David W. Stanley-Samuelson

Long-chain polyunsaturated fatty acids: Patterns of occurrence in insects

Abstract Most published analyses of the fatty acid compositions of insect tissue do not record long-chain polyunsaturated fatty acids beyond C18:3. When this is set against recent discoveries of important nutritional and prostaglandinogenic roles for arachidonic and other long-chain polyunsaturates in several insects of diverse taxa, the issue of a possible general occurrence of such material in insects assumes compelling interest. We used gas chromatographic methods to investigate the presence of fatty acids of more than 18 carbon atoms in lipid extracts from whole-animal and specific tissue preparations, and in particular fractions of lipid extracts. Results indicate that such compounds generally occur as a low proportion of the total fatty acids in whole-animal extracts and in neutral acyl fractions. Larger numbers and higher proportions of long-chain fatty acids were detected in extracts of certain tissues and in the phospholipid fractions of whole animal extracts than were found in the unfractionated whole-animal extracts. These findings show that long-chain polyunsaturated fatty acids are a regular component of the tissues of the insects examined and, in the light of their known nutritional, prostaglandinogenic and presumed structural roles are of considerable biological significance.

Arachidonic and other long-chain polyunsaturated fatty acids in spermatophores and spermathecae of Teleogryllus commodus: Significance in prostaglandin-mediated reproductive behaviour

Abstract Estimates of weights of fatty acids, especially arachidonic acid, in spermathecae from virgin and mated T. commodus indicate substantial elevation in all fatty acids and particularly arachidonic acid following mating. Analysis of spermatophore lipids suggests that this can be in part accounted for by the contents of one or several spermatophores. Fractionation of total lipids from spermatophores showed that arachidonic acid comprised 24% of phosphotidylcholine and 4% of phosphotidylethanolamine, but was not detected in neutral lipids whereas it was approximately equally distributed over phosphotidylcholine and phosphotidylethanolamine in lipids from spermathecae. These data indicate that in addition to prostaglandin synthetase, the spermatophore contains a physiologically significant quantity of prostaglandin precursor, arachidonic acid, esterified to phospholipid and presumably unavailable for enzymatic action during mating transfer. We also note that proportions of arachidonic acid in the phosphotidylcholine of spermatophores are the highest recorded for this fatty acid in the insect literature, which in conjunction with recent work emphasizes the likely physiological significance of long-chain polyunsaturated fatty acids in insects generally.

Biosynthesis of linoleic acid in insects

Abstract The de novo biosynthesis of linoleic acid (9,12-octadecadienoic acid), generally considered an essential dietary nutrient for animals, has been demonstrated in 15 insect species representing four orders. Two of these species were also shown to further elongate and desaturate linoleic acid to form arachidonic acid, implying the ability to insert double bonds on both sides of the Δ position of oleic acid (9-octadecenoic acid).

Polyunsaturated fatty acids in the lipids from adult Galleria mellonella reared on diets to which only one unsaturated fatty acid had been added

Abstract Gas chromatographic analysis of tissue phospholipid and triacylglycerol fatty acids derived from adult moths, Galleria mellonella , reared on semi-synthetic basal medium supplemented with various single C18, C20 and C22 polyunsaturated fatty acids suggests that C20:2n6, C20:3n6 and C20:3n3 can be shortened to their C18 analogues. Only C18:2n6 and C18:3n3, or higher analogues convertible to these, can alleviate the pupal/adult ecdysis failure which results from essential fatty acid deficiency. C18, C20 and C22 polyunsaturates with double bonds on the carboxyl side of the n-9 carbon are all inadequate nutritionally. C18:3n6 is not saturated to the nutritionally active C18:2n6, and it appears that double bonds towards the carboxyl group interfere with the shortening of the higher polyunsaturated fatty acids. In addition to chain shortening, dietary C18:3n3 and C20:3n3 may be elongated/desaturated to C20:5n3, which is elevated in tissues of all moths reared to normal adult eclosion, and also is substantially accumulated in phospholipid of moths reared with dietary C20:5n3 even though their emergences are imperfect. Since the data, overall, indicate n3 and n6 fatty acids are not interconvertible, elevated C20:5n3 obtained with high dietary levels of the nutritionally adequate n6 acids, not so high as for dietary active n3 acids, probably derives from efficient sequestration of n3 fatty acids, possibly formed by symbiotic gut microorganisms. These results are discussed in terms of metabolic modifications of long-chain polyenoics by insects generally.

