Fatty acid composition of four microsporidian species compared to that of their host fishes

ABSTRACT. The fatty acid composition of four microsporidian species (Glugea atherinae, Spraguea lophii, Glugea americanus , and Pleistophora mirandellae) and their host fishes has been determined using gas chromatography. Twenty-four fatty acids were identified with differences in relative abundance of fatty acids among the four parasites. Certain even-saturated fatty acids were found in a very high proportion: palmitic acid (16:0) represented one-third of total fatty acids in Pleistophora mirandellae. The level of docosahexaenoic acid (22:6ω3) attained 26–28% in Glugea atherinae, Spraguea lophii , and Glugea americanus , but only 8–9% in P. mirandellae. With respect to fatty acid compositions of host organs, some significant differences were evident between marine and freshwater fishes. Palmitic acid was prevalent in the marine fishes, Atherinae boyeri and Lophius piscatorius , and oleic acid (18:1ω9) in the freshwater fish Leuciscus cephalus. The proportion of docosahexaenoic acid in marine fishes was two or three times as great as in freshwater fish Leuciscus. The high polyunsaturated fatty acid content in both parasites and host fishes may be related to the scavenging of these fatty acids by the parasites rather than a microsporidia-specific fatty acid biosynthesis pathway.     

Identification and Phylogenetic Relationships of Microsporidia by Riboprinting

ABSTRACT. The SSUrDNA and the ITS of different microsporidia from eight fishes, four insects and a shrimp were amplified and digested with restriction enzymes. The generated riboprints suggest a close evolutionary relationship between Glugea americanus and Spraguea lophii suggesting that Glugea americanus should be renamed Spraguea americanus and that the tissue infected and host origin should be considered of greater taxonomic importance for defining a genus than previously considered. Phylogenetic analysis of the riboprints demonstrates an unidentified microsporidium from a bumper fish ( Chloroscombrus chrysurus ) is related although not identical to Microgemma oviodea , a parasite from red band fish. We were also able to distinguish between Glugea anomala and Glugea atherinae and Glugea stephani but were not able to differenciate among the latter two. Insects isolates, Nosema costelytrae, N. bombycis, N. trichoplusiae, Nosema sp. and a shrimp isolate, Agmasoma penaei , are not related to the fish isolates.     

Ribosomal DNA sequences of Glugea anomala, G. stephani, G. americanus and Spraguea lophii (Microsporidia): phylogenetic reconstruction

The microsporidian species Glugea anomala, G. stephani, G. americanus and Spraguea lophii were compared by using sequence data derived from their small subunit rDNA genes which were amplified by polymerase chain reaction and directly sequenced. These sequence data and published data of G. atherinae were analyzed and were used to infer a phylogenetic tree. The 5 microsporidian fish parasites appeared to be closely related. The higher sequence similarities demonstrated among G. anomala, G. stephani and G. atherinae suggest that these 3 parasites are in fact only 1 species of Glugea. Moreover, the higher sequence similarities between S. lophii and G. americanus support the transfer of the latter Glugea species into the genus Spraguea.     

Effects of selenium deficiency on fatty acid metabolism in rats fed fish oil-enriched diets.

The hepatic fatty acid metabolism was investigated in rats stressed by selenium deficiency and enhanced fish oil intake. Changes in the composition of lipids, peroxides, and fatty acids were studied in the liver of rats fed either a Sedeficient (8 microg Se/kg) or a Se-adequate (300 microg Se/kg) diet, both rich in n-3 fatty acid-containing fish oil (100 g/kg diet) and vitamin E (146 mg alpha-tocopherol/kg diet). The two diets were identical except for their Se content. Se deficiency led to a decrease in hair coat density and quality as well as to changes in liver lipids, individual lipid fractions and phospholipid fatty acid composition of the liver. The low Se status did reduce total and reduced glutathione in the liver but did not affect the hepatic malondialdehyde level. In liver phospholipids (PL), Se deficiency significantly reduced levels of palmitic acid [16:0], fatty acids of the n-3 series such as DHA [22:6 n-3], and other long-chain polyunsaturates C-20-C-22, but increased n-6 fatty acids such as linoleic acid (LA) [18:2 n-6]. Thus, the conversion of LA to arachidonic acid was reduced and the ratio of n-6/n-3 fatty acids was increased. As in liver PL, an increase in the n-6/n-3 ratio was also observed in the mucosal total fatty acids of the small intestine. These results suggest that in rats with adequate vitamin E and enhanced fish oil intake, Se deficiency affects the lipid concentration and fatty acid composition in the liver. The changes may be related to the decreased levels of selenoenzymes with antioxidative functions. Possible effects of Se on absorption, storage and desaturation of fatty acids were also discussed.     

