Rico L Rana

Eicosanoids mediate microaggregation and nodulation responses to bacterial infections in black cutworms, Agrotis ipsilon, and true armyworms, Pseudaletia unipuncta

Nodulation is the first, and quantitatively predominant, cellular defense reaction to bacterial infection in insects and other invertebrates. Inhibition of eicosanoid biosynthesis in true armyworms, Pseudaletia unipuncta, and black cutworms, Agrotis ipsilon, immediately prior to intrahemocoelic injections with heat-killed preparations of the bacterium, Serratia marcescens, severely impaired the nodulation response. Five eicosanoid biosynthesis inhibitors, including dexamethasone (a phospholipase A(2) inhibitor), indomethacin, ibuprofen (cyclooxygenase inhibitors), phenidone (dual lipoxygenase/cyclooxygenase inhibitor) and eicosatetraynoic acid (an arachidonic acid analog that inhibits all arachidonic acid metabolism) severely reduced nodulation in infected insects. The dexamethasone effects were reversed by treating true armyworms with arachidonic acid immediately after infection. In addition to these pharmacological findings, we demonstrate that an eicosanoid biosynthesis system is present in these insects. Arachidonic acid is present in fat body phospholipids at about 0.4% of total phospholipid fatty acids. Fat body expressed a phospholipase A(2) that can hydrolyze arachidonic acid from the sn-2 position of cellular phospholipids. Fat body preparations were competent to biosynthesize prostaglandins, of which PGE(2) was the major product. These findings support the hypothesis that eicosanoids mediate cellular immune reactions in insects.

Eicosanoids mediate nodulation reactions to bacterial infections in adults of the cricket, Gryllus assimilis

Nodulation is the temporally and quantitatively most important cellular defense reaction to bacterial infections in insects. Inhibition of eicosanoid biosynthesis in adults of the cricket, Gryllus assimilis, immediately prior to intrahemocoelic injections of the bacterium, Serratia marcescens, sharply reduced the nodulation response. Separate treatments with specific inhibitors of phospholipase A(2), cyclooxygenase, and lipoxygenase reduced nodulation, supporting our view that nodule formation is a complex process involving lipoxygenase and cyclooxygenase products. The inhibitory influence of dexamethasone was apparent within 2h of injection, and nodulation was significantly reduced, relative to control crickets, over 22h. The dexamethasone effects were reversed by treating bacteria-injected insects with the eicosanoid-precursor polyunsaturated fatty acid, arachidonic acid. Low levels of arachidonic acid were detected in fat body phospholipids, and fat body preparations were shown to be competent to biosynthesize eicosanoids from exogenous radioactive arachidonic acid. These findings in a hemimetabolous insect broaden our hypothesis that eicosanoids mediate cellular immune reactions to bacterial infections in most, if not all, insects.

Eicosanoids mediate nodulation reactions to bacterial infections in adults of two 17-year periodical cicadas, Magicicada septendecim and M. cassini.

Nodulation is the first and quantitatively most important cellular defense reaction to bacterial infections in insects. Treating adults of the 17-year periodical cicadas, Magicicada septendecim and M. cassini, with eicosanoid biosynthesis inhibitors immediately prior to intrahemocoelic injections of the bacterium, Serratia marcescens, sharply reduced the nodulation response to bacterial challenges. Separate treatments with specific inhibitors of phospholipase A(2), cyclooxygenase, and lipoxygenase reduced nodulation, supporting our view that nodule formation is a multi-step process in which individual steps are separately mediated by lipoxygenase and cyclooxygenase products. The inhibitory influence of dexamethasone was apparent by 2 h after injection, and nodulation was significantly reduced, relative to control insects, over the following 14 h. The dexamethasone effects were reversed by treating bacteria-challenged insects with the eicosanoid-precursor polyunsaturated fatty acid, arachidonic acid. Low levels of arachidonic acid were detected in fat body phospholipids. These findings in adults of an exopterygote insect species with an unusual life history pattern broaden our hypothesis that eicosanoids mediate cellular immune reactions to bacterial infections in most, if not all, insects.

