1. INTRODUCTION
⌅In order to meet the increased demand for seafood, aquaculture has experienced significant progress over recent decades. According to the latest FAO report, the world aquaculture production reached 114.5 million tonnes and was valued at 263.6 billion USD in 2018 (FAO, 2020FAO 2020. The State of World Fisheries and Aquaculture 2020. Aquaculture Paper No. 634. Rome, pp 16.). According to the same statistics, fish farming accounted for almost half (47%) of the global aquaculture production. Mullets are members of the Mugilidae family and are among the most ubiquitous fish resources worldwide. Their farming has been practiced for centuries (FAO,2015aFAO 2015a. Mediterranean coastal lagoons: sustainable management and interactions among aquaculture, capture fisheries and the environment, in: S. Cataudella, D. Crosetti, F. Massa (Eds.), Studies and Reviews, General Fisheries Commission for the Mediterranean, No. 95, Rome.). In the Mediterranean basin, the thick-lipped grey mullet Chelon labrosus is cultured in natural earthen ponds under extensive or semi-intensive regimes (De las Heras et al., 2015De las Heras V, Martos-Sitcha JA, Yúfera M, Mancera JM, Martínez-Rodríguez G. 2015. Influence of stocking density on growth, metabolism and stress of thick-lipped grey mullet (Chelon labrosus) juveniles. Aquaculture. 448, 29-37. https://doi.org/10.1016/j.aquaculture.2015.05.033.). It has been described as an easily cultivable species and constitutes a promising species for aquaculture diversification (Zouiten et al., 2008Zouiten D, Ben Khemis I, Besbes R, Cahu C. 2008. Ontogeny of the digestive tract of thick lipped grey mullet (Chelon labrosus) larvae reared in mesocosms. Aquaculture. 279, 166-172. https://doi.org/10.1016/j.aquaculture.2008.03.039 ; Ben Khemis et al., 2013Ben Khemis I, Gisbert E, Alcaraz C, Zouiten D, Besbes R, Zouiten A, Masmoudi AA, Cahu C. 2013. Allometric growth patterns and development in larvae and juveniles of thick-lipped grey mullet Chelon labrosus reared in mesocosm conditions. Aquac. Res. 44, 1872-1888. DOI: 10.1111/j.1365-2109.2012.03192.x ). Indeed, as other mullet species, C. labrosus is a low trophic level feeder, obtaining its energy directly from the first trophic level (Brusle, 1981Bruslé J .1981. Food and feeding in grey mullet. In: Oren, O. H. (Ed.), Aquaculture of the Grey Mullet. Cambridge Univ. Press, Cambridge.). In addition, the eurytherm and euryhaline species, C. labrosus is able to tolerate wide ranges of temperature and salinity (Cardona, 2006Cardona L .2006. Habitat selection by grey mullets (Osteichthyes: Mugilidae) in Mediterranean estuaries: the role of salinity. Sci Marpp. 70, 443-455.; Rabeh et al., 2013Rabeh I, Khaoula, Telahigue k, Gazali N, Chetoui I, Boussoufa D, Besbes R, Cafsi M. 2013. Time course of changes in fatty acid composition in the osmoregulatory organs of the thicklip grey mullet (Chelon labrosus) during acclimation to low salinity. Mar. Freshwater Behav. Physiol. 46, 59-73. https://doi.org/10.1080/10236244.2013.793470 ). Furthermore, several authors have reported that C. labrosus’ osmoregulation abilities appear early during in their development, allowing them to maintain elevated growth rates even under hyposaline conditions (Nordlie et al., 1982Nordlie FG, Szelistowski WA, Nordlie WC. 1982. Ontogenesis of osmotic regulation in the striped mullet, Mugil cephalus L. J. Fish. Biol. 20, 79-86. https://doi.org/10.1111/j.1095-8649.1982.tb03896.x ; Cardona, 2006Cardona L .2006. Habitat selection by grey mullets (Osteichthyes: Mugilidae) in Mediterranean estuaries: the role of salinity. Sci Marpp. 70, 443-455.). In most North African regions, the production of C. labrosus may be carried out in a variety of ecosystems, such as coastal lagoons with brackish to hyper saline waters (Crosetti, 2016Crosetti D. 2016. Current state of capture fisheries and culture of Mugilidae, in: D. Crosetti, S.J.M. Blaber (Eds.), Biology, Ecology and Culture of Grey Mullets (Mugilidae), CRC Press, Boca Raton, FL, pp. 398-450.) or reservoirs and ponds with fresh waters (Losse et al., 1991Losse GF, Nau W, Winter M. 1991. Le développement de la pêche en eau douce dans le nord de la Tunisie Projet de coopération technique Tuniso-Allemande, Projet “Utilisation de barrages pour la pisciculture,” GTZ et CGP, pp 418.). For many years, Tunisia has opted to breed fish in reservoirs and artificial lakes as a strategy for suppling aquatic products to the interior regions and for providing work opportunities for local communities (Besbes et al., 2020Besbes R, Besbes Benseddik A, Kokokiris L, Changeux T, Hamza A, Kammoun F, Missaoui H. 2020. Thicklip (Chelon labrosus) and flathead (Mugil cephalus) grey mullets fry production in Tunisian aquaculture. Aquacult Rep. 17, 100-380. https://doi.org/10.1016/j.aqrep.2020.100380 ). For instance, natural water flow from artesian sources is used directly as a culture medium in the south of Tunisia (Béchima locality) to facilitate in the raising of some freshwater and marine fish species. In this respect, recent investigation has shown the aptitude of Oreochromis niloticus and C. labrosus to live in geothermal waters (26 to 30°C) and to display good growth rates (Azaza et al., 2008aAzaza MS, Besbesbenseddik A, Besbes R, Kraiem MM, M’rabet R. 2008a. Adaptation du mulet d’élevage Chelon labrosus (poisson, téléostéen) aux eaux géothermales. Dixièmes Journées Tunisiennes des Sciences de la Mer et de la Première Rencontre Tuniso-Française d’Ichtyologie.; Azaza et al., 2008bAzaza MS, Dhraïef MN, Kraiem MM. 2008b. Effects of water temperature on growth and sex ratio of juvenile Nile tilapia Oreochromis niloticus (Linnaeus) reared in geothermal waters in southern Tunisia. J. Therm. Biol. 33, 98-105. https://doi.org/10.1016/j.jtherbio.2007.05.007 ).
