The study of Indian shrimp larvae feeding by enriched rotifer with vitamin C and unsaturated fatty acid (epa, dha) and its effects on growth factors, survival rate and environmental stresses

In this experiment, the feeding of Indian white shrimp larvae by unenriched rotifers (treatment 1) and enriched with highly unsaturated fatty acid (treatment 2) and highly unsaturated fatty acid along with vitamin C (treatment 3) on the growth factors, survival and resistance against salinity and formalin stress tests were studied and their differences with control treatment including newly hatched Artemia nauplii is compared. In this the study four treatments in a completely randomized design with 3 replicates per treatment were used. Farming of shrimp larvae of Zoea II to postlarvae 5 was done in 20 liter plastic bucket. Present results indicated that growth factors and survival rate of stage Zoea II to postlarvae 1 in treatments 1, 2 and 3 improve rather than control in which this case was due to optimal size rotifer rather than Artemia nauplii. Also, treatments 2 and 3 feeding with oil liver cod emulsion enriched rotifer have the highest concentration of DHA (mg/g DW) and the ratio DHA/EPA in which due to have shown the highest growth factors and a significant difference (P<0.05) with treatments 1 and control. The highest survival at stage PL1 were observed in treatment 3 that was enriched with ascorbyl palmitate in which have to the synergistic properties of vitamin C rather than treatments 2, 1 and control and showed a significant difference (P<0.05). But in stage PL5 the highest amount of growth and survival rates were related to control treatment which showed a significant difference (P<0.05) with other treatments that control has higher size rather than treatments 1, 2 and 3. Also, among experiment treatments that the two treatments 2 and 3 due to enrichment had higher growth and survival rates compared with treatment 1 in which their differences have also been significant (P<0.05). In the case of stress tests, results indicated that the highest survival rate has been reported when specimens were offered a diet containing high levels of highly unsaturated fatty acids with vitamin C. So that in stage PL1 in the salinity stress tests 10 and 20 ppt the highest survival rate was observed in treatment 3. As for the second, treatment 2 showed a significant difference (P<0.05) with treatment 3. It is worth mentioning that treatment 3 showed a higher survival rate compared to treatment 2 due to the synergistic properties of vitamin C. The difference between these two treatments with treatment 1 and control was also significant. No significant difference was observed in formalin stress test 100 ppm in this stage between treatments 3 and 2 which shows the highest survival rate. But their difference with treatments 1 and control was significant (P<0.05). Also, in stage PL5 in the salinity stress tests 10 and 20 ppt the highest survival rate was observed in treatment 3 which showed no significant difference (P<0.05) with control treatment. While their difference in the amount of survival rate with treatment 1 and 2 was significant (P<0.05). In this stage, the highest observed survival rate in formalin stress test 100 ppm included treatments control, 3 and 2 among which there were no significant differences (P<0.05). While the difference between these three treatments with treatment 1 was significant.

The effect of artisanal preservation methods on nutritional security of “Mukene” Rastrineobola argentea caught from Lakes Victoria and Kyoga in Uganda

The artisanal fish preservation methods in Uganda are characterized by extreme operating conditions. Consequently, vital nutritional components diminish in value and quantity which renders fish consumer nutritionally insecure. To establish the magnitude of nutritional loss, duplicate samples of Mukene Rastrineobola argentea were collected from Kiyindi landing site on L. Victoria and Moone landing site on L. Kyoga. Each set of duplicate samples was divided into five portions and kept on ice. For each preservation method a portion was processed into respective products at Food Bioscience and Agri-Business Laboratories aside from the control (fresh) sample. Both preserved and control samples were analysed for nutrient loss at Department of Chemistry, Makerere University using AOAC methods. The composition of fatty acids was determined by methanolysis gas chromatography and Mass spectrophotometry of the resultant methyl esters. The results indicate that nutrients of all preserved samples did not vary significantly from the control except for some fatty acids. The Eicosapentaenoic acid (EPA) in fresh samples declined from 6.72% to 1.08% in deep-fried samples constituting 83.93% nutrient loss. The sum ratio w3:w6 as well as EPA: DHA (Docosahexaenoic) ratio in fried samples also varied significantly (p<0.5) lower than 0.668 and 0.20 for the average of either preservation methods and experts recommended ratio respectively. Further research has been recommended to ascertain the causative factor, since Mukene frying is being promoted in the Great lakes region as alternative method to sun-drying. In conclusion, regular consumers of fried Mukene do not benefit much from the nutritional and health attributes of Omega 3 and 6.

