Egyptian Journal of Aquatic Research (2016) xxx, xxx–xxx H O S T E D BY National Institute of Oceanography and Fisheries Egyptian Journal of Aquatic Research http://ees.elsevier.com/ejar www.sciencedirect.com FULL LENGTH ARTICLE Induction of the synthesis of bioactive compounds of the marine alga Tetraselmis tetrathele (West) Butcher grown under salinity stress Hala Yassin El-Kassas a,*, Mostafa M El-Sheekh b a b National Institute of Oceanography and Fisheries, Hydrobiology, Alexandria 21556, Egypt Botany Department, Faculty of Science, Tanta University, Tanta, Egypt Received 14 July 2016; revised 13 October 2016; accepted 31 October 2016 KEYWORDS Phytochemicals; Tetraselmis tetrathele; Carotenoids; Salinity; Fatty acids Abstract This work aims at the induction of the synthesis bioactive compounds in microalgae which are used in aquacultures Experiments were done using Tetraselmis tetrathele in batch culture for days under different salinity levels The growth of the alga at salinity 20 ppm was increased by fivefold and synthesis of carotenoids by 20-fold in comparison to the controlled Increasing NaCl concentration resulted in increasing the fatty acid accumulation in T tetrathele cells Saturated fatty acids were the main constituent in the fatty acid methyl esters (FAMEs) (3.48 mg/g) at salinity 25 ppm The predominated fatty acids were tridecylic, myristic and pentadecanoic which have potential antimicrobial activities GC–MS analyses of the alga acetone extract grown under different NaCl concentrations were established The results showed the presence of 18 bioactive compounds: 9-octadecenamide; in addition to the different esters of some fatty acids: hexanedioic, 1,2-cyclohexanedicarboxylic, phthalic, oleanitrile, hexanedioic and 1,2-cyclohexanedicarboxylic (71.5%; 64.9%; 55.4%; 49.6%; 18.7%; 25.2% and 14.5%, respectively) The study suggested that the alga biosynthesized various bioactive compounds under different salinity levels as defense mechanisms Accordingly, the growth of T tetrathele under salinity stress before being used in aquacultures is recommended Ó 2016 National Institute of Oceanography and Fisheries Hosting by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Introduction Tetraselmis is a prasinophyte commonly used in feeding of marine animals in aquacultures due to its nutritional value * Corresponding author E-mail addresses: halayassin12@yahoo.com (H.Y El-Kassas), mostafaelsheikh@science.tanta.edu.eg (M.M El-Sheekh) Peer review under responsibility of National Institute of Oceanography and Fisheries (Fa´bregas et al., 2001) It is also used coincidence with Nannochloropsis for producing rotifers In addition finfish hatcheries which produce algae for rotifers feeding or Artemia enrichment typically use different algal species including Tetraselmis tetrathele (Hemaiswarya et al., 2011) Dhont and Van Stappen (2003) in their study on the nutritional value of food sources, stated that the biochemical constituents (polyunsaturated fatty acids, vitamins, sterols, carbohydrates and proteins) are important indicators for any http://dx.doi.org/10.1016/j.ejar.2016.10.006 1687-4285 Ó 2016 National Institute of Oceanography and Fisheries Hosting by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Please cite this article in press as: El-Kassas, H.Y., El-Sheekh, M.M Induction of the synthesis of bioactive compounds of the marine alga Tetraselmis tetrathele (West) Butcher grown under salinity stressTetraselmis tetrathele –> Egyptian Journal of Aquatic Research (2016), http://dx.doi.org/10.1016/j.ejar.2016.10.006 food quality It is also documented that, the nutritional value of some microalgae depends on the biochemical composition of algae (Durmaz, 2007) Additionally, Ponis et al (2003), compared feeding with non-living and living microalgae diets, and they concluded that non-living diets give lower growth and higher mortalities, however live microalgae diets give higher growth and lower mortalities They attributed this due to the live diets that are free from contamination and toxins Microalgae have wide and diverse nutritional uses in aquaculture especially the fish feeding by enrichment of zooplankton and also their uses to improve the colour of the flesh of salmonids (Hemaiswarya et al., 2011) It is well known that microalgae are rich in colour pigments, the transfer of such colored pigments to zooplankton increasestheir nutritional values when fish feed on these zooplankton which represents the main food in aquatic systems (Raja et al., 2008) The copepod Temora sp showed a high content of the pigments such as lutein and astaxanthin where as, the dominant pigments in Artemia