Characterization and comparison of fatty acyl Delta6 desaturase cDNAs from freshwater and marine teleost fish species.

Fish are the most important dietary source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), that have particularly important roles in human nutrition reflecting their roles in critical physiological processes. The objective of the study described here was to clone, functionally characterize and compare expressed fatty acid desaturase genes involved in the production of EPA and DHA in freshwater and marine teleost fish species. Putative fatty acid desaturase cDNAs were isolated and cloned from common carp (Cyprinus carpio) and turbot (Psetta maximus). The enzymic activities of the products of these cDNAs, together with those of cDNAs previously cloned from rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), were determined by heterologous expression in the yeast Saccharomyces cerevisiae. The carp and turbot desaturase cDNAs included open reading frames (ORFs) of 1335 and 1338 base pairs, respectively, specifying proteins of 444 and 445 amino acids. The protein sequences possessed all the characteristic features of microsomal fatty acid desaturases, including three histidine boxes, two transmembrane regions, and N-terminal cytochrome b(5) domains containing the haem-binding motif, HPGG. Functional expression showed all four fish cDNAs encode basically unifunctional Delta6 fatty acid desaturase enzymes responsible for the first and rate-limiting step in the biosynthesis of HUFA from 18:3n-3 and 18:2n-6. All the fish desaturases were more active towards the n-3 substrate with 59.5%, 31.5%, 23.1% and 7.0% of 18:3n-3 being converted to 18:4n-3 in the case of turbot, trout, sea bream and carp, respectively. The enzymes also showed very low, probably physiologically insignificant, levels of Delta5 desaturase activity, but none of the products showed Delta4 desaturase activity. The cloning and characterization of desaturases from these fish is an important advance, as they are species in which there is a relative wealth of data on the nutritional regulation of fatty acid desaturation and HUFA synthesis, and between which substantive differences occur.

Cloning and nutritional regulation of a Delta6-desaturase-like enzyme in the marine teleost gilthead seabream (Sparus aurata).

Marine fish are presumed to have a lower capacity than freshwater fish for the bioconvertion of 18C fatty acids into 20-22C highly unsaturated fatty acids (HUFA). The present work investigated the first step of this pathway, the Delta6-desaturation, in gilthead seabream. A full-length desaturase-like cDNA was identified from total RNA extracted from viscera of juvenile fish fed for 96 days on an experimental HUFA-free diet containing olive oil as the sole lipid source. The open reading frame encodes a 445-amino acid peptide that contains two membrane-spanning domains, three histidine-rich regions, and a cytochrome b(5) domain, which are characteristic of Delta6- and Delta5-desaturases. Predicted protein sequence of seabream desaturase-like indicated a high percentage of identity with mammalian Delta6-desaturases (approx. 65%). Northern analysis showed two transcripts of approximately 3.7 and 1.8 kb which were highly expressed in fish fed on HUFA-free diet and slightly expressed in fish fed on HUFA-rich diet. The fatty acid profile of the former group was characterized by high levels of Delta6-desaturation products (18:2 n-9 and 20:2 n-9) with no detectable levels of Delta5-desaturation product (20:3n-9). These results demonstrate for the first time the presence and nutritional modulation of a Delta6-desaturase-like cDNA in a marine fish.

Cloning, tissue distribution and nutritional regulation of a Delta6-desaturase-like enzyme in rainbow trout.

This report describes the cloning, nutritional regulation and tissue distribution of a desaturase-like enzyme in rainbow trout (Oncorhynchus mykiss). The open reading frame of the trout desaturase-like cDNA encodes a 454-amino acid peptide that contains two membrane-spanning domains, three histidine-rich regions and a cytochrome b5 domain, which all align perfectly with the same domains located in other recently identified vertebrate Delta5- and Delta6-desaturases. Nutritional regulation of trout desaturase-like gene expression, as well as the tissue expression profile, are also similar to those observed in other vertebrate Delta5- and Delta6-desaturases. Finally, the sequence alignments between the predicted protein sequence of rainbow trout desaturase-like and other Delta6- and Delta5-desaturases revealed a high percentage identity with Delta6-desaturases (64-66% identity with vertebrate Delta6-desaturases). These results demonstrate for the first time the presence and nutritional modulation of a Delta6-desaturase-like cDNA in rainbow trout.

Ontogenesis of hexokinase I and hexokinase IV (glucokinase) gene expressions in common carp (Cyprinus carpio) related to diet.

The expressions of hexokinase IV (glucokinase, GK) and hexokinase (HK)-I genes were analysed during early ontogenesis of common carp (Cyprinus carpio). Unlike HK-I gene, which was expressed during all the stages of the development, GK was only induced by the first feeding with high levels of dextrin as a source of carbohydrate. This study confirms the high capacity of common carp to use glucose even at the very early stages of development.

Effects of environmental temperature on the development of the myotomal white muscle in larval carp (Cyprinus carpio L.).

