ Corresponding author. E-mail address: [email protected] (A. Pombo).
http://dx.doi.org/10.1016/j.aquaculture.2016.08.022 0044-8486/© 2016 Elsevier B.V. All rights reserved.
role in functioning ecosystems (Duport et al., 2006), once these individuals increase the flux of oxygen and nutrients over the sediment-water interface (Hedman et al., 2011). This is called bioturbation and leads to a modification of the physical, chemical and biological sediment properties. This species can grow and reproduce in different sediment's types, being able to tolerate extreme variations of temperature and salinity and to survive to drastic conditions of hypoxia. Moreover, it has relatively generalist feeding habits and a wide adaptation capacity to the food size, being able to behave as both deposit-feeder and suspension-feeder (Fidalgo e Costa et al., 2006). This high adaptability suggests that it is a suitable species for aquaculture.
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The increasing commercial importance of polychaetes as fishing bait and feed source in aquaculture and, consequently, their massive harvesting, are causing disturbance to the benthic community and the ecosystem (Fidalgo e Costa et al., 2000). For these reasons it is important the increase of polychaete production in aquaculture, to avoid the depletion of a natural resource and minimise the negative impacts in the environment (Nesto et al., 2012; Omena et al., 2012). Furthermore, H. diversicolor might be a suitable organism for integrated aquaculture since it can be a detritivorous feeder, producing valuable dietary compounds such as fatty acids from waste products from a primary fish aquaculture system. Bischoff et al. (2009) demonstrated that this species is able to recycle feed nutrients such as fatty acids when reared in an integrated recirculating aquaculture systems. The development and optimisation of new rearing techniques for H. diversicolor at industrial scale is crucial and one of the key steps for successful rearing of this species. In the last years, several studies have been conducted with polychaetes using different feeds, such as marine vascular and macroalgal plant sources (Nesto et al., 2012; Oliver et al., 1996), dried feed for ornamental fish Tetramin Mikromin (Tetra, Blacksburg, USA) (Fidalgo e Costa et al., 2000; Prevedelli and Vandini, 1998, 1999), phytoplankton (Riisgård et al., 1996), faeces of the carpet shell clam Ruditapes decussatus (Batista et al., 2003a), shrimp meat (Nielsen et al., 1995), decapsulated Artemia sp. cysts, sea bream dry feed and Lansy (diet for shrimp, INVE Aquaculture, Salt Lake City, USA) (Fidalgo e Costa et al., 2000). The best results were obtained with sea bream dry feed, shrimp meat and Tetramin. During the last decades, lipids and fatty acids in particular have been more studied as they are of great importance for the food webs, and are in high demand for both human and animal nutrition (Bischoff et al., 2009). It is clear that marine fish are not capable of converting shortchain PUFA to LC-PUFA (long-chain polyunsaturated fatty acids) (Tocher et al., 1989; Tocher and Sargent, 1990; Sargent et al., 2002; Glencross, 2009; Tocher, 2010; Monroig et al., 2013; Tocher, 2015) and, thereby, LC-PUFA are considered to be essential dietary nutrients. Modes of feeding, gametogenesis and environmental temperature are some of the factors influencing the fatty acid composition of marine animals (Luis and Passos, 1995). The polychaete H. diversicolor is a suitable source of high quality lipids, since the fatty acid content of marine polychaetes seems to play an important role in stimulating gonad development and spawning in several