was canthaxanthin (Gentsch et al., 2009) In aquaculture, the poly-unsaturated fatty acids (PUFAs) which derived from microalgae are essential for various larvae feeding (Sargent et al., 1997) Microalgae differ in their biochemical and nutritional constituents as they differ in their size, growth rate and environmental requirements (Helm et al., 2004) In aquaculture, it is forbidden for producers from using synthetic growth promoters or antibiotics for cost-effective intensive production, because the European Union prohibited producers to use such synthetic compounds and in order to overcome this problem, various alternative non-antibiotic feed are allowed in order to improve economic production (Anadon, 2006) Taking all of these parameters into consideration is essential when choosing a species for culture The microalgal strain used in aquaculture should have some criteria such as lack of toxicity, ease of culturing, a non-rigid cell wall which can be easily digested to make nutritional constituents available, high nutritional value (Patil et al., 2007) Several studies investigated the visibility of microalgal production in aquaculture for its uses as nutritional food for fish, and other aquatic organisms (Hemaiswarya et al., 2011) They also concluded that the pigments of such algae enhance the color of flesh salmonods Different algae have the ability to develop defense mechanisms to compete in adverse competitive environments by induction and synthesis of different compounds through different metabolic pathways Algae are promising aquatic microorganisms that can synthesize active compounds (Cardozo et al., 2007) Austin et al (1992) investigated the anti-fouling activity of some microalgae species El-Sheekh et al (2006, 2008, 2014) explained the antimicrobial activity of microalgae by different specific bioactive substances secreted either internal or extracellular of the algal cells Furthermore, Olsen et al (2000) concluded that the incubation of brine shrimp, Artemia franciscana with the marine alga Tetraselmis sp reduced the number and the percentage counts of associated bacteria, as well as hemolytic bacteria, which was correlated to algal components In spite of the large diverse numbers of microalgae species, a low number has been investigated for its biochemical and nutritional constituents and also for investigating the different mechanisms of such algae to survive in diverse conditions such as salinity stresses (Adarme-Vega, 2014) In aquaculture systems the salinity responses are good indicators for controlling H.Y El-Kassas, M.M El-Sheekh the environmental conditions for optimizing cell growth and multiplications (Walsh et al., 1987) Therefore, the present study aims at: (a) Monitoring the growth of T tetrathele at different salinity levels (b) Estimating the bioactive compounds synthesized by the alga grown under the halo stress in addition to pigments composition as well as fatty acids contents in T tetrathele grown under these conditions Materials and methods The alga strain and development conditions Tetraselmis tetrathele was obtained from Phycology laboratory (Department of Botany), Faculty of Science, Tanta University, Egypt The alga was cultivated in a batch culture in L Erlenmeyer flasks with Walne’s medium (Walne, 1970) at an initial inoculum of  105 cells mLÀ1 For the production of biomass, the algae cells in the exponential growth phase were transferred into fresh sterile medium [10% (v/v) of inoculum] Cultures were illuminated by tubular fluorescent lamps (PHILIPS Master TL-D 85 W/840) The light intensity at the surface of the culturing vessels was 100 l mol photons mÀ2 sÀ1 with a photo-period of 16:8 h light: dark at 25 ± °C (Teo et al., 2014) For induction of bioactive compounds biosynthesis, the alga was grown under different salinity values (25, 30, 35 and 40 ppm), where 30 ppm is the control Growth assessment Optical density (OD) method The microalga growth was measured as optical density (OD) at 540 nm using a spectrophotometer Spekol 1300, Analytika Jena, Spain at 540 nm (Rocha Jorge et al., 2003) Cell count The cultures were sampled at a 24 h interval and microalga growth was monitored by counting the cell number with a hemocytometer slide Pigments estimation A known volume of algal culture was centrifuged at 8000 rpm for 10 and the pellet was extracted with known volume of ethyl alcohol and kept in water bath for 30 at 60 °C, and then centrifuged again Absorbance of the pooled extracts was measured using UV-6800 UV/Vis Spectrophotometer (JENWAY-Germany) at 650, 665 and 452 nm Calculations were made following the formulae described by Sengar (1970) for