It is well established that the capacity of an organism to efficiently adapt environmental changes is predominantly dependent on its metabolic flexibility (Smith et al., 2018Smith RL, Soeters MR, Wust RCI, Houtkooper RH. 2018. Metabolic flexibility as an adaptation to energy resources and requirements in health and disease. Endocr. Rev. 39, 489-517. https://doi.org/10.1210/er.2017-00211 ). This phenomenan includes a series of metabolic reorganizations and biochemical adjustments allowing the animal to meet increases in energy requirements and to maintain homeostasis under new environmental condition (Soengas et al., 2007Soengas JL, Sangiao-Alvarellos S, Láiz-Carrión R, Mancera J. 2007. Fish osmoregulation. In: Kapoor BG, editor. Energy metabolism and osmotic acclimation in teleost fish. Enfield: Science Publishers; p. 277-308. ). As the densest form of energy in marine ecosystems and fundamental components of the cell membrane, lipids and their key components, fatty acids (FA), play key roles in the adaptation of aquatic organisms to new environmental conditions (Fokina et al., 2017Fokina NN, Ruokolainen TR, Nemova NN .2017. Lipid composition modifications in the blue mussels (Mytilus edulis L.) from the White Sea. In: RAY S (ed.) Organismal and Molecular Malacology, Intech, Rijeka. http://dx.doi.org/10.5772/67811.). In this regard, several studies have reported high variability in the FA composition of fish depending on different abiotic and biotic factors such as the type and amount of food available, water temperature, pH, salinity, and reproduction cycle (Shirai et al., 2002Shirai N, Terayama M, Takeda H. 2002. Effect of season on the fatty acid composition and free amino acid content of the sardine Sardinops melanostictus. Comp. Biochem. Physiol. 131B, 387-393. https://doi.org/10.1016/S1096-4959(01)00507-3 ; Kaushik et al., 2006Kaushik SJ, Corraze G, Radunz-Neto J, Larroquet L, Dumas J. 2006. Fatty acid profiles of wild brown trout and Atlantic salmon juveniles in the Nivelle basin. J. Fish Biol. 68, 1376-1387. https://doi.org/10.1111/j.0022-1112.2006.01005.x ). Changes in FA composition are likely to induce conformational remodeling of membrane proteins, including receptors and channels, and ultimately affect cell responses to extracellular challenges (Brown, 1994Brown MF. 1994. Modulation of rhodopsin function by properties of the membrane bilayer. Chem. Phys. Lipids 73, 159-180.). FA are distributed into two major sub-classes which represent an essential and integral part of these compounds. Lipid classes can be broadly divided into neutral, mainly triacylglycerol (TAGs), and polar lipids (such as phospholipids). Phospholipids (PLs), are the major structural constituents of biological membranes. They are involved in the maintenance of membrane integrity and permeability and provide the matrix for the function of a large variety of catalytic processes (Dowhan et al., 2008). The most biologically important phospholipids of organisms are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS). These lipid classes are particularly known for their role in the maintaining of membrane integrity and fluidity and in the ensuring of the fish acclimatization process to changes in environmental conditions (Murzina et al., 2020Murzina SA, Pekkoeva SN, Kondakova EA, Nefedova ZA, Filippova KA, Nemova NN, Orlov AM, Berge J, Falk-Petersen S. 2020. Tiny but Fatty: Lipids and Fatty Acids in the Daubed Shanny (Leptoclinus maculatus), a Small Fish in Svalbard Waters. Biomolecules. 10, 368. https://doi.org/10.3390/biom10030368 ). Neutral lipids which includeTAG, serve mainly as a depot for lipids and provide most of the energy consumed (Sargent et al., 1976Sargent JR, Lee RF, Nevenzel JC. 1976. Marine waxes. In Kolattukudy, P. (ed.), Chemistry and Biochemistry of Natural Waxes. Elsevier Press, Amsterdam, pp. 50-91.). These molecules, which mainly come from food sources, tend to directly accumulate in fish muscle (Sushchik et al., 2020Sushchik NN, Makhutova ON , Rudchenko AE, Glushchenko LA , Shulepina SP, Kolmakova AA and Gladyshev MI.2020 Comparison of Fatty Acid Contents in Major Lipid Classes of Seven Salmonid Species from Siberian Arctic Lakes. Biomolecules. 10 (3), 419 https://doi.org/10.3390/biom10030419 ). The accumulation of these lipids can be differentiated by either external factors, such as fluctuations in environmental conditions, temperature, and food availability, or by internal factors, such as metabolic and physiological activities.
Despite their commercial importance, there are few studies on the biochemical composition of grey mullets (Rabeh et al., 2015Rabeh I, Telahigue K, Boussoufa D, Besbes R, El Cafsi MH. 2015. Comparative analysis of fatty acids profiles in muscle and liver ofTunisian thick lipped grey mullet Chelon labrosus reared inseawater and freshwater. J. Tunisian. Chem. Soc. 17, 95-104.; Ben Khemis et al., 2019Ben Khemis I, Hamza N, Sadok, S. 2019. Nutritional quality of the fresh and processed grey mullet (Mugilidae) products: a short review including data concerning fish from freshwater. Aquat. Living Resour 32, 2. https://doi.org/10.1051/alr/2018026 ). According to our hypothesis, lipids could indicate the ability of C. labrosus to adapt to abiotic factors, including water temperature and salinity. Hence, the objective of the present study was to identify and compare the FA composition of individual PL (PC, PE, PI and PS) and NL (mainly TAG) classes in C. labrosus reared in geothermal and seawater conditions in order to gain further insight into the physiological fitness of mullets.