reared species, such as common sole (Solea vulgaris), Senegalense sole (Solea senegalensis) and penaeid shrimp (Penaeus kerathurus) (Bischoff et al., 2009; Fidalgo e Costa et al., 2000; Meunpol et al., 2005; Luis and Passos, 1995) and in wild caught broodstock of Penaeus monodon (Vijayan et al., 2005). The ragworm fatty acid content is important for reared fish, like sole, and for shrimp species as stated before and previous studies suggested that diet could be a relevant factor affecting the fatty acid composition of H. diversicolor (Luis and Passos, 1995). This study aimed to assess the effect of different diets on growth and survival of common ragworms (H. diversicolor), as well as the fatty acid profile and protein content in their tissues, aiming to find an appropriate feed to be used in commercial aquaculture. 2. Material and Methods 2.1. Broodstock conditioning, spawning and juvenile production Adult H. diversicolor were collected from a natural population on Óbidos Lagoon, Portugal (39°24′52.7″N 9°13′13.2″W). Thirty two ragworms were placed in rearing system. The rearing system consisted of two 80 L tanks (0.175 m2), filled with water and 15 cm of sand (250– 500 μm). This was maintained with constant aeration, while temperature and salinity were kept at 25 ± 1 °C and 15, respectively. A partial water exchange was made two times a week, in order to preserve the water quality. Ragworms were reared for one month, until they matured, and then they were induced to spawn by thermal shock,
decreasing temperature (5 ± 1 °C). They were fed with semi-wet pellets for cultured sole (Moist Sole, Sparos Lda, Olhão, Portugal) supplemented with Ergosan, an immunostimulant based on the macroalgae Laminaria digitata and Ascophyllum nodosum, with 0.65 g d−1 and 1 g d−1, before and after reproduction, respectively. 1.1. Growth experiment with H. diversicolor juveniles Juveniles of H diversicolor with similar size were selected from the reproduction assay. Ragworms were weighted (fresh weight 0.17 ± 0.03 g, mean ± SD) and placed into the tanks at a density of around 170 ind m− 2. The rearing system consisted of nine 80 L tanks (0.175 m2) (three replicates per treatment), filled with 15 cm of sand (250–500 μm) and water and 30 polychaetes were stocked per tank. This system was maintained with constant aeration, whereas temperature and salinity were kept at 25 ± 1 °C and 15, respectively (Fidalgo e Costa et al., 2000; Nielsen et al., 1995). Mortality was recorded daily and a partial water exchange was made every week. The ammonium concentration was monitored during the experiment with multiparameter photometer HANNA HI 83203. The ammonium concentration was the highest in the tanks of ragworms fed on mackerel's fillets and Moist Sole diet treatments (4.0 mg L−1). On the contrary, in tanks where Aquagold were applied, the ammonium concentration achieved was lower (2.0 mg L−1). Ragworms were fed with one of the three types of food for 60 days. The diets consisted of two commercial diets, seabream dry feed (Aquagold, Sorgal SA, Ovar, Portugal) and semi-wet pellets for cultured sole (Moist Sole, Sparos Lda, Olhão, Portugal), and a non-processed diet of mackerel's fillets (Trachurus trachurus). It was used a daily feeding rate of 3% total tank biomass, adjusted throughout the trial. The commercial diets, Aquagold (protein: 46.00%; Lipids: 18.00%) and Moist Sole (protein: 52.12%; Lipids: 20.03%), were developed by Sorgal SA and Sparos Lda, respectively. The biochemical composition for mackerel's fillets (Trachurus trachurus) (protein: 19.00%; Lipids: 2.50%) was similar to the results reported by Batista et al. (2008) (19.70%; Lipids: 2.90%). To further