Effect of the water-soluble fraction of petroleum on microsomal lipid metabolism of Macrobrachium borellii (Arthropoda: Crustacea).

The effect of the water-soluble fraction of crude oil (WSF) on lipid metabolism was studied at critical metabolic points, namely fatty acid activation, enzymes of triacylglycerol and phospholipid synthesis, and membrane (lipid packing) properties in the freshwater prawn Macrobrachium borellii. To determine the effect of the contaminant, adults and embryos at different stages of development were exposed to a sublethal concentration of WSF for 7 days. After exposure, microsomal palmitoyl-CoA synthetase (ACS) showed a two-fold increase in adult midgut gland. Embryo's ACS activity was also affected, the increment being correlated with the developing stage. Endoplasmic reticulum acylglycerol synthesis was also increased by WSF exposure in adults and stage 5 embryos, but not at earlier stages of development. Triacylglycerol synthesis was particularly increased (18.5%) in adult midgut gland. The microsomal membrane properties were studied by fluorescent steady-state anisotropy, using the rotational behavior of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Microsomes from midgut gland of WSF-exposed prawn showed no differences in fluidity. Nevertheless, microsomes incubated with WSF in vitro increased their fluidity in a temperature- and WSF concentration-dependent fashion. Both, aliphatic and aromatic hydrocarbons individually tested elicited an increase in membrane fluidity at 10 mg/l, but at 4 mg/l only nC10-C16 aliphatics did. In vivo results indicate that WSF increased the activity of microsomal enzymes that are critical in lipid metabolism, though this change was not due to direct alterations in membrane fluidity, suggesting a synthesis induction, or an enzyme-regulatory mechanism. Nevertheless, hydrocarbons elicited membrane fluidity alterations in in vitro experiments at concentrations that could be found in the environment after an oil spill.

Egg carotenoproteins in neotropical Ampullariidae (Gastropoda: Arquitaenioglossa).

Carotenoid-binding proteins are commonly found in invertebrates. Their carotenoids form non-covalent complexes with proteins giving tissues a variety of colors. In molluscs they have been described in only a few species. In particular, the egg perivitellin fluid of those Ampullariid species which deposit eggs above the waterline is provided with carotenoproteins playing several roles ranging from photoprotection, antioxidant or antitrypsin actions to nutrient provision for development. These molecules form complex glyco-lipo-carotenoproteins of high molecular weight where either free astaxanthin (3,3'-dihydroxy-beta, beta'-carotene- 4,4'dione) or astaxanthin esterified with fatty acids, occur more frequently. This review compiles the current knowledge on the biochemical composition and biophysical data on the chemical and thermal stability of egg carotenoproteins in ampullariid. In addition, recent data on their metabolism, their cellular site of biosynthesis during perivitellogenesis, as well as their carotenoid binding properties are reviewed, highlighting the physiological significance of carotenoproteins in the context of the reproductive biology of these molluscs.

Pallial oviduct of Pomacea canaliculata (Gastropoda): ultrastructural studies of the parenchymal cellular types involved in the metabolism of perivitellins.

Seasonal variations in the morphology of the parenchymal mass and function of the albumen gland/capsule gland complex have been studied in Pomacea canaliculata, together with the cellular types involved in the synthesis and secretion of perivitellin fluid components. The two major parenchymal cell types, albumen secretory cells (AS) and labyrinthic cells (LC), undergo seasonal variations throughout the annual reproductive cycle, which is divided into three periods. Both cellular types show maximal development and structural complexity during the reproductive period (spring and summer). AS cells have a well-developed Golgi complex and rough endoplasmic reticulum and their secretory granules show electron-dense particles of about 20 nm (probably galactogen). These cells are uniquely involved in ovorubin and PV2 perivitellin synthesis and their secretory granules are the single storage site for these two major perivitellins, as revealed by immunoelectron microscopy. AS also possess calcium deposits that infiltrate the cytoplasmic matrix. The luminal surfaces of LC exhibit long cilia intermingled with sparce short microvilli. Basally, the plasma membrane shows deep irregular folds that extend through the cytoplasm up to the subapical region. Calcium deposits infiltrate the cytoplasm and accumulate in the extracellular space of the basal labyrinth. Nerve terminals seem to be involved in the regulation of parenchymal cell secretion. At the post-reproductive period, AS markedly change their aspect following the release of most of the secretory granules into the acinar lumen. LC decrease in volume, the number of their cilia decreases, their cytoplasmic folds are much thinner and their extracellular spaces lack calcium particles. At the pre-reproductive period (winter), AS and LC recover and prepare for the subsequent period.