chlorophyll a and carotenoids Fatty acids Preparation of fatty acid methyl ester from total lipid was performed according to Radwan (1978) All analyses for identification of fatty acid were performed using gas chromatography (GC system Hp, Germany, serial No 6890 D 1530 A serial DE 00000348) equipped with a flame ionization detector; SP-2340 Please cite this article in press as: El-Kassas, H.Y., El-Sheekh, M.M Induction of the synthesis of bioactive compounds of the marine alga Tetraselmis tetrathele (West) Butcher grown under salinity stressTetraselmis tetrathele –> Egyptian Journal of Aquatic Research (2016), http://dx.doi.org/10.1016/j.ejar.2016.10.006 Synthesis of bioactive compounds of the marine alga Tetraselmis tetrathele 16 Statistical analysis All values presented in this study are the mean of triplicate trials for each treatment; error bars in the figures depict the standard deviations (SD) of these triplicates Statistical analysis [One-way Analysis of Variance (ANOVA)] was carried out to assess if there were significant differences (P 0.05) in cell densities and phytochemical concentrations between the cultures at different salinity values by using the SPSS Statistical program Results Growth assessment Optical density (OD) method Cell no mL-1X105 Chemical analysis of algal extracts using gas chromatography– mass spectrometry (GC/MS) The GC–MS analysis was done using GC–MS model Hewlett Packard HB 5890 gas liquid chromatography (GLC) coupled with 5989 B series mass spectrometer (MS) Identification of the individual components was performed by comparison of mass spectra with the profiles from the Wiley GC–MS 275 libraries and was compared with those in the mass spectrum library of the corresponding organic compounds (Pandey et al., 2010) All compounds are reported with their corresponding probabilities at their retention time 14 25ppm 30ppm 12 35ppm 40ppm 10 0 Time (days) Figure Growth patterns of Tetraselmis tetrathele cells growing at different salinity values expressed as cell no mLÀ1  105 growth of the tested alga was inhibited in salt stress as compared to control The cell counts increased by nearly 2.8 folds at 25 ppm However, the growth maxima were recorded at 30 ppm and 35 ppm Accordingly, algal cell division showed good adaptation to the highest salinities and the cells not go into osmotic shock and die By the end of the incubation period, the cell counts increased by times On the contrary, the growth of the alga at 40 ppm presented some cellular death and consequently the smallest values 13.9  105 cells mLÀ1 A visible change in the colour of the culture was observed from green to yellowish brown The comparison changes in salinity primarily altered algal biomass with 30 and 40 ppm having the highest count Optimum growth of T tetrathele ranged in the salinity levels between 30 and 35 ppm The results (Fig 1) show the OD540 of T tetrathele obtained during the growth under different salinity concentrations The cultures were sampled at 24 h interval with starting optical density reading around 0.20 From the overall trend of growth, T tetrathele grows better under salinity ranged between 30 ppm and 40 ppm During the growth in the medium at 25 ppm, the OD540 of the algal culture increased five folds by the end of the study period, as compared with the same culture at zero time However, the culture grown at 30 ppm and 35 ppm increased by relatively times,similar to the algal growth with salinity value of 25 ppm, when the alga grown at 40 ppm, the OD540 increased by only times The amount of chlorophyll a of T tetrathele cells exposed to different salinities for days are presented in Fig The results show that growth of the alga in a medium with salinity values of 25 ppm, 30 ppm and 35 ppm increased the chlorophyll content by nearly 2, 2.5, and 2.5 folds; respectively Similar to the growth of the alga at 25 ppm, increasing the salinity concentrations to 40 ppm have caused the chlorophyll a content to increase by only folds Cell count Carotenoids The results (Fig 2) show the growth curves of T tetrathele expressed as cells mLÀ1  105 The results revealed that the Exposure of T tetrathele cells to high salinities resulted in a pronounced increase in carotenoids concentrations (Fig 4) Pigments estimation Chlorophyll a 1.4 14 25ppm 30ppm 35ppm 40ppm 0.8 0.6 0.4 0.2 25ppm 35ppm 12 Chlorophyll a ( μgL-1) OD540 1.2 30ppm 40ppm 10 2 Time (days) 0 Time (days) Figure Growth pattern of Tetraselmis tetrathele cells growing at different salinity values expressed as OD540 ± slandered deviation Figure Temporal variability of chlorophyll a content (lg LÀ1) of Tetraselmis tetrathele at different