Ricardo J. Pollero

BIOCHEMICAL COMPOSITION AND ENERGY SOURCES DURING EMBRYO DEVELOPMENT AND IN EARLY JUVENILES OF THE SNAIL POMACEA CANALICULATA (MOLLUSCA: GASTROPODA)

Energy partitioning within the developing eggs and early juveniles of the gastropod Pomacea canaliculata was investigated from the time of fertilization to newly hatched snails. The forms and locations of the energy stored as well as their changes while development proceeded were studied in six stages (from morula to 3-day hatchlings). The rates of utilization of different lipid classes, protein classes, and total carbohydrates were measured for the first time in embryos and perivitelline fluid. n n n nPerivitelline fluid is the primary energy store. Its biochemical composition at stage I is represented by carbohydrates, proteins, and lipids with 34.8%, 13.0%, and 1.5% dry weight, respectively. Dry weight of eggs showed no significant increase during development, indicating there was no important mass exchange with the medium. n n n nCarbohydrates and proteins were identified as the major energy-providing components, and lipids as the minor one, contributing 142, 56, and 12 cal/100 mg egg, dry weight, respectively. The overall conversion efficiency (CE) was 32.8% (calculated as percentage of perivitellus energy transformed into embryonic tissues). Carbohydrates were the most important energy reserve supplying virtually all the catabolic demand (CE 14.6%). n n n nProtein electrophoretic profiles during embryogenesis showed three distinct phases: An accumulation period (up to stage IV); a more active accumulation and selective utilization phase (stage V), and a selective consumption and protein synthesis period (hatchlings). Structural lipids from perivitellus (phospholipids and free sterols) were selectively conserved in embryos and juveniles, whereas triacylglycerols, hydrocarbons, and esterified sterols were preferentially depleted by hatchlings. n n n nTherefore, protein and lipid reserves in P. canaliculata perivitellus provide structural precursors during embryogenesis, while they partially contribute to the energy supplied by carbohydrates. J. Exp. Zool. 280:375–383, 1998.

Lipid characterization and distribution among tissues of the freshwater crustacean Macrobrachium borellii during an annual cycle

Abstract 1. 1. Lipid and fatty acid seasonal distribution from tissues of the freshwater shrimp Macrobrachium borelli was studied. 2. 2. Female gonads and hepatopancreas were the organs with the highest lipid percentages. They were mainly composed of triacylglycerols, with oleic acid as the major fatty acid. 3. 3. Total lipids and triacylglycerols from ovarian tissue increased during sexual maturation and they presented a peak during winter in hepatopancreas. 4. 4. Phospholipids were the dominant lipids in muscle. They were mainly constituted by eicosapentaenoic, oleic and arachidonic acids. 5. 5. The w3:w6 acid ratio was more than 1, which is very high compared to other freshwater invertebrates.

Fluorescein diacetate hydrolysis as a measure of fungal biomass in soil.

The fatty acid methyl esters of lipids extracted from an agricultural soil in the preharvest period of soybean or middle growth cycle from wheat were characterized and quantified by gas-liquid chromatography. The fatty acids 18:2ω6 and 16:1ω5 were used as markers of saprotrophic and arbuscular mycorrhizal fungi. In parallel, biomass estimation through plate counts in selective media for cellulolytic and saprotrophic fungi was also performed all throughout a soybean crop or middle growth cycle of wheat. As an enzymatic method, the fluorescein diacetate (FDA) hydrolytic activity of the samples was determined. Owing to the high relationship exhibited by FDA hydrolysis with organic carbon and total nitrogen content of soil, the enzymatic activity was correlated with the microbial biomass estimated through marker lipids or plate counts. The results obtained point out that FDA hydrolysis may be used as a rapid, cheap, and reliable estimator of fungal biomass.

Triacylglycerol consumption during spore germination of vesicular-arbuscular mycorrhizal fungi

Lipids and fatty acids of spores of the vesicular-arbuscular mycorrhizal fungusGlomus versiforme were identified and quantitatively determined at different times of germination. Triacylglycerols were, by far, the most abundant lipid (38% on a wet-weight basis). Phosphatidylethanolamine, together with minor quantities of other phospholipids, was the main polar lipid. Palmitoleic, palmitic and oleic acids were quantitatively the most important fatty acids in total lipids, and even more so in the triacylglycerol fraction. Minor percentages of fatty acids, identified as ω3 and ω6 polyunsaturated, completed the fatty acid spectra. Germination ofG. versiforme spores evokes a continuous decrease of triacylglycerols and an increase of phospholipids. The balance of fatty acids during germination suggests either a degradation or a transference of fatty acids from triacylglycerols to phospholipids.

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 ω5 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 60xa0days. 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 90xa0days.

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.