2. MATERIALS AND METHODS
⌅2.1. Experimental material
⌅Immature thick-lipped grey mullets C. labrosus (30 to 40 g body mass) were supplied by an experimental fish culturing center (National Institute of Marine Science and Technology (INSTM)- Tunisia). The specimens of C. labrosus provided by INSTM-Center de Monastir were reared in seawater conditions with a salinity of 35 ppt and an ambiant temperature ranging from18 to 20 °C. While the specimens supplied by INSTM-Center de Béchima were reared in geothermal water conditions with a salinity of 2 ppt and a temperature varying between a minimum of 23 °C and the maxima of 28 °C. During the period from June 2006 to May 2007, all fish were hand fed six times between 08.00 and 18.00 h every 2 h until satiation. To reduce the sampling differences or external influences, six individuals from each habit were randomly selected at the same age and all fed the same local diet. The diet was made of 45% fresh sardines, 40% soybean meal, 10% fish meal, 4% vegetable oil and 1% vitamin premix. The moisture, crude protein, crude lipid, crude ash and lipid composition of the diet are shown in Table 1. They were fasted for 24 h before sampling, and six specimens form each group were sacrificed and the dorsal muscles (without skin) were sampled and conserved at -30 °C until analysis.
Ingredients | |
---|---|
Fatty acids composition % | |
C14:0 | 4.59±0.19 |
C15:0 | 0.05±0.03 |
C16:0 | 28.80±0.90 |
C16:1 | 0.55±0.00 |
C16:2 | 1.25±0.01 |
C16:3 | 2.78±0.46 |
C16:4 | 0.39±0.01 |
C17:0 | 0.10±0.10 |
C18:1n-9 | 2.43±0.25 |
C18:2n-6 | 29.60±2.36 |
C18:3n-6 | 16.11±0.87 |
C18:3n-3 | 1.18±0.06 |
C18:4n-3 | 0.73±0.01 |
C20:0 | 0.83±0.01 |
C20:1n-9 | 25.32±2.07 |
C22:1n-11 | 0.71±0.07 |
C20:3n-6 | 0.18±0.02 |
C20:3n-3 | 0.44±0.03 |
C20:4n-3 | 0.07±0.05 |
C20:5n-3 | 0.37±0.02 |
C20:4n-6 | 0.55±0.07 |
C22:5n-3 | 2.74±0.55 |
C22:5n-6 | 1.06±0.18 |
C22:6n-3 | 1.25±0.01 |
C21:5 | 0.02±0.01 |
C24:1n-9 | 0.59±0.12 |
Proximate composition | |
Humidity | 35 |
Protein | 37 |
Crude fat | 13 |
Ash | 2 |
Gross energy (kcal·g-1) | 4.34 |
Note: Results are expressed as mole % of total FAME based on peak areas. Data are means ± standard deviations from triplicate estimations (n =3 ) using ANOVA (Tukey HSD test).
2.2. Lipid extraction
⌅Lipids were extracted from fillet samples according to the method of Folch et al. (1957)Folch J, Lees M, Sloane-Stanley GA. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497-509. with a mixture of chloroform:methanol (2:1, v/v) containing buthylated-hydroxy-toluene (BHT), which was added to the solvent mixture as an antioxidant. For 1 g of fresh sample (individually analyzed), 30 mL of the solvent mixture were used. After evaporation of the solvent mixture under nitrogen, the extracts were dried overnight in a vacuum desiccator and quantified gravimetrically. Once weighed, the lipids were re-dissolved in the organic solvent and the obtained lipid extracts were conserved in -30 °C until analysis.
2.3. Lipid class separation
⌅The lipid classes from total lipid samples were separated using thin-layer chromatography (TLC) with one dimensional double development following the method of Olsen and Henderson (1989)Olsen R.E, Henderson RJ. 1989.The rapid analysis of neutral and polar marine 1448 lipids using double-development HPTLC and scanning densitometry. J. Exp. Mar.Biol. Ecol. 129, 189-197.. Concisely, 500 μL of lipid extracts from each sample were separated on silica gel plates (20 × 20 cm, Merck, Germany) into neutral and polar fractions. The plates were activated by heating at 105 °C for 1 h and developed with hexane/diethyl-ether/glacial-acetic-acid (80:20:2, v/v) for the neutral lipids (NL), and methyl acetate/isopropanol/chloroform/methanol/0.25% KCl (25:25:25:10:9, v/v) for the polar lipids (PL). The individual lipid categories were detected under UV light after being sprayed with 0.1% 2’-7’dichloro-fluorescein in absolute methanol. Lipid fractions were identified by corresponding standards and scraped from the plate into separate tubes and their constitutive fatty acids were transmethylated.
2.4 Analysis of fatty acids
⌅After evaporation to dryness, the lipid extracts and fractions were trans-esterified for fatty acid analysis according to the method of Cecchi et al. (1985)Cecchi G, Basini S, Castano C. 1985. Méthanolyse rapide des huiles en solvant [Rapid methanolysis of oils in solvent]. R. Franç. Corps Gras. 32, 163-164.. The resulting fatty acid methyl esters (FAME) were extracted using sodium methylate (NaOCH3) in the presence of hexane and sulfuric acid (H2SO4). Methyl nonadecanoate 19:0 (Sigma-Aldrich, St. Louis, MO, USA), which was absent from our samples, was added as an internal standard product.
