Identification and characterization of a testis-specific isoform of a chaperonin in a moth, Heliothis virescens.

Two relatively abundant proteins having subunit molecular weights of 60,000 and 63,000 (p60 and p63, respectively) have been purified as a 16 to 18S complex from sperm mitochondria of a moth. Heliothis virescens. Although the function of these proteins had heretofore not been established, interest in the p63 polypeptide stemmed from its sperm-specific expression and its striking occurrence as a net charge variant among several insect species surveyed, using two-dimensional gel electrophoresis. Genomic and cDNA clones corresponding to the p63 protein have now been isolated and their sequencing has revealed extensive amino acid sequence identity with both the Escherichia coli GroEL protein and its eukaryotic homologues, the chaperonins. Immunoblot studies with a Tetrahymena chaperonin antiserum demonstrated that the p60 protein, which is expressed in all cell types, is structurally related to p63 and is itself a chaperonin subunit. While the chaperonin complex from Heliothis sperm shares certain properties with GroEL, including the ability to hydrolyze ATP and organization of its subunits into a seven-member ring, electron microscopic analysis revealed that its higher-order structure differed from GroEL (and other lower eukaryotic chaperonins) in that the native particle comprises one such ring rather than a doublet. It is not yet known whether the two chaperonin isoforms coexpressed in moth sperm assemble separately or give rise to hybrid particles. In either case, the existence of multiple chaperonin subunits in sperm leaves open the possibility that some aspect of mitochondrial biogenesis that is dependent upon the activity of these proteins is qualitatively or quantitatively different in this cell type.

Isolation and characterization of a lipoprotein receptor from the fat body of an insect, Manduca sexta.

A lipoprotein receptor has been purified from the fat body of Manduca sexta larvae. The purification involves solubilization of membrane proteins in detergent, DEAE-, and hydroxyapatite chromatography, affinity chromatography on a concanavalin A column, and affinity chromatography on a lipoprotein-Sepharose column. An overall purification of 220-fold from the solubilized membranes was achieved. The receptor has an apparent molecular mass of 120 kDa. The receptor has an absolute requirement for Ca2+ and is inhibited by Suramin. The pH optimum of the receptor is 6.5, which is near the pH of the hemolymph. Binding data indicate a single high affinity binding site with a Kd = 4.1 +/- 0.19 x 10(-8) M as measured with the lipoprotein isolated from larval hemolymph. The major neutral lipid carried by insect lipoproteins is diacylglycerol, and it was shown that the affinity of the receptor for lipoprotein ligands correlates with their diacylglycerol content. It is proposed that the decrease in affinity of the receptor for lipoproteins depleted of diacylglycerol plays a key role in facilitating the transport of diacylglycerol from the midgut to the fat body during the larval feeding period. The insect receptor has some properties which are similar to those of vertebrate lipoprotein receptors, viz. molecular weight, requirement for Ca2+, and inhibition by Suramin. However, the insect receptor does not bind human low density lipoprotein.

Distribution of FMRFamide-like immunoreactivity in the brain and suboesophageal ganglion of the sphinx moth Manduca sexta and colocalization with SCPB-, BPP-, and GABA-like immunoreactivity.