Activation of lipid catabolism by the water-soluble fraction of petroleum in the crustacean Macrobrachium borellii.

Little is known about the effect of the water-soluble fraction of crude oil (WSF) on lipid metabolism in invertebrates. The effect of the WSF on the triacylglycerol (TAG) mobilization, fatty acid activation and degradation was evaluated in the decapod Macrobrachium borellii, exposing adult and eggs at different stages of development for 7 days to a sublethal concentration of WSF. Using radioactive tracers, mitochondrial palmitoyl-CoA synthetase (ACS), triacylglycerol lipase (TAG-lipase) and fatty acid beta-oxidation system activities were assayed. Before studying the effect of WSF, the kinetic parameters of ACS were determined in purified mitochondria. Its optimal temperature and pH were 32 degrees C and 8.0, respectively, the apparent K(m) 2.48 micromol l(-1), and its V(max) of 1.93 nmol min(-1) mg protein(-1). These kinetic parameters differed significantly from this shrimp's microsomal isoform. After 7 days exposure to a sublethal concentration of WSF (0.6 mg/l), changes were observed in the enzymatic activity of all enzymes or enzymatic system assayed in adult midgut gland as well as in stage 5 eggs, a period of active organogenesis. An increase in the mobilization of energy stores was detected as early as stage 4, where TAG-lipase activity increased by 27% in exposed eggs. The increase was even more marked in exposed eggs at stage 5 where a three-fold rise (154%) was determined. Exposed adult shrimp also showed an augmented lipase activity by 38%. Fatty acid beta-oxidation increased by 51.0 and 35.5% in midgut gland and eggs at stage 5, respectively, but no changes were observed at less-developed stages. Mitochondrial fatty acid activation by ACS also increased in adults and stage 5 eggs by 7.4 and 52.0%, respectively. A similar response of the lipid catabolic pathways to WSF contamination in both adult and eggs, suggests that the exposure to this pollutant causes an increase in the energy needs of this shrimp. When validated by field studies, these catabolic enzymes could be employed as early pollution biomarkers.

Toxicity, uptake, and release of the water-soluble fraction of crude oil in different developing stages of the prawn Macrobrachium borellii.

The effects of a water-soluble fraction of light crude oil dissolved in freshwater (WSF) on Macrobrachium borellii exposed at three life stages was evaluated. Adults, larvae (PL), and embryos were exposed to five levels of WSF for 96 h. At 48 and 72 h PL were significantly more sensitive to WSF than adults, though values for 96-h LC50 were not significantly different (1.56 and 1.41 mg/L, respectively). Mortality was never high enough to allow LC50 calculation in embryos, but chronic exposure to WSF increased the mortality near hatching and significantly decreased PL survival compared to the control group. The uptake, accumulation, and depuration of WSF were evaluated in adult prawns (lipid content 1.5% wet wt) exposed to a sublethal concentration for 96 h followed by a 10-d depuration period. Hydrocarbons were rapidly taken up, and after 24 h most of them reached an equilibrium concentration. Increases in the number and alkylation of the aromatic rings enhanced both their accumulation and their retention. When returned to clean freshwater, levels of lighter aromatics dropped rapidly over 12 h. From these studies we conclude that early life stages of M. borellii are not very vulnerable to WSF toxicity, while adults have a rapid uptake and release of most of the contaminating hydrocarbons, probably due to their low lipid levels.