evaluate the nutritional value of dietary treatments used during experimental period, it was determined the fatty acid profile of the diets. At the end of the experiment, following a gentle sieving, all individuals were weighted. The specific growth rate (SGR): % d−1 = 100 [ln (final wet weight) − ln (initial wet weight)] / duration and the daily growth rate (DGR): g d−1 = (final wet weight − initial wet weight) / duration were calculated according to Batista et al. (2003b) and Fidalgo e Costa et al. (2000). The crude protein, total lipid content and fatty acid profile were estimate with sub-samples of juveniles fed the experimental diets at the end of the on-growing period (three replicates per dietary treatment). 2.2. Protein content analysis The Kjeldahl method (model Kjeltech 2006, Foss Tecator, Hillerod, Denmark) was used to determine the nitrogen content. The crude protein was estimated based on the equation: Protein content (%) = [(Va − Vb) × NHCl × 6.25 × 0.014] / m × 100, where Va is the titration volume of the sample, Vb is the titration volume of the blank, NHCl is the normality of HCl solution (0.1 N), 6.25 is the nitrogen conversion factor, 0.014 is the milliequivalent weight of nitrogen, and m stands for sample weight (g). 2.3. Lipid extraction and fatty acid profile Total lipid extraction method was adapted from Bligh and Dyer (1959) following a dry matter basis. Fatty acid methyl esters were prepared according to the methods of Lepage and Roy (1986) and Masood et al. (2005). 0.015 mg of crude fat was dissolved in 5 mL acetyl chloride:methanol (1:19 v/v) and heated in a water bath at 80 °C for 1 h. Then, 1 mL ultrapure water and 2 mL n-heptane were added and
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the solution was vortex-stirred for 1 min followed by centrifugation at 1500 × g for 5 min. The organic upper phase was recovered and analysed by gas chromatography (GC). A Finnigan Ultra Trace gas chromatograph equipped with a Thermo TR-FAME capillary column (30 m × 0.25 mm ID, 0.25 μm film thickness), an auto sampler AS 3000 from Thermo Electron Corp. (Boston, Mass., U.S.A.), and a flame ionization detector (FID) were used to quantify the fatty acid methyl esters. Fatty acid methyl esters were identified in comparison to an external standard, fatty acid methyl ester mix (PUFA No. 3 from Menhaden oil) was purchased from Supelco (Bellefonte, Pa., U.S.A.).
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Table 2 Initial and final individual weight (g), specific growth rate (% d−1), daily growth rate (g d−1) and survival (%) (n = 30). Results are presented as mean ± SD.
Initial weight (g) Final weight (g) SGR (% d−1) DGR (g d−1) Survival (%)
Aquagold
Moist Sole
T. trachurus
p-Values
0.18 ± 0.03 0.89 ± 0.10 a 6.49 ± 0.30 a 0.146 ± 0.02 a 96
0.16 ± 0.02 0.79 ± 0.03 b 6.54 ± 0.06 a 0.129 ± 0.01 b 100
0.18 ± 0.04 0.53 ± 0.02 c 5.62 ± 0.20 b 0.085 ± 0.005 c 99
0.23 b0.015 b0.0001 b0.019 0.07
a, b and c represent statistically significant differences between polychaetes fed with different feeds (one-way ANOVA, Tukey HSD).
2.4. Statistical analysis Data were checked for normality and homoscedasticity. All data were subjected to one-way analysis of variance (ANOVA) to assess differences between diet treatments concerning growth rates, survival, total fat and protein content and fatty acid composition. When the conditions were not fulfilled, Kruskal-Wallis non-parametric test was performed. When differences were found, Tukey HSD post-hoc tests were employed. Where applicable, results are presented as mean ± SD. For all statistical tests, the significance level was set at p ≤ 0.05. All statistical tests were performed with IBM SPSS Statistics 20. 