Effect of dietary linolenic acid/linoleic acid ratio on growth performance, hepatic fatty acid profiles and intermediary metabolism of juvenile yellow catfish Pelteobagrus fulvidraco

The present study aimed to evaluate the effect of dietary linolenic acid (LNA)linoleic acid (LA) ratio on growth performance, hepatic fatty acid profile and intermediary metabolism of juvenile yellow catfish Pelteobagrus fulvidraco. Six isonitrogenous and isolipidic diets were formulated to contain incremental levels of LNA from 0 to 5% at the expense of corn oil (rich in LA), resulting in six dietary treatments with LNA to LA ratios ranging from 0.35 to 14.64. The experiment continued for 7 weeks. Best growth and feed intake were obtained in the fish fed the diets containing the LNA/LA ratios of 1.17 and 2.12 (P<0.05). In contrast, feed conversion ratio was the lowest for fish fed the diets containing the LNA/LA ratios of 1.17 and 2.12 (P<0.05). Dietary LNA to LA ratios significantly influenced viscerosomatic index and hepatosomatic index (P<0.05), but not condition factor (P>0.05). Body composition was also significantly influenced by dietary LNA to LA ratios (P<0.05). Generally, liver FA compositions reflected dietary FA profiles. Declining LA and increasing LNA contents in liver were observed with the increasing dietary LNA/LA ratios (P<0.05). Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) increased with the increasing LNA to LA ratios, suggesting that yellow catfish could elongate and desaturate C18 polyunsaturated fatty acids into highly unsaturated fatty acids. As a consequence, the n-6 fatty acids (FA) declined, and total n-3 FA and n-3/n-6 ratios increased with the dietary ratios of LNA/LA (P<0.05). Dietary LNA to LA ratios significantly influenced several enzymatic activities involved in liver intermediary metabolism (P<0.05), such as lipoprotein lipase, hepatic lipase, pyruvate kinase, succinate dehydrogenase, malic dehydrogenase and lactate dehydrogenase, suggesting that dietary LNA/LA ratios had significant effects on nutrient metabolism in the liver. To our knowledge this is the first demonstration of the effects of dietary LNA to LA ratios on the enzymatic activities of liver in fish, which provides information on diet quality and utilization, and can also be used as an indicator of the nutritional status of this fish. (C) 2009 Elsevier B.V. All rights reserved.

Molecular cloning and stress-dependent expression of a gene encoding Delta(12)-fatty acid desaturase in the Antarctic microalga Chlorella vulgaris NJ-7

The psychrotrophic Antarctic alga, Chlorella vulgaris NJ-7, grows under an extreme environment of low temperature and high salinity. In an effort to better understand the correlation between fatty acid metabolism and acclimation to Antarctic environment, we analyzed its fatty acid compositions. An extremely high amount of Delta(12) unsaturated fatty acids was identified which prompted us to speculate about the involvement of Delta(12) fatty acid desaturase in the process of acclimation. A full-length cDNA sequence, designated CvFAD2, was isolated from C. vulgaris NJ-7 via reverse transcription polymerase chain reaction (RT-PCR) and RACE methods. Sequence alignment and phylogenetic analysis showed that the gene was homologous to known microsomal Delta(12)-FADs with the conserved histidine motifs. Heterologous expression in yeast was used to confirm the regioselectivity and the function of CvFAD2. Linoleic acid (18:2), normally not present in wild-type yeast cells, was detected in transformants of CvFAD2. The induction of CvFAD2 at an mRNA level under cold stress and high salinity is detected by real-time PCR. The results showed that both temperature and salinity motivated the upregulation of CvFAD2 expression. The accumulation of CvFAD2 increased 2.2-fold at 15A degrees C and 3.9-fold at 4A degrees C compared to the alga at 25A degrees C. Meanwhile a 1.7- and 8.5-fold increase at 3 and 6% NaCl was detected. These data suggest that CvFAD2 is the enzyme responsible for the Delta(12) fatty acids desaturation involved in the adaption to cold and high salinity for Antarctic C. vugaris NJ-7.