Using an antiserum against the tetrapeptide FMRFamide, we have studied the distribution of FMRFamide-like substances in the brain and suboesophageal ganglion of the sphinx moth Manduca sexta. More than 2000 neurons per hemisphere exhibit FMRFamide-like immunoreactivity. Most of these cells reside within the optic lobe. Particular types of FMRFamide-immunoreactive neurons can be identified. Among these are neurosecretory cells, putatively centrifugal neurons of the optic lobe, local interneurons of the antennal lobe, mushroom-body Kenyon cells, and small-field neurons of the central complex. In the suboesophageal ganglion, groups of ventral midline neurons exhibit FMRFamide-like immunoreactivity. Some of these cells have axons in the maxillary nerves and apparently give rise to FMRFamide-immunoreactive terminals in the sheath of the suboesophageal ganglion and the maxillary nerves. In local interneurons of the antennal lobe and a particular group of protocerebral neurons, FMRFamide-like immunoreactivity is colocalized with GABA-like immunoreactivity. This suggests that FMRFamide-like peptides may be cotransmitters of these putatively GABAergic interneurons. All FMRFamide-immunoreactive neurons are, furthermore, immunoreactive with an antiserum against bovine pancreatic polypeptide, and the vast majority is also immunoreactive with an antibody against the molluscan small cardioactive peptide SCPB. Therefore, it is possible that more than one peptide is localized within many FMRFamide-immunoreactive neurons. The results suggest that FMRFamide-related peptides are widespread within the nervous system of M. sexta and might function as neurohormones and neurotransmitters in a variety of neuronal cell types.

Manduca sexta lipid transfer particle acts upon a lipoprotein to catalyze lipid and apoprotein disproportionation.

A novel reaction, catalyzed by Manduca sexta lipid transfer particle (LTP), transforms low density lipophorin (LDLp) into two distinct lipoprotein species. A population of LDLp particles serves as lipid donor or acceptor in LTP-catalyzed production of a very low density lipophorin (VLDLp) and a high density lipophorin (HDLp) product. The products result from facilitated net transfer of lipid mass from donor LDLp particles to acceptor LDLp particles. Transfer of apolipophorin III (apoLp-III) from donor to acceptor lipoprotein occurs during the reaction to produce a lipid- and apoLp-III-enriched VLDLp species and lipid- and apoLp-III-depleted HDLp species. The VLDLp produced in this in vitro reaction contains more lipid and apoLp-III than any previous lipophorin species reported and further demonstrates the scope of the lipid binding capacity of lipophorin. Lipid analysis and radiolabeling studies confirmed that unidirectional net transfer of lipid mass and apoLp-III from donor to acceptor occurs. When 3H-lipid-LDLp was used as substrate in the LTP-catalyzed disproportionation reaction the density distribution of radioactivity and protein provided evidence of vectorial transfer of diacylglycerol, phospholipid, and free fatty acids. Electron micrographs of the original LDLp population and of the LTP-induced product lipoprotein population provided further support for the interpretation derived from biochemical studies. This LTP-catalyzed disproportionation was observed only with apoLp-III-rich LDLp suggesting that the presence of increased amounts of this apoprotein dramatically affects the properties of the particle and appears to be directly related to the capacity of the lipoprotein to bind lipid.

Serotonin-immunoreactive neurons in the median protocerebrum and suboesophageal ganglion of the sphinx moth Manduca sexta.

Serotonin-immunoreactive neurons in the median protocerebrum and suboesophageal ganglion of the sphinx moth Manduca sexta were individually reconstructed. Serotonin immunoreactivity was detected in 19-20 bilaterally symmetrical pairs of interneurons in the midbrain and 10 pairs in the suboesophageal ganglion. These neurons were also immunoreactive with antisera against DOPA decarboxylase. All major neuropil regions except the protocerebral bridge are innervated by these neurons. In addition, efferent cells are serotonin-immunoreactive in the frontal ganglion (5 neurons) and the suboesophageal ganglion (2 pairs of neurons). The latter cells probably give rise to an extensive network of immunoreactive terminals on the surface of the suboesophageal ganglion and suboesophageal nerves. Most of the serotonin-immunoreactive neurons show a gradient in the intensity of immunoreactive staining, suggesting low levels of serotonin in cell bodies and dendritic arbors and highest concentrations in axonal terminals. Serotonin-immunoreactive cells often occur in pairs with similar morphological features. With one exception, all serotonin-immunoreactive neurons have bilateral projections with at least some arborizations in identical neuropil areas in both hemispheres. The morphology of several neurons suggests that they are part of neuronal feedback circuits. The similarity in the arborization patterns of serotonin-immunoreactive neurons raises the possibility that their outgrowing neurites experienced similar forces during embryonic development. The morphological similarities further suggest that serotonin-immunoreactive interneurons in the midbrain and suboesophageal ganglion share physiological characteristics.