3. Results 3.1. Fatty acid profile of the diets The fatty acid profile of the three diets is present in Table 1. The most abundant saturated fatty acid in the experimental diets was palmitic (C 16:0). The stearic acid (C 18:0) presented a significantly higher value in T. trachurus diet (8.37 ± 0.35 g 100 g−1 FAME). The higher level of linoleic acid (18:2n-6) was obtained in Moist Sole diet. Regarding LCPUFAs, docosahexaenoic, DHA (C 22:6 n3), and arachidonic acid, ARA (C 20:4 n6), were significantly higher in T. trachurus diet (37.49 ± 2.32 and 2.11 ± 0.10 g 100 g−1 FAME, respectively). Also, significant statistical differences (p b 0.05) were found in the abundance of eicosapentaenoic acid, EPA (C 20:5 n3) between Aquagold diet and the other two experimental diets. This fatty acid was significantly higher in Aquagold diet (11.73 ± 1.00 g 100 g−1 FAME). 1.2. Growth experiment with H. diversicolor juveniles
Table 1 Dietary fatty acid composition (g 100 g−1 fatty acid methyl esters, FAME) (n = 9) of the experimental diets. Results are presented as mean ± SE. Fatty acid
Aquagold
Moist Sole
T. trachurus
C 14:0 C 16:0 C 16:1 C 16:3 n3 C 18:0 C 18:1 n7 C 18:1 n9 C 18:2 n6 C 18:3 n6 C 20:1 n9 C 20:2 n6 C 20:3 n9 C 20:4 n6 C 20:5 n3 C 22:5 n3 C 22:6 n3 ∑ SFA ∑ MUFA ∑ PUFA ∑ LC - PUFA
4.97 ± 0.80 19.61 ± 0.40 5.07 ± 0.54 0.32 ± 0.22 3.71 ± 0.23 1.67 ± 0.26 15.32 ± 1.54 6.78 ± 0.19 0.14 ± 0.03 1.53 ± 0.32 2.88 ± 0.49 0.19 ± 0.06 0.64 ± 0.22 11.73 ± 1.00 1.60 ± 0.35 12.22 ± 0.96 28.29 ± 1.43 23.59 ± 2.66 36.5 ± 4.62 26.19 ± 3.63
2.67 ± 0.29 15.41 ± 1.34 1.91 ± 0.39 0.12 ± 0.03 4.24 ± 0.44 2.37 ± 0.20 22.30 ± 1.62 10.22 ± 0.67 0.13 ± 0.04 0.50 ± 0.44 1.35 ± 0.26 0.13 ± 0.01 0.48 ± 0.41 8.10 ± 0.11 1.45 ± 0.20 15.75 ± 1.17 22.32 ± 2.07 27.08 ± 2.65 37.73 ± 2.9 25.78 ± 7.89
1.46 ± 0.11 19.05 ± 0.79 1.21 ± 0.61 0.31 ± 0.10 8.37 ± 0.35 2.53 ± 0.42 8.44 ± 0.18 1.19 ± 0.07 0.04 ± 0.07 0.22 ± 0.19 0.37 ± 0.46 0.16 ± 0.02 2.11 ± 0.10 8.22 ± 0.78 1.83 ± 0.45 37.49 ± 2.32 28.88 ± 1.25 12.4 ± 1.4 51.72 ± 4,37 49.65 ± 3.65
SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; and LC-PUFA, long-chain polyunsaturated fatty acid.
The initial and final weight, SGR, DGR and survival are reported in Table 2. There were no differences (p N 0.05) in the initial weight between treatments. The same was not observed for the final weight, which differed significantly (p b 0.05) between diets. Juveniles fed with Aquagold had the highest final weight (0.89 ± 0.10 g) and DGR (0.146 ± 0.02 g d−1). On the other hand, juveniles fed the diet consisting of mackerel's fillets had the lowest final individual weight (0.53 ± 0.02 g) and DGR (0.085 ± 0.005 g d−1). Regarding the SGR, the highest values were attained with the individuals fed with Aquagold (6.49 ± 0.30% d−1) and Moist Sole (6.54 ± 0.06% d−1). Individuals fed with mackerel's fillets had the lowest SGR (5.62 ± 0.20% d−1), which value differed significantly (p b 0.05) in relation to the other diets. Survival was high for all treatments and not significantly (p N 0.05) affected by the diets (96–100%). In particular, individuals fed with Moist Sole presented a survival of 100%. 3.1.1. Total fat and protein content Regarding the proportion of total fat content (Fig. 1), ragworms fed with Moist Sole and Aquagold presented higher values, 2.25 ± 0.11% and 2.18 ± 0.21%, respectively. On the contrary, 0.85 ± 0.05% was the lowest value corresponding to the individuals fed with mackerel's fillets. Concerning the concentration of crude protein (Fig. 1), ragworms fed with Moist Sole presented the highest value corresponding 8.87 ± 0.82%. The percentage of protein of ragworms fed with Aquagold was 8.65 ± 1.21% and the lowest value was observed for ragworms fed with mackerel's fillets 8.61 ± 1.18%. 3.1.2. Fatty acid profile The studied ragworms presented high values of important LC-PUFAs such eicosapentaenoic acid (EPA, C 20:5 n3), 17.38 ± 2.21%, 16.10 ± 3.97% and 14.80 ± 1.73% in individuals fed with Aquagold, mackerel's fillets and Moist Sole, respectively. Another key LC-PUFAs in aquafeeds, such DHA (C 22:6 n3), presented a higher value in H. diversicolor fed
