Prostaglandin biosynthesis by salivary glands isolated from the lone star tick, Amblyomma americanum.

Salivary glands separated from internal tissues of the lone star tick, Amblyomma americanum, are competent to synthesize prostaglandins (PGs). Using an in vitro PG biosynthesis assay four major PGs, namely, PGA2/PGB2, PGD2, PGE2, and PGF2 alpha were synthesized. Under standard assay conditions PGA2/PGB2 was the predominant product. Salivary tissues as well as non-salivary internal tissues were capable of PG biosynthesis. We observed that storing ticks at -80 degrees C for 3 months resulted in reduced PG biosynthesis. This indicates that the tick preparation, unlike comparable mammalian preparations, is not stable to freezing. Cyclooxygenase inhibitors, indomethacin (> 10 microns) and naproxen (> 15 microns), completely inhibited PG biosynthesis. These results demonstrate the presence of a PG biosynthetic system in salivary glands and other internal tissues of the lone star ticks.

What can we learn from prostaglandins and related eicosanoids in insects?

Eicosanoids are oxygenated metabolites of three C20 polyunsaturated fatty acids (20:3n-6, 20:4n-6, and 20:5n-3). While eicosanoids are very well known in mammalian systems, mostly due to their pharmaceutical interest, there is increasing recognition of the significance of these compounds in insects and other invertebrates. In this paper we consider four major concepts emerging from work on eicosanoids in invertebrates. First, the biological significance of eicosanoids extends far beyond their physiological and pathophysiological actions in human and veterinary medicine. Second, we can greatly improve our understanding of eicosanoids in insects by integrating our work on insects into ongoing studies of other invertebrates. Third, some eicosanoid actions may be fundamental to animals. Fourth, the biochemistry of eicosanoids in insects and other invertebrates can differ from expectations based on the mammalian background. Finally, we point to an uncharted frontier in insect studies--the biochemical mechanisms of eicosanoid action--by drawing attention to some of the work on eicosanoid receptors in mammalian systems.

Prostaglandin biosynthesis and subcellular localization of prostaglandin H synthase activity in the lone star tick, Amblyomma americanum.

We report on prostaglandin (PG) biosynthesis in the lone star tick, Amblyomma americanum. In vitro preparations of whole female ticks and internal tissues were competent to biosynthesize four PGs: PGA2/PGB2, PGD2, PGE2, PGF2 alpha. PGA2/PGB2 was the major product under optimal conditions. PG biosynthesis by whole tick and internal tissues were sensitive to incubation conditions including, protein concentration, time, temperature, pH, and presence of a co-factor cocktail composed of reduced glutathione, hydroquinone, and hemoglobin. Under standard assay conditions, 2 mg/ml protein were incubated at pH 8.0 for 2 min at 32 degrees C. PG biosynthesis was inhibited by indomethacin, a potent cyclooxygenase inhibitor in mammalian systems. Internal tissue preparations were fractionated into cytosolic and microsomal preparations by ultracentrifugation. PG biosynthetic activity was detected in both fractions. The subcellular distribution of PG biosynthetic activity in ticks is similar to other invertebrates, but quite different from mammals, in which PG biosynthetic activity is almost exclusively localized in the microsomal fractions. PGH synthase-2 was detected in the microsomal fraction on western blot analysis. These results suggest that the lone star tick is competent to biosynthesize PGs. These compounds may contribute to the success of tick feeding ecology by attenuating the defense responses of vertebrate hosts during lengthy feeding periods.

Eicosanoid biosynthesis by hemocytes from the tobacco hornworm, Manduca sexta.

We describe eicosanoid biosynthesis by microsomal-enriched preparations of hemocytes from larvae of the tobacco hornworm Manduca sexta. Four major prostaglandins, PGA2, PGE2, PGD2, and PGF2 alpha, and a lipoxygenase product that co-chromatographed with 15-hydroxyeicosatetraenoic acid (HETE) were synthesized under most conditions. The HETE's fraction was the predominant product. Eicosanoid biosynthesis was sensitive to experimental conditions, including incubation time, temperature, and protein concentration. Optimal biosynthesis was observed with 1.5 mg of microsomal-enriched protein, incubated at 30 degrees C for 2 min. The hemocyte preparation is sensitive to low dosages of naproxin and esculetin. As in mammals, most lipoxygenase activity (87%) was localized in the cytosolic fraction of hemocytes. Unlike mammals, in which PGH synthase is associated with intracellular membranes, the hemocytic activity was detected in microsomal (59%), cytosolic (35%) and mitochondrial fractions (5%).

Eicosanoids mediate insect nodulation responses to bacterial infections.