Diet-induced changes in lipid and fatty acid composition of Artemia salina

The influence of diet on lipid and fatty acid composition of the brine shrimp Artemia salina nauplii was investigated. Various diets with different lipid composition and fatty acid profiles were fed to nauplii for 2 weeks. The lipid composition of microalgal diets, Isochrysis galbana, Phaeodactylum tricornutum and Nannochloropsis oculata and baker's yeast was analyzed. Newly hatched nauplii were examined before the feeding experiment. It was shown that Artemia was able to incorporate and selectively concentrate some dietary lipids. Depot lipids were more sensitive to changes in the dietary lipid composition than the main structural lipids, polar lipids and sterols. Variations in the content of the lipid classes correlated with stage of development of the animal. The fatty acid composition of the animal varied with that of diet. The concentrations of saturated fatty acids were apparently supported in the nauplii by biosynthesis de novo. The acid 16:1(n-7) originated from the food. The concentration range of n-6 polyunsaturated fatty acids (PUFAs) remained constant through the accumulation from the diet. The proportion of n-3 PUFAs varied with their level in the diet. The dynamics of alteration of 20:5(n-3) content in Artemia fed on Isochrysis, which is poor in this acid, suggested a limited capacity for elongation and desaturation of 18:3(n-3) to 20:5(n-3). None of the diets provided dietary input of 22:6(n-3). (C) 1998 Elsevier Science Inc. All rights reserved.

Wangia profunda gen. nov., sp nov., a novel marine bacterium of the family Flavobacteriaceae isolated from southern Okinawa Trough deep-sea sediment

An orange-pigmented, Gram-negative, nonmotile, strictly aerobic and oxidase- and catalase-positive bacterium (SM-A87(T)) was isolated from the deep-sea sediment of the southern Okinawa Trough area. The main fatty acids were i15 : 0, i17 : 0 3OH, i15 : 1 G, i17 : 1 omega 9c, 15 : 0, i15 : 0 3OH and summed feature 3 (comprising i-15 : 0 2OH and/or 16 : 1 omega 7c). MK-6 was the predominant respiratory quinone. DNA G+C content was 35.8 mol%. Flexirubin-type pigments were absent. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain SM-A87(T) formed a distinct lineage within the family Flavobacteriaceae, with < 93% sequence similarity to the nearest strain of genus Salegentibacter. Moreover, strain SM-A87(T) could be distinguished from the nearest phylogenetic neighbors by a number of chemotaxonomic and phenotypic properties. On the basis of polyphasic analyses, it is proposed that strain SM-A87(T) be classified in a novel genus and a new species in the family Flavobacteriaceae, designated Wangia profunda gen. nov., sp. nov. The type strain is SM-A87(T) (CCTCC AB 206139(T)=DSM 18752).

Myroides profundi sp nov., isolated from deep-sea sediment of the southern Okinawa Trough

A Gram-negative, nonmotile, aerobic and oxidase- and catalase-positive bacterium,, designated D25(T), was isolated from the deep-sea sediments of the southern Okinawa Trough area. Phylogenetic analyses of 16S rRNA gene sequences showed that strain D25(T), fell within the genus Myroides, with 99.2%, 96.0% and 93.4% sequence similarities to the only three recognized species of Myroides. However, the DNA-DNA similarity Value between strain D25(T) and its nearest neighbour Myroides odoratimimus JCM 7460(T) was only 49.9% ( < 70%). Several phenotypic properties could be used to distinguish strain D25(T) from other Myroides species. The main cellular fatty acids of strain D25(T) were iso-C-15:0, iso-C-17:1 omega 9C, iso-C(17:0)3-OH and Summed Feature 3 (comprising C-16:1 omega 7c and/or iso-C(15:0)2-OH). The major respiratory quinone was MK-6. The DNA G+C content was 33.0 mol%. The results of the polyphasic taxonomy analysis suggested that strain D251(T) represents a novel species of the genus Myroides, for which the name Myroides profundi sp. nov. is proposed. The type strain is D25(T) (=CCTCC M 208030(T) = DSM 19823(T)).