#
#
*
Fig. 1. Values of total fat content and crude protein (%) of the ragworms fed the experimental diets at the end of the on-growing period. Results are presented as mean ± SD. Different symbols were significantly different (p b 0.05).
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with mackerel's fillets (8.72 ± 1.52%), however, arachidonic acid (ARA, 20:4 n6) presented higher values with Aquagold and Moist Sole, 2.42 ± 1.09% and 2.50 ± 0.38%, respectively. The palmitic acid (C 16:0) was the most abundant fatty acid in the ragworms, independently of the diet they were fed; individuals fed with mackerel's fillets presented a value of 22.40 ± 0.11%, and 19.15 ± 0.33% and 16.63 ± 1.71% were the values observed for individuals fed with Aquagold and Moist Sole, respectively. In our study, significant statistical differences (p b 0.05) were found in the abundance of palmitic acid (C 16:0) between the ragworms fed with the three different diets. The abundance of eicosatrienoic acid (C 20:3) showed statistical significant differences between ragworms fed with Moist Sole and mackerel's fillets. It should also be noted that the accumulation of linoleic acid (LOA, 18:2 n6) in organisms fed with the Moist Sole is higher than with the other two dietary treatments, although no statistical significance was observed (Table 3). The mackerel's fillets diet afforded a higher DHA/EPA ratio in ragworms (0.54 ± 0.08). In individuals fed with Moist Sole and Aquagold, the DHA/EPA ratios were 0.45 ± 0.06 and 0.36 ± 0.03, respectively. On the other hand, the Moist Sole diet afforded a higher concentration of polyunsaturated fatty acids (PUFA) in ragworms (10.42 ± 0.02 mg g−1) and individuals fed with mackerel's fillets presented the lowest value (3.99 ± 0.01 mg g− 1). Aquagold and Moist Sole diets afforded higher concentrations of omega 3 (6.96 ± 0.01 mg g−1) and omega 6 (3.82 ± 0.00 mg g−1), respectively (Table 4). 4. Discussion In the present study a sea bream dry feed (Aquagold) was compared with semi-wet pellets for sole (Moist Sole) and mackerel's fillets (T. trachurus). The Aquagold feed led to promising results in previous studies (Fidalgo e Costa et al., 2000) and in experiments conducted by our research group at MARE-IPleiria (unpublished data). However, both Moist Sole and T. trachurus have not been studied before. The results showed that H. diversicolor was able to survive and grow with a single food source. The ragworms fed with mackerel's fillets presented a lower growth rate (Table 2); however, this diet can be used as a complement to other diets. Besides the fact that mackerel is a relatively expensive fish and, therefore, its use as aquaculture feed may be uneconomical, this experiment revealed that its use had a negative impact in water quality, resulting in high concentrations of ammonium. Moreira et al. (2006) reported that toxic concentrations of ammonium may lead to a reduction in feeding activity; that could explain the lower final weight of ragworms fed with mackerel's fillets (Table 2). Nevertheless, the obtained weight at the end of the experimental period
Table 3 Fatty acid composition (% total FAME) of H. diversicolor fed the experimental diets at the end of the on-growing period (n = 9). Results are presented as mean ± SE. Fatty acid