(R)-(Z,E)-9,11-octadecadien-13-olide: an intriguing lactone from Heliconius pachinus (Lepidoptera).

A fourteen-membered lactone, (R)-(Z,E)-9,11-octadecadien-13-olide, was isolated from extruded abdominal glands of a Neotropical, nymphalid butterfly. Heliconius pachinus (Lepidoptera). This compound was obtained from mature adults of both sexes, but was not detected in young adults or pupae.

An insect lipid transfer particle promotes lipid loading from fat body to lipoprotein.

The role of Manduca sexta lipid transfer particle (LTP) in the transport of lipid from fat body to lipophorin was investigated in vitro. Fat body that contained radiolabeled lipid was incubated with either high density lipophorin or low density lipophorin, and it was shown that lipid was transferred from fat body to lipophorins. The transfer of diacylglycerol was blocked by preincubating fat body with LTP antibody. Furthermore, transfer was restored by the addition of LTP, indicating that LTP promotes the transfer of lipid from fat body to lipophorins. Using lipophorins radio-labeled in their lipid moiety, transfer of lipid from lipophorin to fat body was demonstrated. This transfer was not mediated by LTP. The adipokinetic hormone induced diacylglycerol mobilization from the fat body and the concomitant interconversion of high density lipophorin to low density lipophorin were performed in vitro and were shown to require the presence of LTP.

Effects of a purified cecropin D from a Chinese silk moth on growth, function and differentiation of murine hemopoietic cells.

The effects of cecropin D, a small basic peptide isolated from a Chinese oak silk moth, on the functions or differentiation of mammalian hemopoietic cells are described in the present paper. This peptide suppressed lectin-induced DNA synthesis of murine splenocytes in a dose-dependent manner without any significant cytotoxic effects. It also exhibited inhibitory effects on antibody production in lipopolysaccharide-stimulated lymphocytes and on colony formation of hemopoietic progenitor cells in plasma clots culture. These results indicate that cecropin D can regulate growth, function and differentiation of murine hemopoietic cells. The biological significance of this finding is discussed from the comparative immunological point of view.

Alteration of a lepidopteran peritrophic membrane by baculoviruses and enhancement of viral infectivity.

The peritrophic membrane (PM), which lines the midgut of many insect species, has several functions. In particular, it may serve as a mechanical barrier to invading microorganisms. The protein composition of the PM from healthy and baculovirus-treated Trichoplusia ni (cabbage looper) larvae was analyzed by polyacrylamide gel electrophoresis. A specific interaction took place between baculoviruses and the PM of susceptible T. ni larvae. A 68-kDa glycoprotein of the PM disappeared within 15 min postinoculation with occlusion bodies of either Autographa californica nuclear polyhedrosis virus (AcMNPV) or T. ni nuclear polyhedrosis virus (TnSNPV). In contrast, inoculation of larvae with a T. ni granulosis virus (TnGV) resulted in the disappearance of three distinct major glycoproteins with molecular weights of 253, 194, and 123 kDa. PMs of virus-treated larvae were very fragile compared with those of untreated controls, indicative of a physical/chemical change in their structure. T. ni larval bioassays showed that a factor, present in the TnGV granulin or AcMNPV polyhedrin, enhanced the infectivity of AcMNPV. These data showed that a factor present in the occlusion bodies of three distinct baculoviruses can cause specific biochemical and structural changes in the PM. The biological significance of these observations in relation to increased larval infection is not known at this time.

Carboxylic acids from hairpencils of male Amauris butterflies (Lep.: Danainae).