Lipid circulation in spiders. Transport of phospholipids, free acids and triacylglycerols as the major lipid classes by a high-density lipoprotein fraction isolated from plasma of Polybetes pythagoricus

Abstract A high-density lipoprotein (HDL) fraction was isolated from the hemolymphatic plasma of the spider Polybetes pythagoricus by density-gradient ultracentrifugation. Hydrated density (1.13 g/ml), electrophoretic mobility (SDS—PAGE) of apoproteins and lipid classes composition were determined. Lipids were identified by HP-TLC and auxiliary techniques; they were quantified by TLC-FID. The protein moiety is composed of two main apoproteins (250 and 76 kDa, respectively) and several polypeptides of low molecular weight. It resembles the apoliphorins of insects and some other arachnids. The lipid composition differs from most lipophorins. Phospholipids amount to more than 60% of total lipids, while diacylglycerols (2.4%) are supplanted by triacylglycerols (16.5%) as the main circulating energetic lipids.

Partial purification and characterization of a lipolytic enzyme from spores of the arbuscular mycorrhizal fungus Glomus versiforme

The lypolytic activity of extracts from G. versiforme spores was detected and measured in in vitro assays using triolein as a substrate. Among the different subcellular fractions assayed, a membrane- rich one showed the greatest lipolytic activity. Like other membrane-bound enzymes, G. versiforme lipase was well extracted in presence of detergent. The time-course of triolein hydrolysis by several lipase preparations was studied. The partial purification of the lipase from the total homogenate revealed in- creasing enzyme specific activities in the following or- der: homogenate < sediment at 13 000 g for 30 min < acetone powder < fraction obtained by gel per- meation chromatography. The molecular mass of the enzyme was estimated to be 30 kDa as determined by gel filtration column chromatography.

Characterization of lipoprotein fractions with high content of hemocyanin in the hemolymphatic plasma of Polybetes pythagoricus

In order to evaluate the role of hemocyanin in the hemolymphatic transport of lipids, two hemocyanin-containing lipoprotein fractions, a very-high-density lipoprotein (VHDL) and a high-density lipoprotein (HDL) isolated from plasma of the spider Polybetes pythagoricus, were characterized. VHDL concentration was found to be twofold when compared to that of HDL, and both of them transport 70% of total circulating lipids. Their lipid moieties were mainly composed of phospholipids (65% and 55% in VHDL and HDL, respectively), triacylglycerols, and free fatty acids in a minor degree, and diacylglycerols, cholesterol, and hydrocarbons even in lesser amounts. Although hemocyanin was the predominant apoprotein, two other proteins were also present in minor amounts, especially in VHDL. The two lipoproteins were analyzed by gel filtration chromatography and separated into three subfractions. Two of them were composed of the heptamer (500 kDa) and the monomer (70 kDa) of hemocyanin, respectively, and different proportions of binding lipids. Free fatty acids (37% of total subfraction lipids) were the main lipid classes linked to the heptamer, whereas triacylglycerols (24%) and free fatty acids (22%) predominated among lipids associated with the hemocyanin monomer. The third subfraction contained the hexamer of hemocyanin (420 kDa), two minor polypeptides, and 65% of the VHDL total lipids. This subfraction is the principal means of transport of phosphatidylcholine and triacylglycerols in VHDL. It is here suggested that hemocyanin, as a single apoprotein or associated with other polypeptides, is responsible for the transport of the majority of plasma lipids in this invertebrate.

Metabolism of ovorubin, the major egg lipoprotein from the apple snail

The site of synthesis of molluscs lipoproteins is little known and was investigated for the egg lipoprotein perivitellin 1 (PV1) or ovorubin in the freshwater snail Pomacea canaliculata. Tissues (albumen gland, gonad–digestive gland complex and muscle) of vitellogenic females were incubated in vitro at 25°C for 12 h with 14C Leucine. After that, soluble proteins from tissue homogenates and medium samples were analysed for de novo protein synthesis by electrophoresis and HPLC, and radiolabelled proteins quantified by liquid scintillation. Gonad–digestive gland complex did not synthesise ovorubin, in spite its high protein synthesis levels. Three albumen gland radiolabelled proteins (35, 32 and 28 kDa) comigrated with the subunits of ovorubin and represented 1.3% of the total labelled protein of that tissue. Western blot analysis with polyclonal antibodies confirmed that these were ovorubin subunits. In vivo experiments where vitellogenic females were injected with 3H Leucine, revealed that ovorubin was not present in hemolymph. ELISA analysis confirmed ovorubin presence only in albumen gland and developing eggs with levels of 800 and 582 mg/g protein, which represent 30.3 and 28.4 mg ovorubin/g of tissue, respectively. Therefore, albumen gland is the single site of ovorubin synthesis as no extragland synthesis, circulation or accumulation could be detected in the apple snail.