salinity values Please cite this article in press as: El-Kassas, H.Y., El-Sheekh, M.M Induction of the synthesis of bioactive compounds of the marine alga Tetraselmis tetrathele (West) Butcher grown under salinity stressTetraselmis tetrathele –> Egyptian Journal of Aquatic Research (2016), http://dx.doi.org/10.1016/j.ejar.2016.10.006 H.Y El-Kassas, M.M El-Sheekh During the growth of the alga at 25 ppm NaCl, carotenoids increased by nearly 20-fold as compared with the control culture after days Opposite to the growth response, increasing NaCl concentration led to the increase in carotenoids, where it reached 0.12 ± 0.05 lg LÀ1 at salt concentration of 40 ppm fatty acids These were myristic, butanoic, palmitic as well as pentadecanoic and octadecanoic acids Also, x9c (oleic acid) was represented by 0.57 mg/g that was completely absent in the alga grown under the other different NaCl concentrations Gas chromatography–mass spectrometry (GC–MS analyses) Fatty acids Fatty acid accumulation in T tetrathele grown at different salinity concentrations (Table 1) shows that increasing of NaCl concentration in the growth medium led to an increase in the amounts of fatty acids During the growth of T tetrathele at 25 ppm, the alga accumulated more than 50% of its fatty acids content in the saturated form (3.48 mg/g) The predominated fatty acids were Tridecylic, Myristic and Pentadecanoic; represented by 0.63, 0.97 and 0.68 mg/g, respectively Similarly, the alga accumulated 5.49 mg/g at 35 ppm of the total fatty acids Under these conditions 3.02 mg of the fatty acids were monounsaturated (Myristoleic acid and cis-10-pentadecenoic acid) Furthermore, under salinity stress, (40 ppm), T tetrathele accumulated 34.41 mg/g fatty acids More than 90% of the accumulated fatty acids under salinity stress were saturated GC–MS analysis of acetone extract of T tetrathele grown under different NaCl concentrations was carried out to detect the type (s) of compounds present Table 2, showed the presence of 18 compounds as a function of time Each peak represents a discrete chemical compound Retention times of these phytochemicals were 32.0, 34.0, 36.0, 38.0, 28.9, 28.0, 29.38, 32.30, 34.52, 30.69 and 28.70 These compounds include 12 ester compounds in addition to Amides The major constituents of these bioactive compounds were: 9-octadecenamide (71.5%) as well as different esters of some fatty acids including; hexanedioic acid (64.9%); 1, 2Cyclohexanedicarboxylic acid (55.4%); phthalic acid (49.6%); oleanitrile (18.7%); hexanedioic acid (25.2%); and 1, 2-cyclohexanedicarboxylic acid (14.5%) in addition to phthalic acid derivatives Statistical analysis 0.14 Carotenoids (μgL-1) 0.12 25ppm 30ppm 35ppm 40ppm One way analysis of variance (ANOVA) test (Table 3) revealed that there were significant relationships between the cultures at different salinity levels and growth of the prasinophyte T tetrathele (cell numbers & OD540) also between salinity levels and chlorophyll a as well as salinity levels and carotenoids (p 0.05) 0.1 0.08 0.06 0.04 0.02 Discussion Time (days) Figure Temporal variability of carotenoids content (lg LÀ1) of Tetraselmis tetrathele at different salinity values The prasinophyte T tetrathele is a hardy minute alga that can withstand different adverse environmental conditions T tetrathele is also characterized by its high nutritional value and simple requirements for culture It can replace rotifers and Table Fatty acid methyl ester of Tetraselmis tetrathele biosynthesized in response to growth at different salinity levels during days of incubation Total conc of FAs in mg/g of sample Fatty Acids 25 ppm 30 ppm 35 ppm 40 ppm C4:0 (Butanoic) C8:0 (Caproic) C10:0 (Capric) C12:0 (Lauric) C13:0 (Tridecylic) C14:0 (Myristic) C15:0 (Pentadecanoic) C16:0 (Palmitic) C17:0 (Heptadecyclic) C18:0 (Octadecanoic) C14:1 (Myristoleic) C15:1 (cis-10-pentadecenoic) C18:1 x9c (Oleic) P Saturated FAs P Monounsaturated FAs 0.09 0.0 0.03 0.13 0.63 0.97 0.68 0.18 0.00 0.09 0.32 0.37 0.00 3.48 2.79 1.00 0.01 0.00 0.14 0.67 1.03 0.14 0.31 0.00 0.20 0.34 0.40 0.00 4.06 0.74 0.40 0.0 0.00 0.09 0.50 0.76 0.53 0.14 0.02 0.03 0.26 0.29 0.00 2.47 3.02 2.60 0.0 0.06 0.32 1.44 23.56 1.59 1.72 0.00 0.87 0.75 0.96 0.57 32.16 2.28 Total conc of FAs in mg/g of sample 6.27 4.80 5.49 34 41 Please cite this article in press as: El-Kassas, H.Y., El-Sheekh, M.M Induction of the synthesis of bioactive compounds of the marine alga Tetraselmis tetrathele (West) Butcher grown under salinity stressTetraselmis tetrathele –> Egyptian Journal of Aquatic Research (2016), http://dx.doi.org/10.1016/j.ejar.2016.10.006 Synthesis of bioactive compounds of the marine