Effects of single and mixed diatom diets on the reproduction of copepod Calanus sinicus

Under laboratory conditions, the potential influence of diatom diets on reproduction of zoo-plankton Calanus sinicus was studied. Four diatom diet ingredients: Skeletonema costatum (SC), Chaetoceros muelleri (CM), Phaeodactylum tricornutum (PT), diatom mixture (MIX) and a control diet: the flagellate Platymonas subordiformis (PS), were used at the same carbon concentrations of 2.0 mu g mL(-1) C. In a period of 17-day laboratory experiment, the effects of these algae diets on egg production and hatching success of the copepod Calanus sinicus were examined. The diets were analyzed for fatty acid content as an indicator of food quality. The results showed that the female survival of all treatments reached more than 80% except PT. Comparing to the initial value, egg production of Calanus sinicus was reduced in diatom diets (PT, CM), but remained in normal level in SC and MIX, indicating that some single diatom diets had a negative effect on the egg production of Calanus sinicus. Feeding with mixed food however can eliminate this negative effect. Among all the treatments, hatching success in filtered seawater was significantly higher than in algal exudates, indicating that not only diatoms but also other phytoplankton in certain concentration can release extracelluar substance that may inhibit eggs from hatching. Fatty acid analysis showed that both egg production rate and hatching success were negatively correlated to the ratio of 20:5 omega 3 and 14:0 in fatty acid composition.

An examination of the impact of dietary lipids on behaviour and neurochemistry

The molecular and cellular basis of stress pathology remains an important research question in biological science. A better understanding of this may enable the development of novel approaches for the treatment of stress-related disorders. There is a considerable body of scientific evidence suggesting that dietary lipids, phospholipids and omega-3 polyunsaturated fatty acids (n-3 PUFAs), have therapeutic potential for certain psychiatric disorders. Thus, we proposed n-3 PUFAs as a novel strategy for the prevention or amelioration of stress-related disorders. We hypothesised that these compounds would improve behavioural and neurobiological responses and alter gut microbial composition. Furthermore, we proposed a new mechanism of action exerted by n-3 PUFAs using an in vitro model of stress. Lastly, we explored the protective effects of both phospholipids and n-3 PUFAs against neuroinflammation, which has been shown to contribute to the development of stress-related disorders. We provide further evidence that glucocorticoids, inflammation and early-life stress induce vulnerability to psychopathologies. Specifically, we have demonstrated that corticosterone (CORT) alters cortical neuron and astrocyte percentage composition, reduces brain-derived-neuronal factor (BDNF) expression, and induces glucocorticoid receptor (GR) down-regulation in mixed cortical cultures. Interestingly, we found that lipopolysaccharide (LPS) treatment resulted in an over-expression of pro-inflammatory cytokines in cortical astrocyte cultures. Moreover, we demonstrate that early-life stress induces changes to the monoaminergic and immune systems as well as altered neuroendocrine response to stressors later in life. In addition, we found that early-life stress alters the gut microbiota in adulthood. These data demonstrate that n-3 PUFAs can attenuate CORT-induced cellular changes, but not those caused by LPS, within the cerebral cortex. Similarly, phospholipids were unable to reverse LPS-induced inflammation in cultured astrocytes. In addition, this thesis proposes that n-3 PUFAs may prevent the development or lessen the symptoms of mental illnesses, ameliorating anxiety- and depressive-like symptoms as well as cognitive effects, particularly when administered during neurodevelopment. Such effects may be mediated by GR activation as well as by modification of the gut microbiota composition. Taken together, our findings suggest that n-3 PUFAs have therapeutic potential for stress-related disorders and we provide evidence for the mechanisms by which they may exert these effects. These findings contribute to an exciting and growing body of research suggesting that nutritional interventions may have an important role to play in the treatment of stress-related psychiatric conditions.