Effect of phenanthrene and Rhodotorula glutinis on arbuscular mycorrhizal fungus colonization of maize roots.

The effect of the polycyclic aromatic hydrocarbon (PAH) phenanthrene and the yeast Rhodotorula glutinis on the arbuscular mycorrhizal fungus (AMF) Glomus geosporum colonizing maize roots, was studied. During a 90-day experiment, the highest G. geosporum colonization values were found in control plants. Mycorrhiza root length, measured both on the basis of percentage of root colonization and on the activity of succinate dehydrogenase, showed similar patterns in different phenanthrene treatments. The presence of phenanthrene in the substrate reduced G. geosporum intraradical colonization. The presence of R. glutinis did not enhance AMF colonization in the presence of phenanthrene. The biomass of the external mycelium estimated on the basis of the fatty acid 16:1 omega 5 concentration showed a progressive increase through time, and the amounts of this fatty acid differed among treated and untreated substrates. However, this increase was found to be lowest in the phenanthrene and Rhodotorula treatment at 60 days. There was less phenanthrene accumulation in roots of maize inoculated with AMF and the yeast than in roots inoculated only with AMF. A similar pattern was observed in the phenanthrene content of G. geosporum spores collected after 90 days.

Synthesis, distribution, and levels of an egg lipoprotein from the apple snail Pomacea canaliculata (Mollusca: Gastropoda).

The site of synthesis of mollusc lipoproteins is hitherto unknown and was investigated for perivitellin 2 (PV2), an egg lipoprotein found in the freshwater snail Pomacea canaliculata. Tissues (albumen gland, gonad-digestive gland complex, and muscle) from vitellogenic females were incubated in vitro with 14C-leucine at 25 degrees C for 12 hr. At the end of incubation, soluble proteins from tissue homogenates and medium were analyzed for de novo protein synthesis by electrophoresis and HPLC, and radiolabeled proteins were quantified by liquid scintillation. Two albumen gland radiolabeled proteins (67 and 31 kDa) co-migrated with the subunits of PV2, and they represented 6.0% of the total labeled protein in that tissue. Western blot analysis confirmed the presence of PV2 only in the albumen gland. In vivo experiments where adult females were injected with 3H-leucine revealed that PV2 was not present in hemolymph. ELISA analysis in all tissues of the snail confirmed the PV2 presence only in the albumen gland and developing eggs with levels of 26 and 98 mg/g protein, respectively. Therefore, the albumen gland is the only site for PV2 synthesis, and no extra-gland synthesis, circulation, or accumulation could be found. PV2 subunits were further characterized analyzing N-terminal sequences which showed no homology with other proteins.

Lipid and fatty acid composition and energy partitioning during embryo development in the shrimp Macrobrachium borellii.

Energy partitioning, composition of lipids and fatty acids, and their utilization by embryos were determined in the lecithotrophic shrimp Macrobrachium borellii during seven development stages. The biochemical composition at stage I is represented by lipids, proteins, and carbohydrates, with 29.3, 28.7, and 0.2% dry weight, respectively. The former two were identified as the major energy-providing components, contributing 131 and 60 cal/100 mg egg, dry weight, respectively. The overall conversion efficiency (CE) was 45.0% (calculated as percentage of vitelline energy transformed into embryonic tissues). Lipids were the most important energy reserve (CE 39.3%), followed by proteins (CE 57.1%), both being simultaneously utilized during development while carbohydrates were synthesized de novo (CE 587.5%). Variation in the lipid class composition of embryos and vitellus showed an accumulation of triacylglycerols (TAG) and phospholipids (PL) up to stage IV, a more active accumulation and selective utilization phase (stages V and VI), and a consumption and de novo synthesis period until hatching. Structural lipids (PL and cholesterol) and pigment astaxanthin were selectively conserved in embryos, but TAG, hydrocarbons, and esterified sterols were preferentially depleted. Monounsaturated fatty acids (FA) were the major group in TAG, whereas polyunsaturated FA (PUFA) were the major group in PL after organogenesis. Certain PUFA such as 22:6n-3 and 20:5n-3 were selectively accumulated in PL.