Aquagold
Moist Sole
T. trachurus
p-Values
C 14:0 C 16:0 C 16:1 C 16:3 C 18:0 C 18:1 n7 C 18:1 n9 C 18:2 n6 C 18:3 n6 C 20:1 n9 C 20:2 C 20:3 C 20:4 n6 C 20:5 n3 C 22:4 n6 C 22:5 n3 C 22:6 n3
2.02 ± 0.55 19.15 ± 0.33 b 2.46 ± 0.06 4.45 ± 1.81 7.21 ± 1.14 2.54 ± 0.26 10.71 ± 3.16 4.50 ± 0.58 0.88 ± 0.34 3.20 ± 1.42 1.57 ± 0.48 2.70 ± 0.42 a,b 2.42 ± 1.09 17.38 ± 2.21 1.08 ± 0.82 2.66 ± 0.22 6.72 ± 1.41
1.56 ± 0.31 16.63 ± 1.71 c 1.09 ± 0.05 5.80 ± 1.14 6.04 ± 0.72 2.45 ± 0.45 13.45 ± 3.88 8.03 ± 1.45 0.57 ± 0.25 2.90 ± 0.67 1.97 ± 0.25 3.44 ± 0.21 a 2.50 ± 0.38 14.80 ± 1.73 1.04 ± 0.21 1.87 ± 0.28 6.72 ± 0.81
0.97 ± 0.49 22.40 ± 0.11 a 2.23 ± 1.03 4.07 ± 1.62 8.35 ± 0.90 3.31 ± 0.85 5.89 ± 4.72 5.21 ± 4.72 1.00 ± 0.48 2.17 ± 1.05 1.16 ± 0.05 2.23 ± 0.49 b 1.25 ± 0.83 16.10 ± 3.97 2.14 ± 0.33 3.15 ± 0.83 8.72 ± 1.52
0.081 b0.001 0.61 0.409 0.062 0.214 0.299 0.346 0.387 0.535 0.051 b0.024 0.196 0.561 0.071 0.061 0.169
a, b and c represent statistically significant differences between polychaetes fed with different diets, for each fatty acid (one-way ANOVA, Tukey HSD).
Table 4 Abundance of fatty acids (mg g−1 wet weight) of H. diversicolor fed the experimental diets at the end of the on-growing period (n = 9). Results are presented as mean ± SE. Means values with different superscripts were significantly different (p b 0.001). Fatty acids ∑ Saturated ∑ Unsaturated ∑ Polyunsaturated ∑ n3 ∑ n6 n3/n6 DHA/EPA
Aquagold
Moist Sole a
6.91 ± 0.03 3.41 ± 0.01 b 9.29 ± 0.02 a 6.96 ± 0.01 2.19 ± 0.01 3.18 ± 0.66 0.36 ± 0.03
T. trachurus a
5.75 ± 0.02 5.03 ± 0.05 a 10.42 ± 0.02 a 6.43 ± 0.01 3.83 ± 0.00 1.68 ± 0.91 0.45 ± 0.06
3.04 ± 0.01 b 0.89 ± 0.02 c 3.99 ± 0.01 b 3.05 ± 0.01 0.87 ± 0.00 3.49 ± 0.51 0.54 ± 0.08
(approximately 0.5 g per individual) corresponded to H. diversicolor commercial minimum weight that Nesto et al. (2012) observed with a high protein food after 6 weeks. This could be an advantage since, within the same period, the ragworms are available to commercialization and less mature and, subsequently, with less probability to reproduce. Both Aquagold and Moist Sole were efficiently assimilated by the ragworms. By observation during the experimental period, it is known that the most part of individuals had a deposit feeding behaviour, collecting the food near its burrow opening (Scaps, 2002). It is likely that there was also a suspension feeding behaviour by the presence of mucous inside galleries (Fidalgo e Costa et al., 2006). Ragworms fed with Aquagold showed the highest final weight (Table 2). This is in accordance with the studies reported by Fidalgo e Costa et al. (2000) and by Batista et al. (2003b), in which the sea bream dry feed afforded one of the highest growth rates. Moist Sole, with high protein content, afforded high growth rates as well, being a good alternative as polychaete aquaculture's feed. Nesto et al. (2012) suggested that high protein commercial diet determines higher growth rates, which was observed in this experiment (Table 2). However, besides the fact that Moist Soil was the diet with higher protein content, ragworms fed with Aquagold presented higher values of final weight and daily growth rate (Table 2). Regarding the specific growth rate (Table 2), the obtained values were higher than 6% d− 1, being in accordance with several authors that had similar results with commercial feeds (Batista et al., 2003b; Fidalgo e Costa et al., 2000; Nesto et al., 2012; Nielsen et al., 1995). Concerning the survival rates (Table 2), there were no significant differences between treatments, however, it is worth noting that Moist Sole afforded 100% survival, probably