alga Tetraselmis tetrathele Table Phytochemicals recorded in the acetone extract of Tetraselmis tetrathele induced due to growth under different salinity levels identified by GC–MS Compound name M.F M.W Nature Salinity 25 ppm 32.0 49.6 34.0 5.25 36.0 3.2 38.0 2.91 R Time Probability% Phthalic acid, bis(7-methyloctyl) ester 1,2-Benzenedicarboxylic acid, dinonyl ester Phthalic acid, 2-ethylbutyl nonyl ester pyrrolidinecarboxylate Phthalic acid, decylnonyl ester C26H42O4 C26H42O4 C23H36O4 C27H44O4 418 418 376 432 Ester Ester Ester Ester Salinity 30 ppm 28.9 71.5 28.0 18.7 28.9 11.8 28.9 11.5 28.9 7.88 28.9 4.71 28.9 2.34 9-Octadecenamide (Oleamide) Oleanitrile Hexadecanoic acid, 1-(hydroxymethyl)-1,2-ethanediyl ester 2-Hexadecanol Hexadecanoic acid cis-11-Eicosenamide 9-Octadecenamide, (Z)- C18H35NO C18H33N C35H68O5 C16H34O C16H32O2 C20H39NO C18H35NO 281 263 568 242 256 309 281 Amide of oleic acid Salinity 35 ppm 29.38 64.9 29.38 14.6 29.38 10.6 Hexanedioic acid, bis(2-ethylhexyl) ester Hexanedioic acid, mono(2-ethylhexyl)ester Hexanedioic acid, diisooctyl ester C22H42O4 C14H26O4 C22H42O4 370 258 370 Ester compound Ester compound Ester compound Salinity 40 ppm 32.30 55.4 34.52 14.5 30.69 6.6 28.70 4.13 1,2-Cyclohexanedicarboxylic 1,2-Cyclohexanedicarboxylic 1,2-Cyclohexanedicarboxylic 1,2-Cyclohexanedicarboxylic C26H48O4 C24H42O4 C23H42O4 C25H46O4 424 394 382 410 Ester Ester Ester Ester acid, acid, acid, acid, dinonyl ester cyclohexylmethylnonyl ester 2-methylpent-3-yl nonyl ester nonyl 4-octyl ester Artemia nauplii as live diet during the different feeding stage (Ronquillo et al., 1997) Generally, Tetraselmis sp represented a good and simple model for the production of better nutritional quality microalgae strains used in aquaculture (Adarme-Vega, 2014) In this study the growth yields of T tetrathele was high under the salinity concentrations of 30 ppm and 35 ppm and the statistical analyses (ANOVA) results affirmed this point Many previous studies Hieks et al (1989), reported that the alga when grown under different stress conditions, it produce functional proteins which protect the cell against surroundings’ adverse conditions These proteins improve the cell division-machinery and operate the mechanism of gene regulation efficiently On the other hand Vazquez-Duhalt and Arredondo-Vega (1991), during their study on halo adaptation of a green alga reported that the diminished yields of biomass were probably due to non adaptability of the organism to higher salinity However, Rao et al (2007) reported that excess salinity inhibited photosynthesis process, thus reduces the yield of algal biomass in the green alga Botryococcus braunii Considering chlorophyll a content of T tetrathele, increasing the salinity concentrations to 40 ppm have caused lower increases in the alga chlorophyll a content than that when grown under the other NaCl concentrations In this respect Moradi and Ismail (2007), reported that the decreased chlorophyll contents at higher salinities are a direct result of the reduction in photosynthetic rate due to salt osmotic and toxic ionic stress Furthermore, carotenoid concentrations recorded the highest value at 40 ppm NaCl and (ANOVA) results confirmed the significant relationship between salinity concentrations at which the alga grown and its contents of carotenoids (P 0.05) The obtained results in this work are in agreements compound compound compound compound Ester compound Amide Amide compound compound compound compound with those obtained by Pisal and Lele (2005) who reported that the carotene is a secondary metabolite produced by the cells in stress condition as cell protecting mechanism They stated also that compounds such as alkaloids, carotenoids, flavonoids and fatty acids from the alga Chlorococcum humicola grown under higher saline conditions are present in its different organic extracts including acetone extract of the alga Moreover, the antibacterial effect of the pigments: b-carotene and chlorophyll have been reported by Bhagavathy et al (2011) However, Holzinger and Karsten (2013) concluded that algae show different mechanisms for survival in stressed conditions such as altering and adapting the fatty acid content to avoid osmotic stress as a result of rapid salinity changes, which may occur in natural environments Interestingly, this is the first report of production of fatty acids in T tetrathele cultures in response to different salinity concentrations and bioactive compounds as a defense mechanism Tetraselimis tetrathele accumulated 34.41 mg/g fatty acids when grown under salinity stress (i.e., 40 ppm NaCl) and more than 90% of the FA fraction was saturated