Egg‐laying in response to prostaglandin injections in the Australian field cricket, Teleogryllus commodus

ABSTRACT. We report the effects of prostaglandin (PG) injection on egg‐laying by sexually mature virgin crickets, Teleogryllus commodus Walker. High egg‐laying activity is associated with PGE1 PGE2, 6‐keto‐PGE1 and 15‐keto‐PGE2, compounds that share the basic prostanoid backbone (C20 substituted fatty acid, 5 membered ring, a 7‐ and an 8‐carbon aliphatic chain), and 9‐keto, 11‐hydroxyl ring substitutions. PGs without these ring features and other compounds that lack the prostanoid backbone have no or only intermediate egg‐laying effects. Adding oxygen functionalities or an aliphatic double bond tends to increase egg‐laying activity. Hence, 15‐keto‐PGF2 and TxB2, both of which have an additional backbone oxygen compared with their less active analogues, are highly active compounds. Two of the most active PGs, 15‐keto‐PGE2 and 15‐keto‐PGE2α, are the products of inactivating metabolism in mammalian systems, describing a fundamental difference in PG biochemistry with respect to mammals and insects.

Arachidonic and other tissue fatty acids of Culex pipiens reared with various concentrations of dietary arachidonic acid

Abstract Chloroform/methanol extracts were prepared from groups of Culex pipiens reared in synthetic dietary media provided with various concentrations of arachidonic acid. Extracts were analyzed by gas-liquid chromatography to determine the fatty acid composition of whole extracts and also of phospholipid and triacylglycerol fractions separated by thin-layer chromatography from the whole extracts. The same extracts were also tested for their ability to support flight of adult C. pipiens reared in basal synthetic diet containing various concentrations of the extracts: this provided a bioassay for the presence of arachidonic acid or related polyunsaturates in the extracted lipid, since adults can fly only if provided, as larvae, with dietary arachidonic or related fatty acids. For comparison, chromatographic and bioassay data obtained from normal stock mosquitoes, reared in crude septic medium, are also presented. All extracts were shown by gas-liquid chromatography to contain some arachidonic acid and other polyunsaturated fatty acids. The proportions of arachidonic acid in extracts from mosquitoes reared in synthetic media were greater the greater the concentration of dietary arachidonic acid provided; and in the bioassay, extracts induced more flight activity in test mosquitoes the higher the dietary arachidonic acid provided for extracted mosquitoes. Extracts from stock-reared mosquitoes were more active in the bioassay than synthetic dietreared extracts, even though gas-liquid chromatography indicated lower proportions of arachidonic acid in stock-reared extract. However, stock-reared extract contained a substantial proportion of gammalinolenic acid, which is flight active for C. pipiens , as well as more linolenic acid and a large amount of linoleic acid, both of which are semi-active for flight; thus, stock-reared extract contained a higher overall proportion of flight-inducing fatty acids. Proportions of polyunsaturates in the phospholipid fractions of extracts from synthetic diet-reared mosquitoes were much greater than in the unfractionated extracts, whereas polyunsaturates were virtually absent from the triacylglycerol fractions, indicating a sequestering of polyunsaturates into phospholipids.

Biosynthesis of polyunsaturated fatty acids by the australian field cricket, Teleogryllus commodus

Abstract Aspects of testicular fatty acid biochemistry from the Australian field cricket, Teleogryllus commodus, are reported. Over 10% of the phospholipid fatty acids were C20 polyunsaturated fatty acids (PUFAs), with nearly 6% arachidonic acid (20:4). The testes and ovaries accumulated a large proportion of label from radioactive arachidonic acid that was injected into the hemocoel (about 30%). Specificity in the uptake was shown by comparison to a similar study with labelled stearic acid, in which only 1.5% of the radioactivity was taken up by testes. Sixty percent of the radioactivity taken up by testes from [3H]20:4 was incorporated into phospholipids and 30% into triacylglycerols. Fat body of males and females incorporated 27% of the [3H]20:4 into phospholipids and 68% (males) or 55% (females) into triacylglcyerols. Radioactivity from [1-14C]acetate was incorporated into testicular linoleic acid and eicosatrienoic acid, but not eicosatetraenoic acid, suggesting the de novo biosynthesis of both 18:2 and a C20 PUFA by this species. Label from injected [U-14C]linoleic acid was recovered mostly as linoleic acid, with a small portion of the recovered radioactivity in eicosatrienoic acid, but not eicosatetraenoic acid. Very little label from injected linoleic acid occurred as monounsaturated or saturated fatty acids, indicating only slight, if any, β-oxidation of 18:2 to acetate and subsequent lipid synthesis.