Fatty acid methyl esters (FAMEs) were separated by a HP6890 gas chromatograph (Agilent Technologies; Santa Clara, CA) with a split/splitless injector. Nitrogen was the gas carrier at a flow rate of 1.5 mL·min-1 in an Innowax 250 capillary column (0.25 inside diameter × 30m length, 0.25 μm film; Agilent Technologies). The gradient temperature program was set as follows: from 50 °C to 180 °C at a rate of 4 °C·min-1, from 180 °C to 220 °C at a rate of 1.33 °C·min-1, and finally to stabilize at 220 °C for 7 min. The detector and injector were maintained at 250 °C. The identification of fatty acid methyl esters was based on the comparison of their retention times with those of authentic standards (C4 C24 by SUPELCO) and a well-characterized fish oil (Menhaden oil by SUPELCO). FA peaks were integrated and analyzed using the Agilent G2070BA GC Hewlett-Packard Chemstation Software. All fatty acid data were reported as percentage of total fatty acids.
2.5 Calculation of indices and statistical analysis
⌅The equations of unsaturation index (UI) and unsaturated-to-saturated FA ratio (U/S) were calculated according to Snyder and Hennessey (2003)Snyder RJ, Hennessey TM. 2003. Cold tolerance and homeoviscous adaptation in freshwater alewives Alosapseudoharengus. Fish Physiol. Biochem. 29, 117-126. https://doi.org/10.1023/B:FISH.0000035920.60817.11 and Wallaert and Babin (1994)Wallaert C, Babin PJ. 1994. Thermal adaptation affects the fatty acid composition of plasma phospholipids in trout. Lipids. 29, 373-376. https://doi.org/10.1007/BF02537193 .
The unsaturation index (UI):
where monoenes, diened, and trienes were fatty acids containing 1, 2, 3 double bonds, respectively.
The unsaturated-to-saturated FA ratio (U/S):
where %: weight percentage; UFA: unsaturated fatty acids; SFA: saturated fatty acid.
The statistical analyses were performed using R software version 3.3.3 (R Core Team 2017).
The data were checked for normality using the Shapiro-Wilk’s test (Shapiro and Wilk, 1965Shapiro SS, Wilk MB. 1965. An analysis of variance test for normality (complete samples). Biometrika 52, 591-611. https://www.jstor.org/stable/2333709 ) and Levene’s tests, respectively. One-way analysis of variance (ANOVA) and Tukey HSD’s test (at p < 0.05) were performed so as to detect significant statistical differences. The results are presented as means ± standard deviation (SD). Graphs were plotted using Prism. The covariance matrix was computed, and the Principal Component Analysis (PCA) was applied using the FactoMiner R Package (Lê et al., 2008) to evaluate the differences in the compositions of the samples under different conditions.
3. RESULTS
⌅Four major classes of phospholipids: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) in the muscle of C. labrosus with a dominant neutral lipid (TAG) in different rearing conditions were identified. Indeed, C. labrosus from geothermal water was richer in PLs and TAG than those from seawater. A detailed distribution of the fatty acids within the classes is given in Tables 2 and 3. The results show that the amount of neutral and polar lipids of C. labrosus from geothermal water was more important than those from seawater. The predominant FAs in the two groups (seawater and geothermal) were C16:0, C18:0, C18:1, C18:2n-6, C20:4n-6, C20:5n-3 and C22:6n-3 in PL and TAG. Among saturates, palmitic acid (C16:0) was the major fatty acid in all lipid classes and exhibited the highest levels, mainly in the PC, PS, and TAG of the two studied fish groups (Tables 2 and 3). In addition, stearic acid (C18:0) appeared to be particularly abundant in both polar and neutral lipid fractions. The main monounsaturated fatty acid (MUFA) was oleic acid (C18:1) and was found in high amounts in PC as well as in TAG (in both seawater and freshwater groups). Regarding polyunsaturated fatty acids (PUFA), we noted a significant increase in C18:2n-6 in the major polar lipid classes of the geothermal group. C22:6n-3 (DHA) and C20:5n-3 (EPA) were particularly abundant in PI. Indeed, 23 to 25% of PI were made of DHA for both fish groups.
Seawater | |||||
---|---|---|---|---|---|