Enzyme activities involved in lipid metabolism during embryonic development of Macrobrachium borellii.

The activities of the enzymatic systems involved in the activation and degradation of fatty acids, and in the synthesis of triacylglycerols and phospholipids were studied in vitro using total cellular homogenate and subcellular fractions of eggs of the shrimp Macrobrachium borellii at different developing stages. Egg development was divided into seven stages based on morphological features of the embryo. Palmitoyl-CoA ligase activity increased as the embryo developed and showed its maximum at stage V. An increase in the synthesis of triacylglycerols and diacylglycerols was also observed at this stage. Diacylglycerylethers were synthesized more actively during the first stages of development. The higher specific activity observed in total homogenate than in microsomal fraction suggested that their synthesis was not exclusively microsomal. Phospholipid synthesis was very active all along development, reflecting active membrane biosynthesis. The highest activity of the cytosolic triacylglycerol lipase was observed at stage V. Fatty acid degradation, measured as mitochondrial beta-oxidation activity, did not vary significantly during development. We conclude that both the anabolic and catabolic processes concerning lipid metabolism are very active, with values similar to those described for adult hepatopancreas, revealing the major role of lipids during shrimp embryogenesis energetics, and that the highest activities of lipid synthesis-hydrolysis take place at stage V when embryos are under active organogenesis. J. Exp. Zool. 286:231-237, 2000.

Lipoproteins of the egg perivitelline fluid of Pomacea canaliculata snails (Mollusca: Gastropoda).

The lipid and protein composition of the perivitelline fluid of the eggs of Pomacea canaliculata was investigated. Two lipoproteins (PV 1 and PV 2) and one lipoprotein fraction (PV 3) were detected for the first time in gastropods. They represent 57.0, 7.5, and 35.5% of the egg total proteins, respectively. PV 1 is a glyco-carotene-protein complex with characteristics of a very high-density lipoprotein (VHDL). It has 0.33% lipids, mainly free sterols and phospholipids. The particle has a MW of 300 Kd and is composed of three subunits of 35, 32, and 28 Kd, respectively. PV 2 particle is a VHDL of 400 Kd and 3.75% lipids. The major lipid classes are free sterols and phospholipids and also have significant quantities of energy-providing triacylglycerides and free fatty acids. It is composed of two apoproteins of 67 and 31 Kd. PV 3 density corresponds to a high-density lipoprotein (HDL). It was fractionated into two subfractions "h" and "p". Fraction "h" contains 5.16% lipids, mainly free sterols, phospholipids, and free fatty acids, and two particles of 100 and 64 Kd. Dissociating electrophoresis showed two subunits of 34 and 29 Kd. Fraction "p" is composed of a single particle of 26 Kd that contains 9.5% lipids, which represents 30% of the total egg lipids. It has high levels of a carotenoid pigment. Besides it contains free fatty acids, hydrocarbons, sterified sterols, and triacylglycerides. These three fractions are probably the major supply of lipids and amino acids for the developing embryo.

Occurrence of a 22∶2 nonmethylene interrupted dienoic fatty acid and its seasonal distribution among lipids and tissues of the fresh water bivalveDiplodon delodontus from an isolated environment.

Realatively high levels of a non-methylene-interrupted dienoic fatty acid were detected in the freshwater molluscDiplodon delodontus. The (7,13) 22∶2 NMID fatty acid was separated from total fatty acids by TLC, and its structure was determined by GLC and reductive ozonolysis. Its seasonal distribution was investigated in different tissues and lipids of the mollusc. High concentrations of this acid were found in polar lipids. The absence of the 22∶2 NMID fatty acid in the lipids of plankton and sediment in the same habitat suggests that it may be biosynthesized by themollusc. Possible synthesis and functions of the NMID fatty acids are discussed.