63 Carboxylic acids were identified from the male hairpencils of four species of the genus Amauris (Lep.: Danainae), namely A. echeria (Stoll), A. hecate (Butler), A. ochlea (Boisduval) and A. albimaculata Butler. Straight chain saturated as well as unsaturated carboxylic acids, some of which containing an additional oxygen function, contribute to the species-specificity of the odour bouquets. Oxygenated fatty acids form a new class of insect volatiles, 5 of the 10 ketoacids found represent new natural products. (E)-7-Oxo-11-tetradecenoic acid is the main volatile component of the hairpencils of A. echeria, the species with the highest amount of oxygenated fatty acids (70% of the extractable volatiles). 9-Hydroxyoctadecanoic acid is a major compound in both A. ochlea and A. albimaculata while in A. hecate oxygenated carboxylic acids are present in minute amounts only.

Channel-forming properties of cecropins and related model compounds incorporated into planar lipid membranes.

Cecropins, positively charged antibacterial peptides found in the cecropia moth, and synthetic peptide analogs form large time-variant and voltage-dependent ion channels in planar lipid membranes in the physiological range of concentration. Single-channel conductances of up to 2.5 nS (in 0.1 M NaCl) were observed, which suggests a channel diameter of 4 nm. Channels formed by the peptides cecropin AD and MP3 had a permeability ratio of Cl-/Na+ = 2:1 in 0.1 M NaCl. A comparative study of the three cecropins, cecropins A, B, and D, and of six synthetic analogs allowed determination of structural requirements for pore formation. Shorter amphipathic peptides did not form channels, although they adsorbed to the bilayer. A flexible segment between the N-terminal amphipathic region and the C-terminal more hydrophobic region of the peptide was required for the observation of a time-variant, voltage-dependent conductance. Cecropin AD was the most effective voltage-dependent pore-forming peptide and was also the most potent antibacterial peptide against several test organisms. A positive surface charge or cholesterol in the bilayer reduced the conductances caused by cecropin AD or MP3 by at least 5-fold. This behavior is consistent with the known insensitivity of eukaryotic cells to cecropins. Our observations suggest that the broad antibacterial activity of cecropins is due to formation of large pores in bacterial cell membranes.

An insect brain peptide as a member of insulin family.

Amino acid sequencing of bombyxin (previously called 4K-PTTH) isolated from the heads of the silkmoth Bombyx mori has disclosed sequence homology of this insect neuropeptide with insulin. Immunohistochemistry using an antibody against a synthetic bombyxin fragment detected 4 pairs of immunoreactive neurosecretory cells in the dorso-medial region of the Bombyx brain. The same cells were reactive to bovine insulin antibody.

Preliminary characterization and purification of in vitro encapsulation promoting factor: a peptide that mediates insect haemocyte adhesion.

The granular cells and plasmatocytes (PLs) of Heliothis virescens form multicellular aggregations in vitro. This allows capsules to form around suitable targets. Prior trypsinization of the haemocytes abolishes their ability to encapsulate, and this function can be restored by adding plasma to the trypsinized cells. Trypsinized PLs were also unable to spread on a planar glass surface unless normal plasma was present. Plasma was subjected to a variety of treatments to determine the nature of the encapsulation promoting factor (EPF) using the in vitro encapsulation system as a bioassay. The data suggest EPF is a peptide; it is trypsin sensitive and moderately heat stable. Similar results were obtained when using spreading by trypsinized PLs as the bioassay. Dialysis using a 3,500 MW cut-off membrane also abolished encapsulation promoting activity. Protein-free extracts of plasma (crude EPF) has strong activity in both bioassays but does not agglutinate human erythrocytes. A single peak with strong activity in both bioassays was resolved after subjecting crude EPF to reversed-phase HPLC. This active material was purified after additional HPLC with a different solvent system.

Age related occurrence of natural agglutinins in the erisilkworm, Philosamia ricini.