Eicosanoids Mediate Nodulation Responses to Bacterial Infections in Larvae of the Silkmoth, Bombyx mori

Abstract 1) Nodulation is the first, and qualitatively predominant, cellular defense reaction to bacterial infections in insects and other invertebrates; 2) treating silkworms, Bombyx mori, with the eicosanoid biosynthesis inhibitor, dexamethasone, strongly reduced nodulation responses to bacterial infections; 3) the influence of dexamethasone was reversed by injecting the eicosanoid-precursor polyunsaturated fatty acid, arachidonic acid (20:4n-6), into dexamethasone-treated, infected larvae; 4) the presence of an eicosanoid biosynthesis system in silkworms was documented. Demonstrated elements include a digestive phospholipase A2, incorporation of exogenous 20:4n-6 into fat body phospholipids, the presence of 20:4n-6 in cellular phospholipids, a fat body intracellular phospholipase A2 that can hydrolyze 20:4n-6 from cellular phospolipids, and eicosanoid biosynthetic enzymes; and 5) these findings support the hypothesis that eicosanoids mediate cellular immune responses to bacterial infections in silkworms.

Pre-oral digestion: A phospholipase A2 associated with oral secretions in adult burying beetles, Nicrophorus marginatus.

We report on a phospholipase A2 (PLA2) found in the oral secretions, but not midgut contents, of the burying beetle, Nicrophorus marginatus. PLA2 is responsible for hydrolyzing fatty acids from the sn-2 position of dietary phospholipids (PLs), an essential step in digestion and absorption of essential polyunsaturated fatty acids. Like the digestive PLA2s known from mammalian systems, and the one described insect digestive PLA2 the N. marginatus oral secretion PLA2 depends upon Ca2+ for full activity. However, unlike most digestive PLA2s, the N. marginatus enzyme is only partially inactivated in the absence of Ca2+. The PLA2 in N. marginatus oral secretions was influenced by altering the enzyme reaction conditions, including reaction time, protein concentration, pH, and temperature. Standard reaction conditions for assessing enzyme activity include 1.0 μg protein/μl incubated at pH 9.0 for 30 min at 28°C.

A digestive phospholipase A2 in midguts of tobacco hornworms, Manduca sexta L.

We hypothesized that phospholipase A(2) (PLA(2)) is a common feature of insect digestive physiology. PLA(2) hydrolyzes polyunsaturated fatty acids (PUFAs) associated with the sn-2 position of phospholipids (PLs). We describe here a PLA(2) from midgut contents of the tobacco hornworm, Manduca sexta. Our results indicate that the enzyme is sensitive to pH (inactivated at low pH), protein concentration (up to 1.6&mgr;g/&mgr;l), substrate concentration (up to 1.4nmoles/reaction), temperature (up to 30 degrees C), and incubation time. We also found that PLA(2) activity is higher in fed than in starved larvae, and enzyme activity is associated with the midgut contents, rather than the midgut epithelium of fed larvae. All known secretory PLA(2)s, except for a PLA(2) in venom of the marine snail, Conus magus, require high calcium concentrations for catalysis, but the Manduca PLA(2) appears to be calcium-independent, and it exhibits increased PLA(2) activity in the presence of a calcium-chelator, EGTA. In addition, the partially purified Manduca PLA(2) is not inhibited by the phospholipid analog, oleyloxyethylphosphorylcholine. These findings suggest that the Manduca digestive PLA(2) may represent another novel form of PLA(2).

Eicosanoids mediate nodulation reactions to bacterial infections in larvae of the butterfly, Colias eurytheme.

Nodulation is the first, and qualitatively predominant, cellular defense reaction to bacterial infections in insects. Treating larvae of the butterfly Colias eurytheme with the eicosanoid biosynthesis inhibitor dexamethasone, strongly impaired nodulation reactions to bacterial infections. The influence of dexamethasone was reversed by treating infected insects with arachidonic acid, an eicosanoid precursor. An eicosanoid biosynthesis system in C. eurytheme larvae is documented. Specifically, the presence of eicosanoid-precursor polyunsaturated fatty acids in tissue phospholipids was determined, an intracellular phospholipase A2 that can release arachidonic acid from tissue phospholipids was recorded, and eicosanoid biosynthesis, registered as conversion of exogenous radioactive 20:4n-6 into eicosanoids, was observed. These findings support the hypothesis that eicosanoids mediate cellular immune responses to bacterial infections in these butterfly larvae, and more broadly, in most, if not all, insects.