% Fattyacids | PC | PE | PS | PI | TAG |
C14:0 | 0.53±0.07a | 0.19±0.02b | 0.61±0.07c | 0.48±0.01a | 6.95± 0.54d |
C15:0 | 0.03±0.01a | 0.09±0.05b | 0.11±0.03b | 0.13±0.02b | 0.25±0.02c |
C16:0 | 37.28±2.06a | 4.38±0.09b | 33.22±0.28c | 18.12±0.58d | 22.30±1.26d |
C17:0 | 0.41±0.11a | 0.73±0.07b | 0.46±0.24c | 0.39±0.10a | 0.45±0.03c |
C18:0 | 7.00±2.80a | 35.21±0.91b | 2.34±0.15c | 4.69±0.57d | 3.94±0.42d |
C20:0 | 0.09±0.02a | 0.27±0.08b | 0.06±0.01c | 0.07±0.02c | 0.13±0.03a |
C22:0 | 0.02±0.01a | 0.04±0.01b | 0.04±0.01b | 0.05±0.01b | 0.04±0.01b |
C24:0 | 0.11±0.04a | ND | ND | 0.01±0.00b | 0.04±0.01c |
C15:1 | 0.34±0.05a | 0.06±0.02b | 0.34±0.08a | 0.24±0.05c | 0.76±0.02d |
C16:1n-9 | 1.12±0.16a | 0.49±0.16b | 1.51±0.35c | 1.40±0.02c | 9.28±0.17d |
C18:1n-9 | 18.00±1.03a | 7.84±0.11b | 12.85±1.89c | 10.10±0.26c | 21.14±0.66a |
C20:1n-9 | 0.68±0.09a | 0.89±0.05b | 0.42±0.07c | 0.49±0.08c | 1.16±0.05b |
C22:1n-11 | 0.19±0.02a | 0.18±0.03a | 0.22±0.02a | 0.30±0.03b | 0.40±0.02c |
C24:1n-9 | 0.10±0.01a | 0.32±0.07b | 0.21±0.07c | 0.32±0.03b | 0.12±0.01a |
C18:2n-6 | 5.94±0.20a | 1.92±0.23b | 6.08±0.39a | 5.87±0.15a | 10.06±0.02c |
C18:3n-6 | 0.13±0.02a | 0.12±0.03a | 0.14±0.01a | 0.17±0.06a | 0.50±0.12b |
C20:2n-6 | 0.44±0.06a | 0.28±0.03b | 0.26±0.03b | 0.22±0.00b | 0.40±0.03a |
C20:3n-6 | 0.15±0.02a | 0.20±0.03b | 0.19±0.00b | 0.26±0.03c | 0.05±0.01d |
C20:4n-6 | 2.14±0.45a | 8.02±0.15b | 2.81±0.09a | 3.47±0.14c | 0.91±0.16d |
C22:5n-6 | 0.68±0.07a | 1.07±0.24b | 0.96±0.18b | 1.20±0.16b | 0.16±0.01c |
C18:3n-3 | 0.50±0.10a | 0.40±0.28a | 0.95±0.35b | 1.17±0.07b | 2.35±0.06c |
C18:4n-3 | 0.16±0.03a | 0.54±0.05b | 0.35±0.08c | 0.73±0.17d | 0.71±0.02d |
C20:3n-3 | 0.06±0.01a | 0.11±0.07b | 0.08±0.00b | 0.08±0.02b | 0.18±0.01c |
C20:4n-3 | 0.20±0.02a | 0.25±0.03a | 0.48±0.18b | 0.70±0.05c | 0.52±0.13b |
C20:5n-3 | 7.46±0.27a | 8.57±0.68a | 15.10±0.47b | 21.40±0.70c | 5.85±0.33d |
C22:5n-3 | 1.68±0.17a | 3.71±0.27b | 2.41±0.37c | 2.74±0.04c | 1.14±0.07d |
C22:6n-3 | 13.56±0.47a | 23.40±0.86b | 16.62±0.16c | 23.42±0.10b | 5.48±0.01d |
C16:2 | 0.71±0.28a | 0.30±0.04b | 0.80±0.17a | 1.09±0.05c | 1.40±0.07d |
C16:3 | 0.17±0.19a | 0.16±0.08a | 0.23±0.07b | 0.42±0.14c | 1.59±0.08d |
C16:4 | 0.03±0.01a | 0.05±0.01b | 0.04±0.02a | 0.09±0.01b | 0.88±0.06c |
C21:5 | 0.11±0.01a | 0.20±0.04b | 0.14±0.01c | 0.16±0.01c | 0.28±0.01d |
n-3/n-6 | 2.4±0.03a | 1.44±0.05b | 3.45±0.08c | 4.5±0.07d | 1.25±0.12b |
U/S | 1.4±0.1a | 2.8±0.02b | 1.3±0.11a | 3.18±0.04c | 1.09±0.02d |
UI | 1.06±0.01a | 1.7±0.02b | 2.3±0.1b | 2.9±0.02c | 0.9±0.1d |
Total PL (mg·g-1 Fw) | 0.52±0.05 | ||||
TAG (mg·g-1 Fw) | 0.12±0.01 |
Note: Results are expressed as mole % of total FAME based on peak areas. Data are means ± standard deviations from triplicate estimations (n =3). Means followed by different letters in the same line are significantly different (p < 0.05) using ANOVA (Tukey HSD test). SFA, MUFA, and PUFA mean saturated fatty acid, monounsaturated fatty acid, and polyunsaturated fatty acid, respectively. PC, PE, PI, PS and TAG mean phosphatidylcholine; phosphatidylethanolamine; phosphatidylinositol; phosphatidylserine and triacylglycerol, respectively. ND = not detected.
Geothermal water | |||||
---|---|---|---|---|---|
% Fattyacids | PC | PE | PS | PI | TAG |
C14:0 | 0.51±0.19a | 0.29±0.05b | 1.91±0.09c | 1.15±0.06d | 3.23±0.68e |
C15:0 | 0.65±0.03a | 0.68±0.28a | 0.44±0.07b | 0.40±0.06b | 7.92±0.65c |
C16:0 | 33.09±0.90a | 10.99±0.06b | 7.81±0.58b | 6.54±0.29b | 24.08±1.31a |
C17:0 | 0.74±0.10a | 0.19±0.07b | 0.46±0.07c | 1.19±0.04d | 2.60±0.27e |
C18:0 | 5.67±1.66a | 12.84±0.23b | 31.74±1.76c | 13.78±0.59b | 10.38±1.37b |
C20:0 | 0.10±0.01a | 0.47±0.01b | 0.07±0.01c | 0.22±0.04d | 0.10±0.01a |
C22:0 | 0.12±0.00a | 0.61±0.07b | 0.28±0.07c | 0.23±0.02c | 0,29±0.09c |
C15:1 | 0.55±0.00b | 0.29±0.02b | 0.31±0.05a | 0.64±0.14b | 0.75±0.03a |
C16:1n-9 | 2.43±0.25b | 1.94±0.06b | 0.93±0.06b | 1.41±0.08a | 6.03±0.33b |
C18:1n-9 | 25.32±2.07b | 15.54±0.32b | 9.00±0.52b | 11.06±0.14b | 15.41±0.32b |
C20:1n-9 | 0.71±0.07a | 0.83±0.13a | 0.81±0.12b | 0.45±0.13a | 1.20±0.34a |
C22:1n-11 | 0.59±0.12b | 0.53±0.07b | 0.17±0.01b | 0.06±0.01b | 0.37±0.02ab |