These were myristic, butanoic, palmitic as well as pentadecanoic and octadecanoic acids In agreement with our results, Seixas et al (2009) reported that the major FAs found in Tetraselimis suecica were the saturated palmitic acid 16:0 (37%) In this respect, Adarme-Vega (2014) revealed that modifying FA composition may be a mechanism used by the alga to regulate osmotic balance and maintain membrane fluidity under the altered conditions Furthermore, different fatty acids including palmitic, myristic acids are known to have potential antibacterial and antifungal activities as stated by Agoramoorthy et al., (2007) In Dunaliela salina, the stress evoked by the high salinity has sharpened the production of secondary metabolites and consequently, may have increased antimicrobial activity Please cite this article in press as: El-Kassas, H.Y., El-Sheekh, M.M Induction of the synthesis of bioactive compounds of the marine alga Tetraselmis tetrathele (West) Butcher grown under salinity stressTetraselmis tetrathele –> Egyptian Journal of Aquatic Research (2016), http://dx.doi.org/10.1016/j.ejar.2016.10.006 H.Y El-Kassas, M.M El-Sheekh Table SPSS output for ANOVA test between the cultures at different salinity values and cell densities as well as chlorophyll a and carotenoids concentrations (N = 36) Source of variation Sum square Degrees of freedom Mean square F-value P-value F critical Cultures at different salinity values Different variables Error 344.82 2822.75 318.47 35 105 9.85 940.92 3.03 3.25 310.22 0.05 0.05 1.54 2.69 Cakmak et al (2014) examined the antimicrobial potential of fatty acids extracted from D salina against some fish pathogens Vimalavady and Kadavul (2013) reported that the results of the Gas Chromatography Mass spectra (GC–MS) profile can be used as chromatographic tool for the identification of the bioactive components During growth of the algae under different NaCl concentration, it accumulated different bioactive compounds The major constituents of these bioactive compounds were 9-octadecenamide and pthalic acid derivatives in addition to 11 ester compounds as reported by GC–MS technique In this concept, different biochemical studies have considered fatty acids amides as a new family of biologically lipids (Farrell and Merkler, 2008) In addition; phthalic acid derivatives have different activities including antibacterial activities as recommended by Hao et al (2006) Additionally, Gopalakrishan et al (2011) reported that different fatty acid esters possess antibacterial and antifungal activities through inhibition of enzymes (Cowan, 1999), interfere with the binding protein, form a complex with the cell wall, and loss of substrate (Haslam, 1996) Moreover, Ferrer et al (2005) revealed that Fatty acid esters are used as surfactants due to their surface activity Similarly and in accordance with the compounds recorded in this study, El-Sayed et al (2014) reported the reduction in coliforms counts as well as the fish pathogens including Vibrios, Staphylococci and Aeromonas hydrophila which associated with the microalga T chuii cultures showed low counts or disappeared They discussed their results on the basis that the alga produced intra and extra cellular bioactive compounds such as hexa and octadecanoaic acids and diterpene alcohol (like phytol) Moreover, Linolenic acid ester and Fatty acid ester compounds have many different applications including antimicrobial (Rani et al., 2012) Considering other esters compounds, previous studies suggested that 1,2-benzenedicarboxylic acid, mono (2-ethylhexyl) ester has antimicrobial activity (Ezhilan and Neelamegam, 2012) Additionally, 1,2-benzenedicarboxylic acid, mono (2ethylhexyl) ester, a common plasticizer was found to exert antimicrobial activity by Rizwan et al (2012) Moreover, Helal and Sarhan (2006) revealed that hydrocarbons, alcohol, and ketones have biocidal activity against molds, yeast, and bacteria Conclusions This work demonstrates that the prasinophyte T tetrathele is rich in fatty acids and many valuable compounds It is proved that the application of different salinity levels induced the synthesis of active compounds such as carotenoids, flavonoids, steroids, lipids, sterols, triterpenes, and alkaloids as secondary metabolites, which can be exploited in aquaculture Conflict of interest The authors declare that there is no conflict of interest References Adarme-Vega, T.C., 2014 Optimization of microalgal growth conditions for production of eicosapentaenoic acid (EPA) A PhD thesis The University of Queensland 132p Agoramoorthy, G., Chandrasekaran, M., Venkatesalu, V., Hsu, M.J., 2007 Antibacterial and antifungal activities