Tissue polyunsaturated fatty acids and a digestive phospholipase A2 in the primary screwworm, Cochliomyia hominivorax

We report on the presence of arachidonic acid in larval and adult tissues of the primary screwworm, Cochliomyia hominivorax and of the secondary screwworm, C. macellaria. Arachidonic acid is present in the phospholipids of whole animal extracts of both species. This fatty acid appears to be accumulated during the larval stages, because proportions of arachidonic acid were higher in adults than in larvae. These insects probably obtain the arachidonic acid from dietary phospholipids. We also report on a phospholipase A2 activity in midgut preparations from third instars of the primary screwworm. Phospholipase A2 is responsible for hydrolyzing fatty acids from the sn-2 position of dietary phospholipids to release essential fatty acids. The screwworm enzyme is similar to mammalian digestive phospholipase A2s because it depends on calcium for high catalytic activity, it is sensitive to the sitespecific inhibitor oleyloxyethylphosphorylcholine, and it interacts with heparin. We further characterized the screwworm midgut phospholipase A2 by altering the reaction conditions, including reaction time, radioactive substrate concentration, protein concentration, pH and temperature. We speculate that the biological significance of this enzyme relates to acquiring essential fatty acids, including arachidonic acid, from dietary phospholipids.

Arachidonic and eicosapentaenoic acids in tissues of the firefly, Photinus pyralis (Insecta: Coleoptera)

We report on the presence of high proportions of arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3) in the tissue lipids of adult fireflies, Photinus pyralis. Arachidonic acid typically occurs in very small proportions in phospholipids (PLs) of terrestrial insects, ranging from no more than traces to less than 1% of PL fatty acids, while 20:5n-3 is often missing entirely from insect lipids. Contrarily, 20:4n-6 made up approximately 21% of the PL fatty acids prepared from whole males and females, and from heads and thoraces prepared from males. Proportions of 20:4n-6 associated with PLs varied among tissues, including approximately 8% for male gut epithelia, 13% for testes, and approximately 25% for light organs and body fat from males. Substantial proportions of 20:5n-3 were also associated with PLs prepared from male firefly tissues, including 5% for body fat and 8% for light organs. Because 20:4n-6 and 20:5n-3 are precursors for biosynthesis of prostaglandins and other eicosanoids, we considered the possibility that firefly tissues might produce eicosanoids at exceptionally high rates. Preliminary experiments indicated otherwise. Hence, fireflies are peculiar among terrestrial insects with respect to maintaining high proportions of PL 20:4n-6 and 20:5n-3.

In vitro secretion of digestive phospholipase A2 by midguts isolated from tobacco hornworms, Manduca sexta

We report on secretion of phospholipase A(2) (PLA(2)) by in vitro preparations of midguts isolated from tobacco hornworms, Manduca sexta. This enzyme is responsible for hydrolysis of fatty acids from the sn-2 position of phospholipids, a necessary step in fatty acid absorption. The in vitro midgut preparations are competent to secrete PLA(2) into incubation buffer. Secretion began within the first 30 min of incubation and increased to a maximum at 8 h. We selected 2 h incubations because substantial loss of tissue integrity was observed after 8 h incubations. Using 2 h incubations, we recorded increased secretion of digestive PLA(2) from midguts incubated in buffer amended with diet or with yeast as a component of the diet. We also recorded small increases in secretion of PLA(2) from midguts incubated in buffer amended with a specific phospholipid, phosphatidylcholine. Midguts incubated in buffer amended with increased concentrations of phospholipid did not yield higher levels of PLA(2) activity. Lepidopteran midguts can be divided into three regions, and we recorded the highest secretion of PLA(2) from the middle region and lowest secretion from the anterior region. Because isolated midguts responded to food chemicals with increased secretion of digestive PLA(2), we suggest that secretion of digestive enzymes in tobacco hornworms is regulated by a prandial and/or paracrine mechanism, as suggested for digestive proteases in other insect species. Arch. Insect Biochem. Physiol. 42:179-187, 1999.Copyright 1999 Wiley-Liss, Inc.