C24:1n-9 | ND | ND | ND | 1.31±0.24b | ND |
C18:2n-6 | 16.11±0.87b | 28.34±0.01b | 3.44±0.25b | 11.29±1.12b | 8.80±0.29b |
C18:3n-6 | ND | ND | 0.11±0.02a | 0.13±0.03a | ND |
C20:2n-6 | 0.18±0.02b | 1.09±0.11b | 0.14±0.02b | 0.26±0.11a | 1.49±0.29b |
C20:3n-6 | 0.55±0.07b | 2.16±0.01b | 0.94±0.06b | 0.76±0.15b | 0.36±0.05b |
C20:4n-6 | 1.06±0.18b | 1.53±0.35b | 9.34±0.91b | 5.22±0.44b | 1.98±0.28b |
C22:5n-6 | 1.18±0.06b | 1.23±0.27a | 1.46±0.21b | 3.17±0.30b | 0.69±0.15b |
C18:3n-3 | 0.73±0.01b | 1.27±0.05b | 0.26±0.04b | 0.56±0.05b | 1.17±0.25b |
C18:4n-3 | 0.44±0.03b | 1.37±0.03b | 0.08±0.01b | 0.49±0.05b | 1.46±0.22b |
C20:3n-3 | 0.07±0.05a | 1.21±0.07b | 0.36±0.03b | 0.37±0.05b | 0.23±0.08a |
C20:4n-3 | 0.37±0.02b | 1.20±0.19b | 0.62±0.09a | 0.69±0.08a | 0.63±0.20a |
C20:5n-3 | 2.74±0.55b | 5.16±0.51b | 5.54±0.55b | 6.44±0.08b | 2.47±0.41b |
C22:5n-3 | 1.25±0.01b | 1.67±0.05b | 5.10±0.45b | 4.12±0.07b | 0.94±0.13a |
C22:6n-3 | 2.78±0.46b | 6.59±0.16b | 17.36±1.45a | 25.63±0.66b | 4.96±0.12b |
C16:2 | 0.39±0.01b | 0.14±0.00b | 0.70±0.11a | 0.94±0.02b | 0.85±0.03b |
C16:3 | 0.84±0.07b | 1.14±0.03b | 0.23±0.03a | 0.87±0.07b | 1.20±0.19b |
C16:4 | 0.02±0.01a | 0.32±0.02b | 0.12±0.02b | 0.04±0.01b | 0.13±0.01b |
C21:5 | 0.83±0.11b | 0.36±0.04b | 0.24±0.03b | 0.42±0.05b | 0.27±0.02a |
n-3/n-6 | 0.44±0.02a | 2.83±0.02b | 1.89±0.03c | 1.7±0.09c | 0.8±0.01d |
U/S | 1.2±0.03a | 1.44±0.1b | 1.7±0.02c | 3.17±0.1d | 1.8±0.05c |
UI | 1.5±0.05a | 2.5±0.06b | 2.6±0.04b | 3.1±0.1c | 1.47±0.08c |
Total PL (mg·g-1 Fw) | 0.64±0.02 | ||||
TAG (mg·g-1 Fw) | 0.27±0.05 |
Note: Results are expressed as mole % of total FAME based on peak areas. Data are means ± standard deviations from triplicate estimations (n =3). Means followed by different letters in the same line are significantly different (p < 0.05) using ANOVA (Tukey HSD test). SFA, MUFA, and PUFA mean saturated fatty acid, monounsaturated fatty acid, and polyunsaturated fatty acid, respectively. PC, PE, PI, PS and TAG mean phosphatidylcholine; phosphatidylethanolamine; phosphatidylinositol; phosphatidylserine and triacylglycerol, respectively. ND = not detected.
To better visualize the differences in FA in the lipid classes, five-line charts referring to the major fatty acid groups, namely satured fatty acids (SFA), MUFAs and PUFAs, are presented in Figure 1. The results showed that in almost all lipid fractions of the seawater fishes, PUFA (representing up to 50% of the total FA of PS species) was by far the major FA group followed by SFA with corresponding increases in UI and U/S. By contrast, in geothermal fish the distribution proportions of the PUFA series substantially differed between polar lipid fractions. Indeed, PUFA n-3 was particularly abundant in PS and PI fractions while the n-6 series dominated the PC and PE subclass. Additionally, it was found that the level of n-3/n-6 ratio was markedly increased in the PE fraction of the geothermal group. It is also important to note that, SFA stood out as the most abundant fatty acid group in TAG mainly in the geothermal group.
The principal component analysis (PCA) was performed to gain better insight into the effects of rearing conditions on the fatty acid composition of lipid classes in the flesh of the thick-lipped grey mullet, C. labrosus. Figure 2 depicts the twoprincipalcomponents that described 57.72% of the total data variability (PC1 34.22% and PC2 23.5%). The PCA biplot of the overall data described a clear separation between samples from the seawater and the geothermal water. The projection of individuals (each fatty acid from each lipid class of the different fish groups) on the same factorial plan (1:2) showed that samples could be clustered into two groups (I and II). Group I, which consisted of individuals sampled from geothermal water, showed a positive contribution to the first component (PC1), which was characterized by the substantial PUFA n-6 in the PE class. Group II was, however, characterized by high PUFA, particularly n-3 PUFA, DHA and EPA of the phospholipid class representing individuals sampled from seawater (Figure 2).