of fatty acid methyl esters of the blind-your-eye mangrove Braz J Microbiol 38, 739– 742 Anadon, A., 2006 The EU ban of antibiotics as feed additives, alternatives and consumer Safety J Vet Pharmacol Ther 29 (Suppl 1), 41–44 Austin, B., Baudet, E., Stobie, M., 1992 Inhibition of bacterial fish pathogens by Tetraselmis suecica J Fish Dis 15, 55–61 Bhagavathy, S., Sumathi, P., Bell, J.S., 2011 Green algae Chlorococcum humicola—a new source of bioactive compounds with antimicrobial activity Asian Pac J Trop Med., S1–S7 Cakmak, Y.S., Kaya, M., Asan-Ozusaglam, M., 2014 Biochemical composition and bioactivity screening of various extracts from Dunaliella salina, a green microalga EXCLI J 13, 679–690 Cardozo, K.H.M., Guaratini, D.E., Barros, M.P., Falcao, V.S., Tonon, A.P., Lopes, N.P., Campos, S., Torres, M.A., Souza, A O., Colepicolo, P., Pinto, E., 2007 Metabolites from algae with economical impact Comp Biochem Physiol C Toxicol Pharmacol 146, 60–78 Cowan, M.M., 1999 Plant products as antimicrobial agents Clin Microbiol Rev 12 (4), 564–582 Dhont, J., Van Stappen, G., 2003 Biology, tank production and nutritional value of Artemia In: Live Feeds in Marine Aquaculture Blackwell Science Ltd., pp 65–121 Durmaz, Y., 2007 Vitamin E (a-tocopherol) production by the marine microalgae Nannochloropsis oculata (Eustigmatophyceae) in nitrogen limitation Aquaculture 272, 717–722 El-Sayed, H.S., Ibrahim, H.A.H., Beltagy, E.A., Khairy, H.M., 2014 Effects of short term feeding of some marine microalgae on the microbial profile associated with Dicentrarchus labrax post larvae EJAR 40, 251–260 El-Sheekh, M.M., Osman, M.E.H., Dyab, M.A., Amer, M.S., 2006 Production and characterization of antimicrobial active substance from the cyanobacterium Nostoc muscorum Environ Toxicol Pharmacol 21, 42–50 El-Sheekh, M.M., Dawa, A.M., Abd-Elrahman, A.M., Eladel, H.M., Abd EL-Hay, R.A., 2008 Antimicrobial activity of the cyanobacteria Anabaena wisconsinense and Oscillatoria curviceps against pathogens of fish in aquaculture Annal Microbiol 58 (3), 527– 534 El-Sheekh, M.M., Daboor, S.M., Swelim, M.A., Mohamed, S., 2014 Production and characterization of antimicrobial active substance from Artrospira platensis Iran J Microbiol (2), 112–119 Ezhilan, B.P., Neelamegam, R., 2012 GC–MS analysis of phytocomponents in the ethanol of Polygonum chinense L Pharmacogn Res 4, 11–14 Please cite this article in press as: El-Kassas, H.Y., El-Sheekh, M.M Induction of the synthesis of bioactive compounds of the marine alga Tetraselmis tetrathele (West) Butcher grown under salinity stressTetraselmis tetrathele –> Egyptian Journal of Aquatic Research (2016), http://dx.doi.org/10.1016/j.ejar.2016.10.006 Synthesis of bioactive compounds of the marine alga Tetraselmis tetrathele Fa´bregas, J., Otero, A., Domı´ nguez, A., Patin˜o, M., 2001 Growth rate of the microalga Tetraselmis suecica changes the biochemical composition of Artemia species Mar Biotechnol (3), 256–263 Farrell, E.K., Merkler, D.J., 2008 Biosynthesis, degradation and pharmacological importance of the fatty acid amides Drug Discov Today 13, 558–568 Ferrer, M., Soliveri, J., Plou, F.J., Lopez-Cortes, N., Reyes-Duarte, D., Christensen, M., Copa-Patino, J.L., Ballesteros, A., 2005 Synthesis of sugar esters in solvent mixtures by lipases from Thermomyces lanuginosum and Candida antarctica B, and their antimicrobial properties Enzyme Microb Technol 36, 391–398 Gentsch, E., Kreibich, T., Hagen, W., Barbara, N., 2009 Dietary shifts in the copepod Temoralongi cornis during spring: evidence from stable isotope signatures, fatty acid biomarkers and feeding experiments J Plankton Res 31, 45–60 Gopalakrishan, S., Saroja, K., Dulcy, E., 2011 GC–MS analysis of methanolic extract of leaves of Dipleracanthus patulus J Chem Pharm Res 3, 477–480 Hao, H., Xin, Z., Biao, J., 2006 Survey in study on chemical constituents from plants of Elaeagnaceae Chin Trad Herb Drugs 37 (2), 307–309 Haslam, E., 1996 Natural polyphenols (vegetable tannins) as drugs: possible modes of action J Nat Prod 59 (2), 205–215 Helal, G.A., Sarhan, M., 2006 Antimicrobial activity of some essential oils against microorganism deteriorating fruit juices Mycobiology 34 (4), 219–229 Helm, M.M., Bourne, N., Lovatelli, A., 2004 Hatchery culture of bivalves: A Practical Manual FAO Fisheries Technical Paper Number 471 Rome, Italy 200p Hemaiswarya, S., Raja, R., Ravi Kumar, R., Ganesan, V., Anbazhagan, C., 2011 Microalgae: a sustainable feed source for aquaculture World J Microbiol Biotechnol 27, 1737–1746 Hieks, G.R., Rayle, D.L., Lomax, T.L., 1989 The diageotropica mutant of tomato lacks high specific activity auxin binding sites Science 