Lipids and fatty acids of two pelagic cottoid fishes (Comephorus spp) endemic to Lake Baikal

Matured females of two Lake Baikal endemic fish species, Comephorus baicalensis and Comephorus dybowski, have been investigated for lipid of the whole body and specific tissues (liver, muscles, ovaries), phospholipid classes and fatty acids of neutral and polar lipids. Total lipid in the body (38.9% fresh weight), liver (23.5%) and muscles (14.5%) of C. baicalensis were greater than those of C. dybowski (4.7, 8.7 and 2.6%, respectively); only their ovaries were similar (5.3 and 5.6% lipid, respectively). In both species, phosphatidylcholine and phosphatidylethanolamine were the major phospholipids, ranging from 60.7 to 75.1% of total phospholipid and 14.5–25.7%, respectively. In most cases, monounsaturated fatty acids (MUFA) were the major fatty acid group in C. baicalensis, whereas polyunsaturated fatty acids (PUFA) were the major group in C. dybowski. The MUFA 18:1(n-9) prevailed over other fatty acids in C. baicalensis and varied from 19% in polar lipids of muscles to 56.1% in neutral lipids of muscles. In polar lipid of C. dybowski, the PUFA 22:6(n-3) prevailed over other fatty acids in muscles and ovaries, while 16:0 dominated polar liver lipids and neutral lipids of all tissues. Other major fatty acids included 16:1(n-7), 18:1(n-7), and 20:5(n-3). Values of the (n-3)/(n-6) fatty acid ratio for neutral lipids of C. baicalensis (0.5–0.9) are well below the range of values characteristic either for marine or freshwater fish, while these values for polar lipids (1.6–1.8) are in the range typical of freshwater fish. Neutral lipid fatty acid ratios in C. dybowski (2.5–3.1) allow it to be assigned to freshwater fish, but polar lipids (2.8–3.7) leave it intermediary between freshwater and marine fish.     

Lipids and fatty acids in muscle, liver and skin of three edible fish from the Senegalese coast: Sardinella maderensis, Sardinella aurita and Cephalopholis taeniops

Lipid content and fatty acid composition were determined in three species of edible fish caught in Senegalese waters during the upwelling season (January, 1993). Sardinella maderensis and Sardinella aurita are fat fish containing more than 5% (fresh wt.) of lipids, whereas Cephalopholis taeniops is a lean fish with approximately 1% of lipids. Skin, liver and muscle were studied for each fish species. About 40 fatty acids were identified by GC and GC/MS as methyl esters and N-acyl pyrrolidides. Palmitic acid was the main acid in the muscle and skin of all samples studied (20-33% of total fatty acids). Oleic acid was the main fatty acid in the liver of S. maderensis (27.2%+/-0.1) and S. aurita (44.7%+/-0.1). Arachidonic acid was a minor component in all samples. The flesh (muscle) of the three fish species contained high concentrations of omega3 polyunsaturated fatty acids (PUFA), ranging from 16.0 to 29.1% and including 20:5 omega3 (eicosapentaenoic acid, EPA) and 22:6 omega3 (docosahexaenoic acid, DHA) acids as major components. These two acids together accounted for 24.7%+/-0.1 and 12.9%+/-0.1 of total acids in the skin of S. maderensis and S. aurita, respectively. The percentages of PUFA found in the fish studied were very similar to those in fish used commercially as sources of PUFA. Muscle sterols, which accounted for 9-11% of total lipids, consisted mainly of cholesterol (up to 97% of total sterols).     