We propose that nodule formation is mediated by eicosanoids in insects. Nodulation is the temporally and quantitatively predominant cellular defense response to bacterial infection in insects and other invertebrates. Inhibition of eicosanoid biosynthesis in larvae of the tobacco hornworm Manduca sexta immediately prior to intrahemocoelic infections with the bacterium Serratia marcescens strongly reduced the nodulation response. Inhibition of eicosanoid biosynthesis also reduced formation of cellular aggregates at 1 hr postinfection, which indicates that eicosanoids mediate early stages of nodulation. Separate treatments with specific inhibitors of phospholipase A2, cyclooxygenase, and lipoxygenase reduced nodulation, which supports the view that nodule formation is a complex process involving prostaglandins and lipoxygenase products. The inhibitory effects of the phospholipase A2 inhibitor dexamethasone on nodulation were apparent by 1 hr after infection, and the effects increased, relative to controls, over 24 hr. The dexamethasone effects were expressed in a dose-dependent manner, and they were reversed by treating infected insects with eicosanoid-precursor polyunsaturated fatty acids. Treatments with the saturated fatty acid 16:0, which is not an eicosanoid precursor, did not reverse the dexamethasone effects on nodulation. These findings strongly support the identification of nodulation as a specific insect cellular defense mechanism that is mediated by eicosanoids.

Incorporation of polyunsaturated fatty acids into phospholipids of hemocytes from the tobacco hornworm, Manduca sexta.

We examined the incorporation of four radioactive fatty acids, 18:1n-9, 18:2n-6, 20:4n-6 and 20:5n-3, into cellular lipids of hemocytes from tobacco hornworms, Manduca sexta. Most of the radioactivity associated with 18:1n-9 was recovered from triacylglycerols (TGs), and the radioactivity associated with 18:2n-6 was heavily incorporated into phospholipids (PLs) and TGs. Most of the radioactivity associated with the two eicosanoid-precursor polyunsaturated fatty acids (PUFAs), 20:4n-6 and 20:5n-3, was incorporated into PLs. The incorporated fatty acids were redistributed among the lipid classes during 2 h incubations. The two C20 PUFAs were moved from PLs to TGs. While 18:2n-6 underwent little change, 18:1n-9 was redistributed from TGs to PLs. Within PLs, each of the fatty acids were incorporated into phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PtG) and phosphatidylserine/inositol (PS/PI). The incorporation patterns changed over time, indicating that the incorporated fatty acids were redistributed among the four PL fractions. The radioactivity associated with 18:1n-9 was mostly recovered from the sn-1 position of PC (59%) and PE (83%). Most of the radioactivity associated with 18:2n-6 was found in the sn-2 position of PC (88%) and PE (67%). Over 90% of the radioactivity associated with 20:5n-3 was recovered from the sn-2 position of PC and PE. Incorporation of 20:4n-6 differed from 20:5n-3 because more radioactivity was recovered from the sn-2 position of PC (93%) than PE (69%). These findings are in line with the general background of lipid biochemistry, from which incorporation of 20:4n-6 into PE marks a notable departure: 31% of the radioactivity associated with this acid was recovered from the sn-1 position of PE. These findings indicate that hemocytes from the tobacco hornworm elaborate a fatty acid incorporation system, which exhibits specificity with respect to fatty acid structure and lipid class.

Prostaglandin biosynthesis by fat body from the tobacco hornworm, Manduca sexta.

We describe prostaglandin (PG) biosynthesis by microsomal-enriched preparations of fat body from larvae of the tobacco hornworm Manduca sexta. Four major PGs were synthesized under most experimental conditions, PGA2, PGE2, PGD2 and PGF2 alpha. PGA2, was the predominant product under most conditions. Unlike mammals, in which PGA2, is generally thought to arise from non-enzymatic rearrangements of PGE2, the fat body preparations did not convert exogenous PGE2 into PGA2. These findings suggest that PGA2 is an important fat body product that is synthesized by a route that does not involve PGE2. The PG synthase activity and the overall profile of PG synthesis were sensitive to experimental conditions, including incubation time, temperature, and protein concentration. Optimal PG biosynthesis was observed with 1 mg of microsomal-rich protein, incubated at 30 degrees C for 1-2 min. The fat body preparations is sensitive to two non-steroidal anti-inflammatory drugs, indomethacin and naproxen, both of which inhibited PG synthesis at low dosages.

Fatty acid compositions of phospholipids and triacylglycerols from selected terrestrial arthropods.

The fatty acid compositions of phospholipids and triacylglycerols from selected orthognath spider tissues, scorpion body segments, whole millipedes, and whole labidognath spiders were determined. The major components were C16 and C18 saturated and unsaturated fatty acids as well as C20 polyunsaturated fatty acids. The eicosanoid precursor polyunsaturated fatty acids 20:4n-6 and 20:5n-3 were present in substantial proportions of phospholipids in all tissues examined. The fatty acid profiles of these terrestrial arthropods differ markedly from corresponding profiles of terrestrial insects in that substantially greater proportions of C20 polyunsaturated fatty acids are present in the organisms in this study. This finding supports the idea that maintaining low proportions of C20 polyunsaturated fatty acids is a special condition of terrestrial insects.

Arachidonic acid and prostaglandin E2 in Malpighian tubules of female yellow fever mosquitoes.