due to the immunostimulant Ergosan included in this feed that contained red algae extracts. This immunostimulant was already used with other species, such as trout. Heidarieh et al. (2012) reported that diet supplementation with Ergosan improved growth parameters, feed intake and digestive enzymes, and decreased pathogenic bacteria of rainbow trout (Oncorhynchus mykiss). The overall results of survival are also in accordance with Fidalgo e Costa et al. (2000) and Batista et al. (2003b). Moreover, these results may have been favoured by the low stocking density used during the experimental period, since high density is associated with high polychaete mortality (Nesto et al., 2012; Safarik et al., 2006). The results of protein content of H. diversicolor (Fig. 1) suggested that the protein assimilation by ragworms reflected the protein content of diet. Individuals fed diet with the highest content of protein (Moist Sole) presented the highest content in their tissues and, contrariwise, ragworms fed with mackerel's fillets, the diet with the lowest content of protein had the lowest content in their tissues. The results of other authors for other worm's species are not similar with those obtained in this experiment. Lemieux et al. (1997) obtained a maximum value of 41.4 mg g−1 for Nereis virens; Neuhoff (1979) observed a mean value of 458 mg g−1 for Nereis succinea but in natural conditions. As observed for the protein content, the results of fat content also suggested that the nutritional composition of ragworms reflected the dietary composition. Ragworms fed with Moist Sole had a higher fat content, followed by the ragworms fed with Aquagold. Ragworms fed with mackerel's
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fillets, the diet with the lowest fat content, also had the lowest fat content in their tissues. However, Techaprempreecha et al. (2011), reported the highest values of fat content for the sandworm P. nuntia, farmed with a commercial shrimp diet, that presented a fat content of 340 mg g−1 and wild individuals presented a fat content of 273 mg g−1. There are still few studies regarding the fatty acids abundance and profile in ragworms. The present study, where diets with different profiles were administered, may give a better comprehension about the subject. Also, according to Fidalgo e Costa et al. (2000), the fatty acid profile of the diets seems to play a significant role in the survival and growth rates of H. diversicolor under laboratorial conditions. Regarding the saturated fatty acids, the palmitic acid (C 16:0) was the most abundant, being the highest value observed for ragworms fed with mackerel's fillets, followed by ragworms fed with Aquagold and Moist Sole (Table 3). This possibly reflected the diets composition, since this fatty acid was abundant in all diets (Table 1). Palmitic acid may be important, since it is a precursor of many types of molecules with physiological relevance (membrane lipids, fats and waxes) (García-Alonso et al., 2008). In their studies, Bischoff et al. (2009); García-Alonso et al. (2008) and Luis and Passos (1995) also observed that the palmitic acid was one of the most abundant fatty acids in H. diversicolor. The ragworms also presented high concentrations of the stereatic acid (C 18:0), especially those who were fed with mackerel's fillet, with a value of 8.37 ± 0.35 %. The abundance of this fatty acid in ragworms reflected its abundance in the diet. However, the obtained value was higher than the maximum values reported by other authors, 6%, 6.1% and 7.6%, respectively (Bischoff et al., 2009; García-Alonso et al., 2008; Luis and Passos, 1995). It was observed that the