Fatty acid composition of wild anthropoid primate milks.

Fatty acids in milk reflect the interplay between species-specific physiological mechanisms and maternal diet. Anthropoid primates (apes, Old and New World monkeys) vary in patterns of growth and development and dietary strategies. Milk fatty acid profiles also are predicted to vary widely. This study investigates milk fatty acid composition of five wild anthropoids (Alouatta palliata, Callithrix jacchus, Gorilla beringei beringei, Leontopithecus rosalia, Macaca sinica) to test the null hypothesis of a generalized anthropoid milk fatty acid composition. Milk from New and Old World monkeys had significantly more 8:0 and 10:0 than milk from apes. The leaf eating species G. b. beringei and A. paliatta had a significantly higher proportion of milk 18:3n-3, a fatty acid found primarily in plant lipids. Mean percent composition of 22:6n-3 was significantly different among monkeys and apes, but was similar to the lowest reported values for human milk. Mountain gorillas were unique among anthropoids in the high proportion of milk 20:4n-6. This seems to be unrelated to requirements of a larger brain and may instead reflect species-specific metabolic processes or an unknown source of this fatty acid in the mountain gorilla diet.

Extracellular Loop 2 of the Free Fatty Acid Receptor 2 Mediates Allosterism of a Phenylacetamide Ago-Allosteric Modulator

Allosteric agonists are powerful tools for exploring the pharmacology of closely related G protein-coupled receptors that have nonselective endogenous ligands, such as the short chain fatty acids at free fatty acid receptors 2 and 3 (FFA2/GPR43 and FFA3/GPR41, respectively). We explored the molecular mechanisms mediating the activity of 4-chloro-alpha-(1-methylethyl)-N-2-thiazolylbenzeneacetamide (4-CMTB), a recently described phenylacetamide allosteric agonist and allosteric modulator of endogenous ligand function at human FFA2, by combining our previous knowledge of the orthosteric binding site with targeted examination of 4-CMTB structure-activity relationships and mutagenesis and chimeric receptor generation. Here we show that 4-CMTB is a selective agonist for FFA2 that binds to a site distinct from the orthosteric site of the receptor. Ligand structure-activity relationship studies indicated that the N-thiazolyl amide is likely to provide hydrogen bond donor/acceptor interactions with the receptor. Substitution at Leu(173) or the exchange of the entire extracellular loop 2 of FFA2 with that of FFA3 was sufficient to reduce or ablate, respectively, allosteric communication between the endogenous and allosteric agonists. Thus, we conclude that extracellular loop 2 of human FFA2 is required for transduction of cooperative signaling between the orthosteric and an as-yet-undefined allosteric binding site of the FFA2 receptor that is occupied by 4-CMTB.

Selective Orthosteric Free Fatty Acid Receptor 2 (FFA2) Agonists IDENTIFICATION OF THE STRUCTURAL AND CHEMICAL REQUIREMENTS FOR SELECTIVE ACTIVATION OF FFA2 VERSUS FFA3