Sexual transfer of prostaglandin precursor in the field cricket, Teleogryllus commodus

ABSTRACT. When injected into the haemolymph of newly emerged adult males of the field cricket, Teleogryllus commodus (Walker), [3H]arachidonic acid is incorporated into tissue phospholipids. Two reproductive tissues, testes and accessory glands, incorporate and preserve the labelled arachidonic acid for at least 49 days. With onset of circadian rhythm‐controlled spermatophore production, radioactive arachidonic acid is incorporated into spermatophore phospholipids. After mating with untreated females, radioactivity was detected in spermathecae and in haemolymph from females. Some of the transferred radioactive arachidonic acid is subsequently converted into prostaglandins. Hence, males of T. commodus transfer prostaglandin synthetase activity and substrate by way of spermatophores during mating

Polyunsaturated fatty acids of mosquitos reared with single dietary polyunsaturates

Abstract Stock adults of Culex pipiens and tarsalis reared in crude media had a third of their phospholipid fatty acids as polyunsaturates, mainly 18C but including prominent proportions of arachidonic (20:4n6) and eicosapentaenoic (20:5n3) acids. Adults reared with synthetic media devoid of polyunsaturated fatty acids and therefore unable to fly at emergence contained no more than trace amounts of any polyunsaturate. With synthetic media containing single polyunsaturates the following findings emerged. Of four polyunsaturates known to be highly effective essential fatty acids individually 20:4n6 or 20:5n3 appeared unchanged in tissue phospholipids in proportions reflecting dietary concentrations; dietary 22:4n6 or 22:6n3 (docosahexaenoic acid) appeared also as 20:4n6 or 20:5n3, respectively, retroconverted from the administered dietary fatty acids, which were detected only in traces. Two moderately effective dietary fatty acids, 18:3n6 (γ-linolenic) and 20:3n6 (homo-γ-linolenic), which support weak flight at emergence, appeared in tissue phospholipids respectively as 18:3n6 only, or as similar proportions of 18:3n6 and 20:3n6, this latter indicating shortening to the 18C analogue as well as accumulation of the dietary 20C acid. Six other polyunsaturates [18:2n6 (linoleic), 18:3n3 (linolenic) and their 20C and 22C analogues], all considered slightly effective as essential fatty acids although unable to support proper flight, appeared in tissue phospholipid in dose-related proportions as the 18C basal n6 or n3 family analogues, with only traces of the higher analogues when these were the dietary fatty acids provided, indicating sequential chain shortening within each series, n6 or n3, no interconversion of n6 and n3 members (also shown by all other data), and efficient accumulation of the resultant 18C polyunsaturates. These findings show no capability for de novo synthesis of polyunsaturated fatty acids, afford an insight into the metabolic interrelations of diet-derived polyunsaturates and indicate a primary importance for endogenous arachidonic and eicosapentaenoic acids in mosquito essential fatty acid physiology.

De novo biosynthesis of arachidonic acid and 5,11,14-eicosatrienoic acid in the cricket Teleogryllus commodus

The de novo biosynthesis of 5,11,14-eicosatrienoic acid (5,11,14-20:3), arachidonic acid (20:4(n - 6] and eicosadienoic acid (20:2(n - 6] and the elongation/desaturation of linoleic acid (18:2(n - 6] to 20:4(n - 6) and alpha-linolenic acid (18:3(n - 3] to eicosapentaenoic acid (20:5(n - 3] were demonstrated in adult males of the field cricket Teleogryllus commodus. Sodium [1-14C]acetate, [1-14C]18:2(n - 6) and [1-14C]18:3(n - 3) were injected into adult male crickets and after an incubation period, the testes and remaining tissues were extracted and the methyl esters obtained from the phospholipid and triacylglycerol fractions were analyzed. After 5 days of daily injections of [1-14C]acetate, the methyl esters of the triene and tetraene fatty acids from the testicular phospholipid fraction were purified by AgNO3-TLC and HPLC and analyzed by GLC, radio-HPLC, and radio-GLC of ozonolysis products. The results demonstrate the de novo biosynthesis of 20:2(n - 6), 20:4(n - 6) and an isomer of 20:3(n - 6) with double bonds in the 5,11,14 positions. the elongation/desaturation of 18:2(n - 6) to 20:4(n - 6) and 18:3(n - 3) to 20:5(n - 3) was demonstrated by analysis of the methyl esters derived from the testicular phospholipid fraction by radio-HPLC after injecting crickets with radiolabeled substrates.