Pharmacology of octopamine-, dopamine-, and 5-hydroxytryptamine-stimulated cyclic amp accumulation in the corpus cardiacum of the american cockroach,Periplaneta americana L

A variety of potential receptor agonists were incubated with isolated corpora cardiaca from the American cockroach Periplaneta americana to determine their effects on cyclic AMP production in this gland. Octopamine, dopamine, and 5-hydroxytryptamine elevated cyclic AMP levels in a dose-dependent manner with estimated K_a values of 15.8, 1.7, and 1.1 m?M, respectively, and their stimulation was found to be additive. Several vertebrate receptor antagonists were tested against the three amines and a preliminary pharmacological profile developed.     

Cyclic AMP does not mediate the action of synthetic hypertrehalosemic peptides from the corpus cardiacum of Periplaneta americana

Two synthetic peptides identical to those present in the corpus cardiacum of the American cockroach, Periplaneta americana, were tested for their effect on the production of cyclic AMP and the activation of glycogen phosphorylase in cockroach fat body. The peptides activate glycogen phosphorylase and promote trehalose production in incubated tissue when calcium is included in the incubation medium, but have no obvious effect on cyclic AMP levels. The lack of effect of the peptides on cyclic AMP production was confirmed in a fragmented membrane preparation. By contrast, an aqueous extract of corpus cardiacum activates glycogen phosphorylase, promotes trehalose production and elevates cyclic AMP levels in incubated tissue; the extract also enhances cyclic AMP production in the fragmented cell membrane preparation. Observations on the nature of cyclic AMP production in cockroach fat body indicate that the adenylate cyclase has a requirement for GTP and magnesium ions, is stimulated by fluoride and forskolin and, therefore, is similar to the adenylate cyclase complex of other eukaryotes.The results suggest that increases in intracellular calcium concentrations may mediate the expression of hypertrehalosemic effects by the synthetic peptides.     

Binding of juvenile hormone III to lipophorin from the american cockroach Periplaneta americana

Whole hemolymph from the American cockroach, Periplaneta americana, efficiently binds juvenile hormone (JH) III and to a lesser extent JH-I and 10, 11-epoxyfarnesyl diazoacetate (EFDA). The dissociation constants for racemic JH-III and EFDA are 30 ± 2 nM and 1.0 μM, respectively. Isolated lipophorin also binds [³H]JH-III and to a lesser extent JH-I. Other proteins from the hemolymph do not bind JH-III. Binding of JH-III to lipophorin is enantioselective. The dissociation constant, measured with a 92% 10R and 8% 10S mixture, is 21 ± 2 nM. Each lipophorin molecule contains one specific binding site for JH-III. It is concluded that lipophorin is the JH-III-specific transport protein in the hemolymph of the American cockroach. By a combination of photoaffinity labelling and gradient electrophoresis with sodium dodecyl sulphate on polyacrylamide gel, we showed that the JH-III-specific binding site is probably located on apolipophorin I.     

Control of hexose phosphate and citrate in fat body of the cockroach (Periplaneta americana) by the corpus cardiacum

1. Fat body of the American coackroach (Periplaneta americana) treated with CC extract (0.01 gland equivalents per ml) releases approximately 100% more trehalose into physiological saline in 1 hr than paired tissue controls.2. The rate is highest during the first 5 min and thereafter declines.3. Phosphorylaseactivity in CC treated fat body is more than double that in control tissues, the difference being maintained throughout the entire period of incubation.4. G-6-P, the product of glycogenolysis and substrate for trehalose synthesis, increases more than 100% in fat body treated with CC extract.5. The concentration of F-6-P also rises by the same percentage after treatment with CC extract but that of F-1, 6-bisP increases by not more than 50%.6. Within 5 min after treatment with CC extract the concentration of citrate doubles, rising to 3-fold that in the controls after 30 min.7. The data suggest that the CC play an important role in the coupling of carbohydrate metabolism to lipolysis.     