4. DISCUSSION
⌅Teleosteen fish are exposed to varying and occasionally extreme environmental conditions that can produce potent effects on their physiology (Somero, 2004Somero GN. 2004. Adaptation of enzymes to temperature: searching for basic strategies. Comp. Biochem. Physiol B. 139, 321-333. https://doi.org/10.1016/j.cbpc.2004.05.003 ). The Thick-lipped grey mullet C. labrosus, is an euryhaline and eurytherm teleost that presents the ability to live under different environmental conditions of salinity and temperature (Azaza et al., 2008; Cardona, 2006Cardona L .2006. Habitat selection by grey mullets (Osteichthyes: Mugilidae) in Mediterranean estuaries: the role of salinity. Sci Marpp. 70, 443-455.; Rabeh et al., 2013Rabeh I, Khaoula, Telahigue k, Gazali N, Chetoui I, Boussoufa D, Besbes R, Cafsi M. 2013. Time course of changes in fatty acid composition in the osmoregulatory organs of the thicklip grey mullet (Chelon labrosus) during acclimation to low salinity. Mar. Freshwater Behav. Physiol. 46, 59-73. https://doi.org/10.1080/10236244.2013.793470 ). It is known that temperature and salinity are the key factors in teleost fish that cause fluctuations in the fluidity of cell membranes (Soengas et al., 2007Soengas JL, Sangiao-Alvarellos S, Láiz-Carrión R, Mancera J. 2007. Fish osmoregulation. In: Kapoor BG, editor. Energy metabolism and osmotic acclimation in teleost fish. Enfield: Science Publishers; p. 277-308. ) which largely depends on their lipid components. In this context, changes in membrane lipid composition is a key molecular mechanism of adaptation that is commonly called homeoviscous adaptation (Hazel,1995Hazel JR. 1995. Thermal adaptation in biological membranes: is homeoviscous adaptation the explanation? Ann. Rev. Physiol. 57, 19-42. doi: 10.1146/annurev.ph.57.030195.000315.).
In the current study, lipid class composition and the fatty acid profiles of the two fish groups analyzed were shown to respond selectively and significantly to environmental conditions. Our data revealed that the lipids of the examined fractions from the seawater fishes were characterized by higher values for PUFA n-3, particularly in the PI fraction. However, a high content of PUFA n-6 was recorded in the PI fraction of the geothermal group. In addition, a substantial amount of SFA was observed in the TAG for the two groups. Our findings corroborate with those recorded by some authors who have reported that long-chain fatty acids were probably important for PI to successfully conduct particular cellular functions, including the role in cell growth, signal transduction processes and the membrane anchoring of proteins in plants (Riekhof and Benning, 2009Riekhof WR, Benning C. 2009. Glycerolipid biosynthesis. In: Stern D (ed.) The chlamydomonas source book: organellar and metabolic processes. Academic Press, Amsterdam, pp 41-68). Consequently, we hypothesize that an increase in PUFA n-6 in the PE of geothermal water fish might compensate for the effects of warm and lower salinity water, which tends to increase lipid rigidity.
The detailed investigation of C. labrosus groups considered in this study indicated that among the major fatty acids of the different lipid classes are C16:0, C18:1, C22:5n3, C18:2n6, C20:4n6, EPA, and DHA. First, with respect to saturates, the highest levels are usally contained in PE and TAG in the two C. labrosus groups. Among SFA, the major components were C16:0, followed by C18:0. Previous studies have reported that SFA, mainly palmitic acid, which is the main fatty acid synthesized de novo in fish, is classically associated with HUFAs in the diacyl phospholipids of fish (Steffens, 1997Steffens W. 1997. Effects of variation in essential fatty acids in fish feeds on nutritive value of freshwater fish for humans. Aquaculture. 151, 97-119. https://doi.org/10.1016/S0044-8486(96)01493-7 ; Li et al., 2011Li EC, Arena L, Lizama G, Gaxiola G, Cuzon G, Rosas C. 2011. Glutamate dehydrogenase and Na+-K+ ATPase expression and growth response of Litopenaeus vannameito different salinities and dietary protein levels. Chin. J. Oceanol. Limnol. 29, 343-349. http://dx.doi.org/10.1007/s00343-011-0093-8 ). In fact, the important levels of SFA may result from a lipogenic activity (Dias et al., 1998Dias J, Alvarez MJ, Diez A, Arzel J, Corraze G, Bautista JM, Kaushik SJ. 1998. Regulation of hepatic lipogenesis by dietary protein/energy ratio in juvenile European seabass (Dicentrarchuslabrax). Aquaculture 161, 169-186. https://doi.org/10.1016/S0044-8486(97)00268-8 ) that can be biosynthesized by fish through a conventional pathway catalyzed by cytosolic fatty acid synthetase (Sargent et al., 2002Sargent JR, Tocher DR and Bell JG. 2002. The lipids. pp. 181-257. In: Fish Nutrition, 3rd Edition, Ch.4. (Halver, J. E., Ed.). San Diego: Academic Press.). In addition, the TAG fraction in fish can also contain high levels of monoenoic C16-C18 fatty acids that are intensively synthesized in so-called “fatty” fish species to provide energy reserves. Likewise, the findings of Arts et al. (2009)Arts MT, Brett MT, Kainz MJ. 2009. Lipids in Aquatic Ecosystems, Springer, New York, p. 377. revealed that triacylglycerol synthesis notably begins with a polar lipid that mostly has one of five common FA (e.g., myristic, C14:0; palmitic, C16:0; palmitoleic, C16:1n-7; stearic, C18:0; or oleic, C18:1n-9) in the sn-1 position.
The highest MUFA detected in the lipid class of both fish groups was C18:1 with markedly impotant levels in the PC of the two studied groups. This is presumably due to its dominance in the commercial feed used in this survey. Another aspect to be taken into account is that C18:1 is a typical MUFA in fish which is most often considered from the standpoint of its energy importance (Sargent et al.,1989Sargent JR, Henderson RJ, Tocher DR. 1989. The lipids. pp.153-218. In: Fish Nutrition, second edition. (Halver, J. E., Ed.). New York: Academic Press. ). It is also interesting to point out that PC in fish tissues is commonly rich in C18:1 and appears to be more easily influenced by dietary fatty acids than other phosphoglycerides (Tocher, 2003Tocher DR. 2003. Metabolism and functions of lipids and fatty acids in teleost fish. Rev. Fish. Sci. 11, 107-184. https://doi.org/10.1080/713610925 ) and it should be the precise reason for the preponderance of these fatty acids.