A study was conducted on common carp (Cyprinus carpio L.) to determine the effects of environmental temperature experienced by embryos and larvae on the development of myotomal white muscle. Eggs from one female were divided into two groups following fertilisation and incubated at constant pre-hatch temperatures of 18 or 28 degrees C. At hatching, larvae from the 18 degrees C-incubated eggs were divided into two groups and either reared at the same temperature of 18 degrees C ('cold' group) or transferred over a period of 5 days (at 2 degrees C per day) to 28 degrees C ('transferred' group). Larvae hatched from eggs incubated at 28 degrees C were reared at the same temperature of 28 degrees C ('warm' group). Larvae were sampled at two developmental stages (stage 1, inflation of the back chamber of the swimbladder; stage 2, inflation of the front chamber of the swimbladder) and at 26 days post-hatching. The maturation of myotome shape during larval life was studied in parallel with the changes occurring in the organisation of white fibres. At stage 1, the epaxial part of the myotomes surrounding the vent had the shape of lamellae inclined backwards, and only one central layer of white fibres was present. At stage 2, the epaxial part of the myotomes began to acquire a V-shape, which was well developed at 26 days post-hatch. At stage 2 and at 26 days post-hatch, two layers of white fibres were identified: the initial central layer and a second apical layer. These differ in their orientation, the initial central layer being orientated backwards and the apical layer forwards, and in the mean fibre diameter, which is greater in the initial central layer. Studies on the effects of temperature (constant 18 degrees C, constant 28 degrees C, transfer from 18 to 28 degrees C at hatching) were carried out according to both the developmental stage and the length of the larvae. At stage 1, no significant differences were found between the three groups for larval standard length and muscle variables. The number of fibres in one quadrant of epaxial white muscle sectioned at the level of the vent was 100-111. At stage 2, there were significant differences between groups. Larval standard length and mass were higher in the cold group than in the warm group. The transferred larvae were of intermediate standard length but had a significantly higher cross-sectional area of white muscle than either of the other two groups. This increase in surface area was related to a 50 % greater fibre number (233) in the transferred larvae compared with the cold (165) or the warm (152) larvae. The increase in fibre number was more marked for large-diameter ( >20 microm) white fibres located in the initial central fibre layer (+58-72 % in transferred larvae) than in small-diameter ((less than equal to) 10 microm) white fibres mainly located in the apical layer (+18-35 %). In 26 days post-hatch samples, transferred larvae still showed a higher total number of white fibres than warm larvae, but the difference was no longer significant when the total number of white fibres was regressed against larval standard length, suggesting that this stimulation may be temporary.

Tricaproin, tricaprin and trilaurin are utilized more efficiently than tricaprylin by carp (Cyprinus carpio L.) larvae.

We investigated the effect of chain length of dietary medium-chain fatty acids (MCFA) on growth performance and fatty acid composition of first-feeding carp larvae. In a first trial, five semi-purified isolipidic (23-24 g/100 g of dry matter) diets were formulated to contain either 10 g/100 g triolein (control diet) or 5 g/100 g triolein and 5 g/100 g medium-chain triacylglycerols (MCT) supplied as tricaproin, tricaprylin, tricaprin or trilaurin. After 21 d, survival and growth rates were significantly greater in larvae fed diets containing triolein, tricaproin, tricaprin and trilaurin (final survival: 92 +/- 7% and mean larval weight: 42 +/- 15 mg) than in larvae fed tricaprylin (final survival: 56 +/- 12% and mean larval weight: 15 +/- 1 mg). The recovered levels of the fed MCFA in larval total lipids were respectively 0, 1.3, 7.3 and 8.1 g/100 g of total fatty acids. In a second trial, two isolipidic (18 g/100 g) diets containing 10 g/100 g triolein or tricaprylin were tested. High amounts of capric acid (up to 25 g/100 g of total fatty acids) were found in neutral lipids of carp larvae fed tricaprylin for 11 d, suggesting an unusual elongation of caprylic acid. This study underlines the peculiarity of tricaprylin among other MCT which seem well utilized up to 20-30 g/100 g of total dietary fatty acids. The exception of tricaprylin raises the question of the metabolic pathways followed by this MCT, especially for the suggested direct elongation of caprylic acid into capric acid.

Estimation of essential fatty acid requirements of common carp larvae using semi-purified artificial diets.

Two trials were conducted with duplicate groups of (first feeding) carp larvae fed artificial dry diets based on casein and dextrin over 21 or 25 days. One control diet based on yeast was also tested. Survival, growth and fatty acid profiles of larvae were studied. In trial 1, (n-3) fatty acid requirement was estimated using diets supplemented or not with methyl linolenate or cod liver oil. After 21 days, the best survival and growth were observed in larvae fed the unsupplemented diet [(n-3) fatty acid level: 0.05%]. Survival and growth were not improved by higher levels of (n-3) fatty acids. In trial 2, (n-6) fatty acid requirement was estimated using diets with graded levels of methyl linolenate or peanut oil. After 25 days, the best survival and growth were obtained with diets supplemented with 0.25% methyl linolenate (total (n-6) fatty acid level: 1%) or with 1.25% peanut oil (total (n-6) fatty acid level: 0.89%). Survival and growth were not improved by higher levels of (n-6) fatty acids. Fatty acid composition of carp reflected that of the diets and also showed that carp larvae are capable of elongating and desaturating linolenic acid and linoleic acid in longer chain fatty acids.

Specific distribution of fatty acids in the triglycerides of rainbow trout adipose tissue. Influence of temperature.

In the trout, the unsaturated fatty acids are preferentially located in the beta-position and the saturated fatty acids in the alpha-position of triglycerides. This fatty acid distribution is retained even with diets containing lard. The fish are, therefore, able to modify completely the fatty acid distribution of dietary triglycerides. There is no retention of the beta-monoglyceride structure during the biosynthetic processes. However, the modification of the dietary fatty acid distribution by the trout seems to be more difficult at 18C than at 10C.