Effect of the environment and fasting on the lipid and fatty acid composition ofDiplodom patagonicus.

Some effects of food, habitat and temperature on the lipid composition of a freshwater mollusk,Diplodom patagonicus, were studied. Animals kept and fasted up to 60 days in an aquarium at 9 C and 20 C showed a decrease of the total lipid content that corresponded to a decrease of triacylglycerols and diacylglycerol ethers. This decrease evoked an increase of polar-to-nonpolar lipid ratio. However, no significant change in the total fatty acid composition was shown. Moreover, a decrease of temperature from 20 C to 9 C decreased the incorporation of labeled linoleic and α-linolenic acid into the lipids, but did not modify the unsaturated: saturated acid ratio of the mollusk lipids during this period. A change of habitat from lake to estuary changed very significantly the fatty acid composition of the animal. The ω6 acids, linoleic and arachidonic, typical ofD. patagonicus living in the lake, were partially replaced by ω3 acids. That this change was due to a change of food composition was indicated by the fatty acids of corresponding sediments. Therefore, the fatty acid composition ofD. patagonicus is highly sensitive to food composition and varies little with temperature and seasonal changes.

Comparative biosynthesis of polyethylenic fatty acids in Acanthamoeba castellanii and Ochromonas danica.

Acanthamoeba castellanii were incubated in vivo with 1(-14)C linoleic and 1(-14)C-alpha-linolenic acids. The incorporation of the acids into lipid fractions was studied. Labeling was found mainly in triglycerides and phospholipids. Homogenized cells and subcellular fractions separated by centrifugation were incubated with 1-14C-linoleic. 1-14C -alpha-linoleic and 1-14C eicosa-8,11-dienoic acids in the presence of NADH, ATP, and CoA. Different metabolic routes were demonstrated. omega3 and omega6 desaturases of the vegetal type, as well as a delta6 desaturation of alpha-linolenic acid of the animal type were present. The supernatant of 100000 x g contained both types of desaturating enzymes, whereas the corresponding particulated fraction was inactive. The ultrastructure of Acanthamoeba showed the endoplasmic reticulum with a poorly developed membrane component. The metabolic pathways found with Acanthamoeba were compared to Ochromonas danica incubated with linoleic acid in the light and in darkness. Desaturases typical of "vegetal" and "animal" pathways were found in both organisms. In both of them, alpha-linolenic and arachidonic acids could be synthetized. However, alpha-linolenic acid, typical of vegetal synthesis, was only stored in Ochromonas due to the presence of a photosynthetic machinery.

Comparative biosynthesis of polyethylenic fatty acids in Acanthamoeba castellanii and Ochromonas danica

Acanthamoeba castellanii were incubated in vivo with 1(-14)C linoleic and 1(-14)C-alpha-linolenic acids. The incorporation of the acids into lipid fractions was studied. Labeling was found mainly in triglycerides and phospholipids. Homogenized cells and subcellular fractions separated by centrifugation were incubated with 1-14C-linoleic. 1-14C -alpha-linoleic and 1-14C eicosa-8,11-dienoic acids in the presence of NADH, ATP, and CoA. Different metabolic routes were demonstrated. omega3 and omega6 desaturases of the vegetal type, as well as a delta6 desaturation of alpha-linolenic acid of the animal type were present. The supernatant of 100000 x g contained both types of desaturating enzymes, whereas the corresponding particulated fraction was inactive. The ultrastructure of Acanthamoeba showed the endoplasmic reticulum with a poorly developed membrane component. The metabolic pathways found with Acanthamoeba were compared to Ochromonas danica incubated with linoleic acid in the light and in darkness. Desaturases typical of [quot ]vegetal[quot ] and [quot ]animal[quot ] pathways were found in both organisms. In both of them, alpha-linolenic and arachidonic acids could be synthetized. However, alpha-linolenic acid, typical of vegetal synthesis, was only stored in Ochromonas due to the presence of a photosynthetic machinery.