cis-vaccenic (C 18:1 n7) and eicosenoic (C 20:1 n9) acids were found in more abundance in ragworms than in the diets. Regarding the PUFA, the highlight was gamma-linolenic acid (C 18:3 n6) in ragworms fed with mackerel's fillets. In relation to LC-PUFAs, the highlights were DHA (C 22:6 n3) in ragworms fed with mackerel's fillets, the arachidonic acid (C 20:4 n6) in ragworms fed with Moist Sole, and EPA (C 20:5 n3) in ragworms fed with Aquagold. In fact, aquaculture industry value Nereid worms because they are an excellent source of PUFA and, thus, they have potential to supplement fish oil for feeds (Brown et al., 2011). The majority of PUFA was present in higher concentrations in ragworms tissues than in diets. This may be linked with the important role that these fatty acids play in ragworms metabolism and with the ability of these organisms to synthesise PUFAs de novo from acetyl-coenzyme A, using several fatty acid desaturase and elongase enzymes (Vásquez et al., 2014). Also, ragworms fed with mackerel's fillets presented the highest DHA/EPA ratio (Table 4), even though the other diets afforded similar results, wherein all the DHA/EPA ratios were all less than one. This may be related to the omnivorous and opportunistic feeding behaviour of H. diversicolor, since usually carnivorous marine animals have higher ratios. It is known that if the amount of EPA or DHA is low in the diet, the worms can synthesise it de novo. However, when abundant in diet, the worms can retain nearly all the EPA from their diet, while they just can metabolise about 50% of DHA (Fidalgo e Costa et al., 2000). In the overall, the most abundant fatty acids were the palmitic and oleic acids and EPA, in ragworms fed with mackerel's fillets, Moist Sole and Aquagold, respectively. These results are in accordance with Bischoff et al. (2009); García-Alonso et al. (2008) and Luis and Passos (1995), who also observed higher concentrations for these fatty acids. 5. Future prospects In further experiments, the photoperiod should be monitored, which did not happen in the present study. According to several authors, this is a crucial parameter for the control of maturation and reproduction of reared worms (Last and Olive, 1999; Olive et al., 1998; Rees and Olive, 1999).
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Moreover, several studies demonstrated that H. diversicolor is able to reduce the production of waste in integrated multitrophic recirculation systems, while its reproductive fitness is enhanced (Granada et al., 2015). Also, according to the environmental conditions, this species is able to switch its feeding behaviour (Scaps, 2002) and, thus, in these systems fish faeces, uneaten fish feed and sediment borne bacteria are possible sources of different dietary fatty acids to the worms (Bischoff et al., 2009). In order to make, not just the ragworm use as bait, but also its own aquaculture more sustainable it is necessary to develop integrated systems using these organisms as suggested by Carvalho et al. (2013) and “waste” resources should be tested. Additionally, in order to improve this species aquaculture, we consider that is crucial to study how to promote the growth of ragworms and, at the same time, delay the maturation, since the reproduction is followed by the death of the mature worms.
Acknowledgements This study had the support of Fundação para a Ciência e Tecnologia (FCT), through the strategic project UID/MAR/04292/2013 granted to MARE, and the PROMAR Program through the project 31-03-05-FEP42: LIVE BAIT - Annelid polychaetes as live bait in Portugal: harvesting, import and rearing management.
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