245, 52–54 Holzinger, A., Karsten, U., 2013 Desiccation stress and tolerance in green algae: consequences for ultrastructure, physiological and molecular mechanisms Front Plant Sci 4, 327 Moradi, M., Ismail, A.M., 2007 Responses of photosynthesis, chlorophyll fluorescence and ROS – scavenging systems to salt stress during seedling and reproductive stages of rice Ann Bot 99, 1161–1173 Olsen, A.I., Olsen, Y., Attramadal, Y., Christie, K., Birkbeck, T.H., Skjermo, J., Vadstein, O., 2000 Effects of short term feeding of microalgae on the bacterial flora associated with juvenile Artemia franciscana Aquaculture 190, 11–25 Pandey, A., Milind, M., Naik, D.S.K., 2010 Organic metabolites produced by vibrio parahaemolyticus strain An3 isolated from Goan mullet inhibit bacterial fish pathogens, Goa, India Afr J Biotechnol (42), 7134–7140 Patil, V., Kallqvist, T., Olsen, E., Vogt, G., Gislerød, H.R., 2007 Fatty acid composition of 12 microalgae for possible use in aquaculture feed Aquacult Int 15, 1–9 Pisal, D.S., Lele, S.S., 2005 Carotenoid production from microalga, Dunaliella salina Int J Biotechnol 4, 476–483 Ponis, E., Robert, R., Parisi, G., 2003 Nutritional value of fresh and concentrated algal diets for larval and juvenile pacific oysters (Crassostre gigas) Aquaculture 221, 491–505 Radwan, S.S., 1978 Sources of C20 polyunsaturated of fatty acids for use Appl Microbiol Biotechnol 35, 421–430 Raja, R., Hemaiswarya, S., Ashok Kumar, N., Sridhar, S., Rengasamy, R., 2008 A perspective on the biotechnological potential of microalgae Crit Rev Microbiol 34, 77–88 Rani, J.M.J., Chandramohan, G., Renganathan, R., 2012 Antioxidant activity, preliminary phytochemical investication and GC–MS study of Bougainvillea glabra choisy leaves Int J Pharm Sci (2), 12–16 Rao, A.R., Dayananda, C., Sarada, R., Shamala, T.R., Ravishankar, G.A., 2007 Effect of salinity on growth of green alga Botryococcus braunii and its constituents Bioresour Technol 98, 560–564 Rizwan, K., Zubair, M., Rasool, N., Riaz, M., Zia-Ul-Haq, M., de Feo, V., 2012 Phytochemical and biological studies of Agave attenuata Int J Mol Sci 13 (5), 6440–6451 Rocha Jorge, M.S., Juan, E.C.G., Marta, H.F.H., 2003 Growth aspects of the marine microalga Nannochloropsis gaditana Biomol Eng 20, 237–242 Ronquillo, J.D., Matias, J.R., Saisho, T., Yamasaki, S., 1997 Culture of Tetraselmis tetrathele and its utilization in the hatchery production of different penaeid shrimps in Asia Hydrobiologia 358, 237–244 Sargent, J.R., McEvoy, L.A., Bell, J.G., 1997 Requirements, presentation and sources of polyunsaturated fatty acids in marine fish larval feeds Aquaculture 155, 117–128 Seixas, P., Coutinho, P., Ferreira, M., Otero, A., 2009 Nutritional value of the cryptophytes Rhodomonas lens for Artemia sp J Exp Mar Biol Ecol 381, 1–9 Sengar, H., 1970 Characterization of the synchronous culture of Scenedesmus obliquus Planta 90, 243–266 Teo, G., Vogel, C., Ghosh, D., Kim, S., Choi, H., 2014 PECA: A novel statistical tool for deconvoluting time-dependent gene expression regulation J Proteome Res 13 (1), 29–37 Vazquez-Duhalt, R., Arredondo-Vega, B.O., 1991 Haloadaptation of the green alga Botryococcus braunii (A Race) Phytochemistry 30 (9), 2919–2925 Vimalavady, A., Kadavul, K., 2013 Phytocomponents identified on the various extracts of stem of Hugonia mystax L (Linaceae) Eur J Exp Biol (1), 73–80 Walne, P.R., 1970 Studies on the food value of nineteen genera of 611 algae to juvenile bivalves of the genera Ostrea, Crassostrea, 612 Mercenaria, and Mytilis Fish Investment 26, 162 Walsh, D.T., Withstandley, C.A., Ray, K., Petrovits, E.J., 1987 Mass culture of selected marine microalgae for the nursery production of bivalve seed J Shellfish Res 6, 71–77 Please cite this article in press as: El-Kassas, H.Y., El-Sheekh, M.M Induction of the synthesis of bioactive compounds of the marine alga Tetraselmis tetrathele (West) Butcher grown under salinity stressTetraselmis tetrathele –> Egyptian Journal of Aquatic Research (2016), http://dx.doi.org/10.1016/j.ejar.2016.10.006 ... El-Sheekh, M.M Induction of the synthesis of bioactive compounds of the marine alga Tetraselmis tetrathele (West) Butcher grown under salinity stressTetraselmis tetrathele –> Egyptian Journal of Aquatic... El-Sheekh, M.M Induction of the synthesis of bioactive compounds of the marine alga Tetraselmis tetrathele (West) Butcher grown under salinity stressTetraselmis tetrathele –> Egyptian Journal of Aquatic... El-Sheekh, M.M Induction of the synthesis of bioactive compounds of the marine alga Tetraselmis tetrathele (West) Butcher grown under salinity stressTetraselmis tetrathele –> Egyptian Journal of Aquatic