Purification and characterization of trehalose-hydrolyzing enzymes from thoracic musculature of the american cockroach,Periplaneta americana

Trehalose-hydrolyzing enzymes from particulate and cytosolic components of the thoracic musculature of Periplaneta americana were isolated and purified to homogeneity. The molecular weights of the respective enzymes were determined by SDS-polyacrylamide gel electrophoresis and Sephadex G-150 column chromatography and estimated to be 80,000 Da for the cytosolic enzyme whereas the solubilized enzyme of particulate origin has a molecular weight of approx. 110,000 Da. The cytosolic enzyme hydrolyzes a number of α-glycosides in addition to trehalose and, therefore, may be classified as a general α-glucosidase whereas the particulate enzyme exhibits stringent specificity for trehalose. Chemical modification of the particulate trehalase has revealed involvement of carboxyl and imidazole functions in the catalytic mechanism. Of various compounds tested, castanospermine was the most potent inhibitor of the enzyme.     

Characterization of the adipokinetic and hyperglycaemic substances from the locust corpus cardiacum

An extract of locust corpus cardiacum exhibits adipokinetic and hyperglycaemic activities. For purification and characterization of the hormones extracts were subjected to gel filtration, electrofocusing, ultrafiltration, and paper chromatography. Examination of the presence of hormonal activities in the experimental fractions was by bioassay. Throughout the purification procedure both hormone activities remained connected. Acid hydrolysis of the purified fraction yielded the following amino acids in roughly equimolar amounts: aspartic acid, glutamic acid, glycine, leucine, phenylalanine, proline, serine, and threonine. Tryptophan is virtually absent.     

Isolation and characterization of a Chryseobacterium strain from the gut of the American cockroach, Periplaneta americana

A 16S rDNA sequence cloned directly from whole-gut microbiota of the American cockroach, Periplaneta americana, indicated the presence of a member of the Bacteroides/Flavobacterium group most closely related to the genus Flavobacterium. In an attempt to confirm this finding, we isolated a yellow-pigmented bacterium (strain FR2) from the hindgut of this insect. Strain FR2 was phylogentically and phenotypically most similar to species of Flavobacterium and related bacteria, namely Chryseobacterium indologenes. Fifty-four other yellow-pigmented bacteria isolated during a 1-year study shared the salient phenotypic characteristics of Chryseobacterium spp., and thus were considered the same phenotype. This phenotype's abundance was related to the fiber content of the insect diet, being consistently detected only in cockroaches fed a high-fiber diet (30% crude fiber by weight). The highest population density was in the hindgut, ranging from 2 x 10(6) to 1.2 x 10(7) colony forming units ml(-1) during a 1-year period. The nature of the symbiosis between the FR2 phenotype and P. americana is discussed.     

Secretion of hyperglycaemic hormone from the corpus cardiacum of flying blowflies, Calliphora erythrocephala

In flies with an intact brain-corpus cardiacum system, the haemolymph trehalose concentration is kept near the resting level (～23 g/l.) during continuous flight for at least 45 min. Upon cardiacectomy or corpus cardiacum denervation haemolymph trehalose decreases during the first 15 min of flight to one-third of the resting level, and complete flight exhaustion occurs within 45 min. In such flight-exhausted flies a slow, apparently hormone-independent, increase of haemolymph trehalose occurs during subsequent rest. Squeezing of denervated (i.e. non-secreting) corpora cardiaca in situ in flight-exhausted flies leads temporarily to a steep rise of haemolymph trehalose level and restoration of flight performance.     

Purification and characterization of vitellogenin from the American cockroach,Periplaneta americana

• 1.1. Vitellogenin (VG) was isolated and purified from the hemolymph of female American cockroaches.
• 2.2. The purification method used in this study comprises two steps: the first step is based on the method originally developed for purifying lipophorin from hemolymph, and the second step is the separation of VG from lipophorin by a KBr density gradient ultracentrifugation.
• 3.3. The purified VG was characterized according to molecular weight, substructure, shape and size, and lipid composition.
• 4.4. The VG molecule is almost globular in shape with the diameter of about 15.5 nm and is indistinguishable from lipophorin in shape and size.
• 5.5. The native molecular weight determined by light scattering method was 560 kDa.
• 6.6. The VG consists of four subunits with molecular weights of approximately 102, 81, 49 and 40 kDa, respectively.
• 7.7. VG is a lipoprotein and comprises 92% protein and 8% lipid.
• 8.8. Major lipid components were found to be diacylglycerol (25%) and phospholipids (71%).