The fatty acid compositions of Malpighian tubules from adult females of the mosquito Aedes aegypti were determined for total lipids, phospholipids, triacylglycerols and three phospholipid fractions, namely phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol/phosphatidylserine (PI/PS). The prostaglandin precursor arachidonic acid (20:4n-6) occurred in total lipids and phospholipids, but not triacylglycerols. Within phospholipids, nearly all of the 20:4n-6 was detected in PC, with only traces in PE, and none was detected in PI/PS. Isolated Malpighian tubules incorporated exogenous radioactive 20:4n-6 into tissue phospholipids and diacylglycerols, with most of the radioactivity recovered in diacylglycerol. These data indicate selective incorporation of 20:4n-6 into tissue lipids. PGE2 was detected in Malpighian tubule whole mounts by immunohistochemical staining. These findings support the idea that prostaglandins are physiologically active in mosquito Malpighian tubules.

Phospholipase A2 activity in the fat body of the tobacco hornworm Manduca sexta.

We report on phospholipase A2 (PLA2) activity in homogenates prepared from fat bodies of the tobacco hornworm Manduca sexta. PLA2 activity is responsible for hydrolyzing fatty acids from the sn-2 position of phospholipids. The rate of hydrolysis increased with increasing homogenate protein concentration up to approximately 320 micrograms protein/ml reaction volume. Higher protein concentrations did not appreciably increase the rate of PLA2 activity. As seen in some, but not all PLA2s from mammalian sources, hydrolyzing activity increased linearly with time. The fat body activity was sensitive to pH (optimal activity at pH 8-9) and temperature (optimal activity at approximately 40 degrees C). The activity was associated with fat body rather than hemolymph, because no activity was detected in cell-free serum. The fat body PLA2 activity differs from the majority of PLA2s with respect to calcium requirements. Whereas most PLA2s require millimolar calcium concentrations for full activity, a few PLA2s are calcium-independent. A few others are known to require submicromolar calcium concentrations. The fat body activity appears to be calcium independent. These data show that a PLA2 activity that can hydrolyze arachidonic acid from the sn-2 position of phospholipids is associated with the tobacco hornworm fat body. The biological significance of this activity relates to biosynthesis of eicosanoids. Pharmacological inhibition of PLA2 impairs the ability of this insect to respond to bacterial infections. Since the impairment can be reversed by treatment with exogenous arachidonic acid, the PLA2 activity may be an important step in eicosanoid biosynthesis.

Comparative eicosanoid physiology in invertebrate animals.

A comparative survey of eicosanoid physiology in invertebrate animals is presented. Topics include host skin penetration behavior in blood flukes, oocyte maturation in starfishes, prevention of polyspermic fertilizations in sea urchins, ion regulation in bivalves, egg production in snails, egg release in scallops, neurobiology of Aplysia, hatching in barnacles, and release of egg-laying behavior in some insects. Some eicosanoid actions, such as ion regulation, may be common to most vertebrate and invertebrate animals, whereas other actions, such as mediation of whole organism behavior, are not shared. Comparative physiology is revealing that the biological significance of eicosanoids extends far beyond vertebrate animals.

Insect immune response to bacterial infection is mediated by eicosanoids.

Inhibition of eicosanoid formation in larvae of the tobacco hornworm Manduca sexta, using specific inhibitors of phospholipase A2, cyclooxygenase, and lipoxygenase, severely weakened the ability of larvae to clear the bacterium Serratia marscescens from their hemolymph. The reduced capability to remove bacteria is associated with increased mortality due to these bacteria. There is a dose-dependent relationship between the phospholipase A2 inhibitor dexamethasone and both the reduced bacterial clearance and increased larval mortality. The dexamethasone effects on larval survival were reversed by treatment with arachidonic acid. Maleic acid, a nonspecific antioxidant, did not interfere with the insects' ability to remove bacterial cells from hemolymph. The larvae were shown to contain all of the C20 polyunsaturated fatty acids necessary for eicosanoid biosynthesis and to be capable of converting radioactive arachidonic acid into several primary prostaglandins. These results strongly suggest that eicosanoids mediate transduction of bacterial infection signals into the complex of cellular and humoral responses that comprise invertebrate immunity.

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.

Fatty acid composition of whole bodies, specific tissues and cell lines of two lepidopteran insects.

We detail the fatty acid compositions of last larval instars of two lepidopterans, Spodoptera frugiperda and Trichoplusia ni, two tissues from T. ni, a cell line derived from each species and the respective larval and cell culture media. Larval whole-body and specific tissue fatty acid profiles exhibited the major features commonly found in previous lepidopteran analyses, whereas the cell-line fatty acid compositions were substantially different from the compositions of both their growth media and larvae of their respective species. It appears that these cell-line patterns result from increased monoene biosynthesis in response to low levels of exogenous polyunsaturated fatty acids, a commonly observed essential fatty acid deficiency symptom in whole animals.