Growth, fat content and fatty acid profile of South American catfish, surubim (Pseudoplatystoma fasciatum) juveniles fed live, commercial and formulated diets

South American catfish, barred surubim ( Pseudoplatystoma fasciatum ) juveniles (117.6 ± 11.8 mg individual weight; 28.3 ± 2.5 mm total length) were fed various diets: one live ( Tubifex worms), two commercial (Aglo Norse and Bio Kyowa), and one semi-purified formulated diet (75% peptide based protein) over a 2-week period. Fish fed the Aglo Norse diet showed the highest growth performance, but cannibalism also was very high (42%). Fish fed peptide based formulated diet demonstrated the lowest growth rate, with no cannibalism. The highest survival was achieved with fish fed Tubifex worms (100%). Lipid level in the whole body of the fish fed four different experimental diets did not differ significantly, averaging 3.6 ± 0.7%. Fatty acid composition of neutral and phospholipid fractions of whole body lipids of fish reflected the fatty acid composition of the diets. The high level of 20:4 n -6 in Tubifex worms resulted in a high level of this fatty acid in the tissue of fish fed this diet. It remains uncertain how high survival and no cannibalism is related to dietary lipids/fatty acids. In all cases, the increasing ratio of n -3 HUFA (highly unsaturated fatty acids)/ n -6 HUFA in phospholipid fractions suggested the elongation and desaturation of 18:3 n -3 to 22:6 n -3 via 20:5 n -3. Moreover, in respect to the 20:4 n -6 levels in the diets, an increase in the concentration of this fatty acid in phospholipid fraction suggests that South American catfish can transform linoleate into arachidonate.     

Effect of frozen storage on fatty acid composition and changes in lipid content of Scomberomorus commersoni and Carcharhinus dussumieri

Investigated were the changes in fatty acid composition, oxidation and enzymatic deterioration of lipids in frozen (−30°C) fish fillets from the Persian Gulf. The narrow barred Spanish mackerel ( Scomberomorus commersoni ) and white cheek shark ( Carcharhinus dussumieri ) were tested with storage times of 0, 1, 2, 3, 4, 5 and 6 months at −18°C. Statistical results showed that the major fatty acids among the saturated and monounsaturated fatty acids of each fish species were palmitic (C16:0) and oleic (C18:1n-9) acids, respectively. Both linoleic acid (C18:2n-6) and arachidonic acid (AA) (C20:4n-6) were predominant in total n-6 polyunsaturated fatty acids in both mackerel and shark. The EPA (eicosapentaenoic acid; C20:5 n-3) and DHA (docosahexaenoic acid; C22:6 n-3) acids were the major fatty acids among total n-3 acids in both fishes. During frozen storage, the PUFA (40.1 and 23.94%), n-3 (48 and 42.83%), ω 3/ ω 6 (41.36 and 50%), PUFA/SFA (56 and 42.23%) and EPA + DHA/C16 (55.55 and 46.66%) contents decreased in S. commersoni and C. dussumieri , respectively. Also peroxide, thiobarbituric acid (TBA) and free fatty acid (FFA) values significantly increased (P < 0.01) with the time of storage.     

Effect of increasing amounts of dietary fish oil on brain and liver fatty composition

Increasing dietary fish oil in rat had the following effect on brain lipids: Arachidonic acid regularly decreased; eicosapentanenoic acid, normally nearly undetectable, was present; 22:5(n - 3), dramatically increased but remained below 1% of total fatty acids; cervonic acid was increased by 30% at high fish oil concentration. Saturated and monounsaturated fatty acids were not affected regardless of chain-length. In contrast, in the liver, nearly all fatty acids (saturated, monounsaturated and polyunsaturated) were affected by high dietary content of fish oil, but liver function was normal: serum vitamin A and E, glutathione peroxidase, alkaline phosphatase, transaminases were not affected. Serum total cholesterol, unesterified cholesterol and phosphatidylcholine were slightly affected. In contrast, triacylglycerols were dramatically reduced in proportion to the fish oil content of the diet.     