Natural agglutinins against human, guinea pig, and rat erythrocytes (RBC) and bacteria Bacillus thuringiensis were readily detected in hemolymph of final instar larvae with resulting titers of 0-9 (log2). Titers were independent of sex and season but varied conspicuously and reproducibly with age. Moreover, response of hemagglutinins to heating varied with age being heat-labile in younger larvae, yet totally resistant to heating for 30 min at 70 degrees C in older ones. Bacterial agglutinins were uniformly resistant at all larval ages. The study thus reveals that the amounts and physico-chemical properties of P. ricini agglutinins change with larval development. Therefore, larval age should be taken into close consideration before reporting on the occurrence and properties of agglutinins in insects.

Chitin structure and chitinase activity: isolation of structurally intact chitins.

Assessment of chitinase kinetics and mechanism in vitro has been hampered by lack of suitable substrates. We have previously reported rapid linear initial chitinase velocity with chitin substrate isolated from insect larval cuticle. Such chitin is shown to be fibrous in the light microscope. Methods are described for preparing fibrous chitins from any animal source including calcified carapaces. Evidence is given that chitin native fine structure in situ is maintained by structural proteins which in the fibrous chitin isolates are functionally replaced by covalently bound ester groups. Chitin fiber analogues thus reconstructed appear to have retained their native fine structure.

Microanalysis of the amino acid sequence of the eclosion hormone from the tobacco hornworm Manduca sexta.

The amino acid sequence of the eclosion hormone from the tobacco hornworm Manduca sexta has been determined, using less than 500 pmol of protein and microanalytical techniques. The protein contains 62 amino acid residues and has a molecular mass of 6813 Da. The amino-terminal sequence is similar to that of a 13-residue segment at the amino terminus of the eclosion hormone of the silkworm Bombyx mori, but the hormone is not otherwise homologous with other hormones or proteins.

Crystallization, crystal structure analysis and preliminary molecular model of the bilin binding protein from the insect Pieris brassicae.

The bilin binding protein of the butterfly Pieris brassicae has been prepared, crystallized and its crystal structure determined at high resolution using film and FAST area detector intensity data. The crystallographic asymmetric unit contains a tetramer of identical subunits with a molecular weight of about 90,000. The crystal structure was determined by isomorphous replacement. Use was made of the molecular symmetry to improve phases. A molecular interpretation of the electron density distribution and partial tracing of the polypeptide chain was possible without amino acid sequence information, as the fold is very similar to retinol binding protein. It is characterized by a beta-barrel formed by two orthogonal beta-sheets and an alpha-helix. The bilin pigment seems to be bound within the beta-barrel analogously to retinol in retinol binding protein. The tetramer in the crystal has C2 symmetry and is a dimer of dimers of quasi-equivalent subunits.

Retinoid composition in the compound eyes of insects.

Retinoids in the compound eyes of insects in ten orders were extracted by the oxime method and analysed by HPLC. Four geometrical isomers (13-cis, 11-cis, 9-cis and all-trans) of syn and anti retinal oximes, and syn and anti 3-hydroxyretinal oximes were separated in a single analysis by a stepwise eluent condition. The amounts of the two isomers, syn 11-cis and syn all-trans, were quantified. 11-Cis 3-hydroxyretinal was detected in six orders: Lepidoptera, Diptera, Coleoptera, Neuroptera, Hemiptera and Odonata, and retinal and 3-hydroxyretinal were found together in the compound eyes of some species of Coleoptera and Odonata. We conclude that early in their phylogeny, insects had the ability to use 3-hydroxyretinal as the chromophore of visual pigment. Peaks corresponding to syn 9-cis and 13-cis 3-hydroxyretinal oximes were observed on the chromatogram of extracts from fly heads and compound eyes of cicadas. Retinol and 3-hydroxyretinol were also analysed and quantified relative to retinal and 3-hydroxyretinal. Larger amounts of the alcohols than the aldehydes were found in the compound eyes of butterflies, hornets, cicadas and grasshoppers, which are diurnal insects. 3-Dehydroretinal has not been detected in insects.