Free fatty acid receptor 2 (FFA2; GPR43) is a G protein-coupled seven-transmembrane receptor for short-chain fatty acids (SCFAs) that is implicated in inflammatory and metabolic disorders. The SCFA propionate has close to optimal ligand efficiency for FFA2 and can hence be considered as highly potent given its size. Propionate, however, does not discriminate between FFA2 and the closely related receptor FFA3 (GPR41). To identify FFA2-selective ligands and understand the molecular basis for FFA2 selectivity, a targeted library of small carboxylic acids was examined using holistic, label-free dynamic mass redistribution technology for primary screening and the receptor-proximal G protein [S-35] guanosine 5'-(3-O-thio) triphosphate activation, inositol phosphate, and cAMP accumulation assays for hit confirmation. Structure-activity relationship analysis allowed formulation of a general rule to predict selectivity for small carboxylic acids at the orthosteric binding site where ligands with substituted sp(3)-hybridized alpha-carbons preferentially activate FFA3, whereas ligands with sp(2)- or sp-hybridized alpha-carbons prefer FFA2. The orthosteric binding mode was verified by site-directed mutagenesis: replacement of orthosteric site arginine residues by alanine in FFA2 prevented ligand binding, and molecular modeling predicted the detailed mode of binding. Based on this, selective mutation of three residues to their non-conserved counterparts in FFA3 was sufficient to transfer FFA3 selectivity to FFA2. Thus, selective activation of FFA2 via the orthosteric site is achievable with rather small ligands, a finding with significant implications for the rational design of therapeutic compounds selectively targeting the SCFA receptors.

Extracellular ionic locks determine variation in constitutive activity and ligand potency between species orthologs of the free fatty acid receptors FFA2 and FFA3

Free fatty acid receptors 2 and 3 (FFA2 and FFA3) are G protein-coupled receptors for short chain free fatty acids (SCFAs). They respond to the same set of endogenous ligands but with distinct rank-order of potency, such that acetate (C2) has been described as FFA2 selective while propionate (C3) is non-selective. Although C2 was confirmed to be selective for human FFA2 over FFA3, this ligand was not selective between the mouse orthologs. Moreover, although C3 was indeed not selective between the human orthologs it displayed clear selectivity for mouse FFA3 over mouse FFA2. This altered selectivity to C2 and C3 resulted from broad differences in SCFAs potency at the mouse orthologs. In studies to define the molecular basis for these observations marked variation in ligand-independent, constitutive activity was identified. The orthologs with higher potency for the SCFAs, human FFA2 and mouse FFA3, displayed high constitutive activity while the orthologs with lower potency for the agonist ligands, mouse FFA2 and human FFA3, did not. Sequence alignments of the 2nd extracellular loop identified single negatively charged residues in FFA2 and FFA3 not conserved between species and predicted to form ionic lock interactions with arginine residues within the FFA2 or FFA3 agonist binding pocket to regulate constitutive activity and SCFA potency. Reciprocal mutation of these residues between species orthologs resulted in the induction (or repression) of constitutive activity, and in most cases also yielded corresponding changes in SCFA potency.

Synthesis of medium-chain-length polyhydroxyalkanoates in arabidopsis thaliana using intermediates of peroxisomal fatty acid beta-oxidation.

Polyhydroxyalkanoate (PHA) is a family of polymers composed primarily of R-3-hydroxyalkanoic acids. These polymers have properties of biodegradable thermoplastics and elastomers. Medium-chain-length PHAs (MCL-PHAs) are synthesized in bacteria by using intermediates of the beta-oxidation of alkanoic acids. To assess the feasibility of producing MCL-PHAs in plants, Arabidopsis thaliana was transformed with the PhaC1 synthase from Pseudomonas aeruginosa modified for peroxisome targeting by addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. Immunocytochemistry demonstrated that the modified PHA synthase was appropriately targeted to leaf-type peroxisomes in light-grown plants and glyoxysomes in dark-grown plants. Plants expressing the PHA synthase accumulated electron-lucent inclusions in the glyoxysomes and leaf-type peroxisomes, as well as in the vacuole. These inclusions were similar to bacterial PHA inclusions. Analysis of plant extracts by GC and mass spectrometry demonstrated the presence of MCL-PHA in transgenic plants to approximately 4 mg per g of dry weight. The plant PHA contained saturated and unsaturated 3-hydroxyalkanoic acids ranging from six to 16 carbons with 41% of the monomers being 3-hydroxyoctanoic acid and 3-hydroxyoctenoic acid. These results indicate that the beta-oxidation of plant fatty acids can generate a broad range of R-3-hydroxyacyl-CoA intermediates that can be used to synthesize MCL-PHAs.