Effect of temperature acclimatization on the fatty acid composition of goldfish intestinal lipids

1. The fatty acid composition of whole goldfish, whole-intestinal mucosa, intestinal mucosal membranes and individual phospholipids extracted from mucosal membranes were measured, fish adapted to different temperatures being used. 2. Alterations of the adaptation temperature did not noticeably affect the fatty acid composition of the whole-fish lipids, but there were marked changes in the fatty acids of lipids extracted from homogenates of goldfish intestinal mucosa. These changes were more pronounced in a membrane fraction prepared from these homogenates. Raising the adaptation temperature by 20 degrees C halved the percentage of C(20:1), C(20:4) and C(22:6) fatty acids and nearly doubled the percentage of C(18:0) and C(20:3) fatty acids recovered. 3. Choline phosphoglycerides constituted about one-half and ethanolamine phosphoglycerides about one-quarter of the total membrane phospholipids. 4. The fatty acids of choline and ethanolamine phosphoglycerides were more susceptible to temperature-dependent changes than were the phosphoglycerides of inositol or serine. 5. The increase in C(18:0) fatty acid that occurred in membranes of warm-adapted fish was greatest for ethanolamine phosphoglycerides, but increases also occurred in other phospholipid fractions and in membrane neutral lipids.     

Lipids of microorganisms from the family Vibrionaceae causing diseases in fishes]

Lipid fractional and fatty acid compositions of microorganisms from the genera Aeromonas, Pseudomonas, and Vibrio (the family Vibrionaceae), causing diseases of different fish species, were studied. Motile aeromonads and vibrios displayed higher relative contents of membrane lipids and oleic acid and lower relative contents of storage lipids compared with immotile aeromonads and pseudomonads, which is connected with the activities of their movements. Immotile aeromonads and vibrios exhibited higher levels of polyunsaturated fatty acids and higher absolute phospholipid contents compared to motile aeromonads and pseudomonads. This is likely to be related to host specificity of these bacteria and reflects the specific patterns of fatty acid compositions of the infected fish (salmonid and cyprinid) tissues.     

Fatty acids in liver and muscle of farmed and wild sea bass (Dicentrarchus labrax L.)

Growth rate and assimilation of essential fatty acids were analysed in sea bass (Dicentrarchus labrax L.) fed different diets. Gas Chromatographie analyses of total lipids in commercial diets indicated excessive presence of saturated, mono and dienoic fatty acids, while the content of to 3 HUFA was very low. Fatty acid patterns of liver and white muscle farmed sea bass reflected the contents of the dietary lipids. The concentration of polyunsaturated fatty acids, particularly ω3 HUFA was significantly lower when compared with samples from the wild population. The atherogenic and fish lipid quality indices confirmed the higher nutritional quality of wild sea bass when compared with farmed fishes.     

Fatty-Acid Profile of Total and Polar Lipids in Cultured Rainbow Trout (Oncorhynchus mykiss) Raised in Freshwater and Seawater (Croatia) Determined by Transmethylation Method

Fatty acids from total lipids and polar lipids in cultured rainbow trout (Oncorhynchus mykiss) raised in seawater (SW) and freshwater (FW) were identified and quantified from the muscle samples in January, April, and July. The highest total lipid and polar lipid amounts were found in April. July contents of total lipids were low, but percent of the polyunsaturated fatty acids (PUFAs) was high in SW and FW environment (particularly n-3 PUFAs). Variety of 17 fatty acids was identified by GC-FID after transmethylation. The predominant fatty acids in rainbow trout from SW and FW were: docosahexaenoic acid among n-3 PUFAs, palmitic acid among saturated fatty acids (SFAs), and oleic acid among monounsaturated fatty acids (MUFAs). Appreciably higher n-3/n-6 ratio was found in total lipids in April (6.40, FW fish) and in polar lipids in July (18.76; SW fish). High n-3/n-6 ratio in total lipids and polar lipids of rainbow trout from SW and FW, besides beneficial n-3/n-6 ratio in the commercial fish food, could be characteristic for the local environmental conditions (Croatia).     

Lipids of Microorganisms of the Family Vibrionaceae,Causative Agents of Fish Diseases

Lipid fractions and fatty acid compositions of microorganisms from the genera Aeromonas, Pseudomonas, and Vibrio(the family Vibrionaceae) that cause diseases of various fish species were studied. Motile aeromonads and vibrios displayed higher relative contents of membrane lipids and oleic acid and lower relative contents of storage lipids compared with nonmotile aeromonads and pseudomonads, which is connected with the activities of their movements. Nonmotile aeromonads and vibrios exhibited higher levels of polyunsaturated fatty acids and higher absolute phospholipid contents compared to motile aeromonads and pseudomonads. This is likely to be related to the host specificity of these bacteria and reflects the specific patterns of fatty acid compositions of the infected fish (salmonid and cyprinid) tissues.     