It is remarkable that the phospholipids of the geothermal groups were found to contain significantly higher contents of C18:2n-6 compared to the seawater groups. The same conclusion was reached in a experiment in which M. cephalus fry were acclimatized to freshwater (El Cafsi et al., 2003El Cafsi M, Romdhane MS, Chaouch A, Masmoudi W, Kheriji S, Chanussot F, Cherif A. 2003. Qualitative needs of lipids by mullet, Mugil cephalus, fry during freshwater acclimatation. Aquaculture. 225, 233-241. https://doi.org/10.1016/S0044-8486(03)00292-8 ). The drop in salinity in the geothermal habit may have led to an increase in the percentages of PUFAs and particularly the n-6 series in the PE fraction. On the other hand, it is also possible that the mechanism involved in the catabolism of C18:2n-6 was more active in the fresh water fish than those from seawater (Sargent et al., 1989Sargent JR, Henderson RJ, Tocher DR. 1989. The lipids. pp.153-218. In: Fish Nutrition, second edition. (Halver, J. E., Ed.). New York: Academic Press. ; Kheriji et al., 2003Kheriji S, El Cafsi M, Masmoudi W, Castell JD, Romdhane MS. 2003. Salinity and temperature effects onthe lipid composition of mullet sea fry (Mugil cephalus, Linne, 1758). Aquacult Int. 11, 571-582. https://doi.org/10.1023/B:AQUI.0000013321.93743.6d ). It is also noteworthy that C18:2n-6, which is a well-known key dietary compoment constitutes a good energy source from fish (Castell et al., 1972Castell JD, Sinnhuber RO, Wales JH, Lee DJ. 1972. Essential fatty acids in the diet of rainbow trout (Salmo gairdneri): growth, feed conversion and some gross deficiency symptoms. J. Nutr. 102, 77. https://doi.org/10.1093/jn/102.1.77 ; Glencross, 2014Glencross BD. 2009. Exploring the nutritional demand for essential fatty acids by aquaculture species. Rev. Aquacult. 1, 71-124. https://doi.org/10.1111/j.1753-5131.2009.01006.x ). Herein, the dominant FA in the food is C18:2n-6 (29%), which allow us to explain the high levels of PUFA n-6 in the fillet of C. labrosus. Indeed, dietary lipids are a source of fatty acidswhich are required for the synthesis of new cellular lipids and for the turnover of existing lipids.
In the present study, an increased proportion of physiologically significant FAs (C22:6n-3 and C20:5n-3) in the phospholipid class was observed for both groups. It has been well established that fish phospholipids are usually considered as a physiologically crucial lipid classes since they are rich in PUFA, predominately DHA and EPA (Sargent et al., 1993Sargent JR, Bell JG, Bell MV, Henderson RJ, Tocher DR. 1993. The metabolism of phospholipids and polyunsaturated fatty acids in fish. In: Aquaculture: Fundamental and Applied Research. Coastal and Estuarine Studies (Lahlou, B. &Vitiello, P. eds.), vol. 43. pp103-124. American Geophysical Union, Washington, DC.). Typically, EPA occurs in lower proportions than DHA due to the availability of these FA as dietary sources. Interestingly, C22:6n-3 is abundant in PI and PS particularly in geothermal specimens. The importance of DHA in PS was previously attributed to the ability of C. labrosus to assimilate this FA. The abundance of DHA observed in the polar lipids started with recognition that this FA has a unique conformation dictacted by helical or an angle iron shape with an overall length similar to that of C16:0 (Applegate et al., 1986Applegate KR, Glomset JA. 1986. Computer-based modeling of the conformation and packing properties of docosahexaenoic acid. J. Lipid Res. 27, 658-680.). This structure favors the formation of the hexagonal phase in phosphoglycerides, above all in C22:6n-3 phosphoglycerides containing small head groups such as phosphatidylserine and this will facilate very fast conformational changes in membranes (Brown,1994Brown MF. 1994. Modulation of rhodopsin function by properties of the membrane bilayer. Chem. Phys. Lipids 73, 159-180.). In a general way, such essential FA are, for example, involved in the modulation of the properties of the lipid phase of the cell membrane, membrane bound enzymes as well as the precursor of functionally important lipooxygenase products (Koven et al., 2011Koven W, Barr Y, Lutzky S, Ben-Atia I, Weiss R, Harel M, Behrens P, Tandler A. 2001. The effect of dietary arachidonic acid (20:4 n6) on growth, survival and resistance to handling stress in gilthead seabream (Sparus aurata) larvae. Aquaculture 193, 107-122. https://doi.org/10.1016/S0044-8486%2800%2900479-8 ). Previous investigations have shown that variations in polar lipid contents and their individual fractions particularly in muscle are key compensatory mechanisms in organisms that guarantee optimum performance of several membrane-bound enzymes under different environmental conditions (Los, 2001Los DA. 2001. Structure, regulation of expression and functioning of fatty acid desaturases. Biol. Chem. Rev. 41, 163-198.; Cengiz et al., 2012 Cengiz EI, Ünlü1 E, Bashan M, Ali Satar A, Uysal E. 2012. Effects of Seasonal Variations on the Fatty Acid Composition of Total Lipid, Phospholipid and Triacylglicerol in the Dorsal Muscle of Mesopotamian Catfish (Silurus triostegus Heckel, 1843) in Tigris River (Turkey). Tur. J. Fish. Aquat. Sci. 12 33-39. https://doi.org/10.4194/1303-2712-v12105 ).
5. CONCLUSIONS
⌅This study provides initial insight into the lipid class composition of C. labrosus reared under different conditions. From the obtained results we can conclude that variations in the salinity and temperature chiefly affected the PUFA group. Indeed, we noticed that the juvenile C. labrosus reared in seawater conditions were characterized by the predominance of the n-3 series in phospholipid fractions. As for specimens from the geothermal system, high levels of n-3 PUFA were recorded in the PS and PI fraction, while PC and PE were dominated by the n-6 series. This suggests the aibility of C. labrosus to remodel its lipid composition to adapt to extreme environmental conditions. The obtained results can provide useful basic information that can help in the management of inland aquaculture practices. However, futher investigations are needed to better understand the impact of abiotic parameters on the lipid metabolism of of this promising species.