Lipid composition in muscle and liver of sympatric coregonid fishes from Lake Baikal (<Emphasis Type="Italic">Coregonus</Emphasis> spp.) under common garden experiment

The content of total lipids, total phospholipids, and fatty acids of total lipids in muscles and liver of juvenile Lake Baikal sympatric coregonid fishes have been analyzed for the first time under a common garden experiment. Baikal omul, Coregonus migratorius Georgi, is an active migrant of the pelagic zones of the lake. Baikal (lacustrine) whitefish, C. baicalensis Dybowski, is a colonizer of the bottom habitats, which are relevant to the pelagic zones of the littoral and underwater slope. Structural lipids (phospholipids and cholesterol) dominated in total lipids of the tissues of all fish under study. Spare lipids significantly prevailed in omul muscles when compared to whitefish. The highest variability of fatty acid composition was reported in muscles of coregonid fishes. Statistically significant differences were revealed in the content of the ω3 polyunsaturated fatty acids in muscles of lacustrine whitefish and omul. Associations of lipid compositions revealed in tissues of the whitefishes under the study with their respective ecotypes have been discussed.     

Seasonal changes in lipid classes and fatty acid composition in the digestive gland of Pecten maximus

Seasonal variations in lipid classes and fatty acid composition of triacylglycerols and phospholipids in the digestive gland of Pecten maximus were studied over a period of 16 months. Acylglycerols predominated (19-77% of total lipids), in accordance with the role of the digestive gland as an organ for lipid storage in scallops. Seasonal variations were mainly seen in the acylglycerol content, while phospholipids (2.5-10.0% of total lipids) and sterols (1.9-7.4% of total lipids) showed only minor changes. The most abundant fatty acids were 14:0, 16:0, 18:0, 16:1(n-7), 18:1(n-9), 18:1(n-7), 18:4(n-3), 20:5(n-3) and 22:6(n-3) and these showed similar seasonal profiles in both, triacylglycerol and phospholipid fractions. In contrast to the phospholipid fraction, the triacylglycerol fraction contained more 20:5(n-3) than 22:6(n-3). In three phospholipid samples we noted a high percentage of a 22-2-non-methylene-interrupted fatty acid, previously described to have a structural role in several bivalve species. The main polyunsaturated fatty acids displayed important seasonal variations parallel to those of the acylglycerols, suggesting good nutritional conditions. A positive correlation existed between the level of saturated fatty acids and temperature, whereas the levels of polyunsaturated fatty acids correlated negatively with temperature.     

Phospholipid fatty acid composition, vitamin E content and susceptibility to lipid peroxidation of duck spermatozoa

Recent studies on chicken semen have suggested that the lipid and fatty acid composition of spermatozoa may be important determinants of fertility. Phospholipid fatty acid composition, vitamin E content and in vitro susceptibility to lipid peroxidation of duck spermatozoa were investigated using GC-MS and HPLC based methods. The total phospholipid fraction of duck spermatozoa was characterized by high proportions of the n-6 polyunsaturated fatty acids arachidonic (20:4n-6), docosatetraenoic (22:4n-6) and docosapentaenoic (22:5n-6) acids but a substantial proportion of the n-3 fatty acid docosahexaenoic (22:6n-3) acid was also present. Palmitic (16:0) and stearic (18:0) fatty acids were the major saturates in sperm phospholipids. Among the phospholipid classes, phosphatidylserine (PS) had the highest degree of unsaturation due to very high proportions of 22:6n-3, 22:5n-6, 22:4n-6 and 20:4n-6, comprising together more than 75% of total fatty acids in this fraction. Phosphatidylethanolamine (PE) also contained high proportions of these four C(20-22) polyunsaturates, which together formed 60% of total fatty acids in this phospholipid. Spermatozoa and seminal plasma of duck semen were characterized by unexpectedly low content of vitamin E, being more than 4-fold lower than in chicken semen. In duck semen the major proportion of the vitamin E (>70%) was located in the spermatozoa. The very high proportion of 22:6n-3 in PS and PE fractions of duck sperm lipids and the comparatively low levels of vitamin E could predispose semen to lipid peroxidation. Nevertheless the in vitro susceptibilities to Fe2+-stimulated lipid peroxidation of duck and chicken spermatozoa were very similar. The results of the study suggest that increased superoxide dismutase and glutathione peroxidase activity and increased antioxidant activity of seminal plasma may compensate for the low levels of vitamin E to help protect the membranes of duck spermatozoa, which exhibit a high degree of unsaturation from oxidative stress.     

Comparative levels of arachidonic acid and eicosapentaenoic acid in Malaysian fish

Total lipids were extracted from 22 species of Malaysian fish and the constituent fatty acids were analysed by gas chromatography. Malaysian fish generally contained high levels of saturated fatty acids (range 36-55% total fatty acids) and contained variable amounts of monounsaturates, chiefly palmitic and stearic acids, but only trace levels of 20:1 and 22:1. Unlike fish caught in colder northern hemisphere waters, Malaysian fish were found to contain arachidonic acid (20:4 omega 6, range 2-12%) in addition to the expected eicosapentaenoic acid (20:5 omega 3, range 1-13%) and docosahexaenoic acid (22:6 omega 3, range 6.6-40.4%).     

Spores of two fish microsporidia (Pseudoloma neurophilia and Glugea anomala) are highly resistant to chlorine

Pseudoloma neurophilia (Microsporidia) is the most common pathogen found in zebrafish Danio rerio research facilities. The parasite is associated with marked emaciation. Zebrafish laboratories usually disinfect eggs to prevent transmission of pathogens, typically with chlorine at 25 to 50 ppm for 10 min. The ability of chlorine to kill spores of P. neurophilia and 2 other microsporidia, Glugea anomala and Encephalitozoon cuniculi, was evaluated using 2 viability stains. SYTOX Green was used to visualize dead spores, and live spores were identified by their ability to extrude polar tubes in Fungi-Fluor solution following UV exposure. Results with both stains were similar at various chlorine concentrations for P. neurophilia and G. anomala, but Fungi-Fluor was not useful for E. cuniculi, due to the much smaller spore size. Using the SYTOX stain, we found that 5 ppm chlorine for 10 min causes 100% death in spores of E. cuniculi, which was similar to findings in other studies. In contrast, the spores of P. neurophilia and G. anomala were much more resistant to chlorine, requiring >100 or 1500 ppm chlorine, respectively, to achieve >95% spore death. Repeating chlorine exposures with spores of P. neurophilia using solutions adjusted to pH 7 increased the efficacy of 100 ppm chlorine, achieving >99% spore inactivation. We corroborated our viability staining results with experimental exposures of zebrafish fry, achieving heavy infections in fry at 5 to 7 d post-exposure in fish fed spores treated at 50 ppm (pH 9). Some fish still became infected with spores exposed to 100 ppm chlorine (pH 9.5). This study demonstrates that spores of certain fish microsporidia are highly resistant to chlorine, and indicates that the egg disinfection protocols presently used by most zebrafish research facilities will not prevent transmission of P. neurophilia to progeny.     

Bone lipids of marine fishes.

Assessment of candidates for investigation of bone lipid metabolism yielded the following findings. (1) A tropical marine butterflyfish, Chaetodon ornatissimus, had oil-filled bones (66-80% lipid, percent dry weight) hence may be a suitable condidate. (2) The tropical marine fishes Exallias brevis, Pomacentrus jenkensi, and Chromus agilis, and a Canadian fish Sebastes ruberrimus, had intermediate quantities of oil in their bones (12-49% lipid). (3) In all the foregoing species the major bone lipid was triglyceride, usually more abundant in skull than spine. Sterol and phospholipid were also present. (4) The major fatty acids of the triglycerides (and phospholipids) were 16:0, 18:0, 18:1, and C20, C22 acids. Those acids were dominated by 20:4, 20:5, 22:5, and 22:6. (5) There was more total unsaturation in the bone lipids of S. ruberrimus (from 10 degrees C water; 67-72% unsaturation) compared to the tropical fish (from 25 degrees C water; 32-67% unsaturation) with the exception of E. brevis. (6) One of the tropical species (Arothron meleagris) and a Canadian Chimaeran (Hydrolagus colliei) contained only 1-3% lipid in their bones.