     

Separation of the hyperglycemic and adipokinetic factors from the cockroach corpus cardiacum

An extract of corpora cardiaca of Periplaneta americana exhibits hyperglycemic and adipokinetic activities. The carbohydrate mobilizing action can be evoked in the cockroach itself, whereas the lipid mobilizing response is found when the extract is injected into locusts. The two hormonal activities are connected with chemically different entities. Neither hormone is identical with the locust adipokinetic-hyperglycemic hormone. Both cockroach hormones lack electrophoretic mobility.     

Molecular characterization of the ebony gene from the American cockroach, Periplaneta americana

Biogenic amines are an important class of primary messengers in the central (CNS) and peripheral nervous systems and in peripheral organs. These substances regulate and modulate many physiological and behavioral processes. Various inactivation mechanisms for these substances exist to terminate biogenic amine-mediated signal transduction. In vertebrates, the enzymes monoamine oxidase and/or catechol-O-methyl-transferase are involved in these processes. In insects, however, in which both enzymes are low in abundance or absent, biogenic amines are inactivated mainly by N-acetylation or O-sulphation. In Drosophila, beta-alanyl conjugation mediated by the Ebony protein has recently been shown to be a novel and alternative pathway for biogenic amine inactivation. Here, we report the cloning of ebony cDNA (Peaebony) from a brain-specific cDNA library of the cockroach Periplaneta americana. The open reading frame encodes a protein of 860 amino acid residues (PeaEbony). The PeaEbony polypeptide shares homology to Ebony sequences from Anopheles gambiae, Apis mellifera, and Drosophila melanogaster. In addition, PeaEbony exhibits sequence similarity to a family of microbial non-ribosomal peptide synthetases. The mRNA encoding PeaEbony is highly expressed in the cockroach brain and to a lesser extent in the salivary glands. PeaEbony is, therefore, probably involved in the inactivation of various biogenic amines through beta-alanyl conjugation in the cockroach CNS. Since the salivary glands in Periplaneta are innervated by dopaminergic and serotonergic neurons, PeaEbony probably also biochemically modifies dopamine and serotonin in these acinar glands.     

Further purification and characterization of trehalases from the American cockroach,Periplaneta americana

Summary A soluble trehalase was purified more than 200-fold from the male accessory gland of the American cockroach,Periplaneta americana, by CM-cellulose, hydrophobic chromatography, and Sephacryl S-200 gel filtration. The final preparation was homogeneous as judged by polyacryl-amide gel electrophoresis in the absence and presence of SDS, isoelectric focusing, and immuno-diffusion tests. The purified enzyme was maximally active at pH 5.2, and showed high specificity for trehalose with aK _m of 0.98 mM. The isoelectric point was 4.7. The molecular weight of the enzyme (75,000) was determined by molecular sieve chromatography and SDS-polyacrylamide gel electrophoresis. The amino acid composition was determined and compared with those of trehalases purified from other sources. The trehalase could be stained for carbohydrate with the periodic acid-Schiff's reagent following SDS-polyacrylamide gel electrophoresis, indicating that it was a glycoprotein. Another soluble trehalase and two types of fat body trehalases could be highly purified by the method described. A comparison of the properties of trehalases from the accessory gland and the fat body showed some resemblance.     

Classical olfactory conditioning in the cockroach Periplaneta americana

We established a classical conditioning procedure for the cockroach, Periplaneta americana, by which odors were associated with reward or punishment. Cockroaches underwent differential conditioning trials in which peppermint odor was associated with sucrose solution and vanilla odor was associated with saline solution. Odor preference of cockroaches was tested by allowing them to choose between peppermint and vanilla sources. Cockroaches that had undergone one set of differential conditioning trials exhibited a significantly greater preference for peppermint odor than did untrained cockroaches. Memory formed by three sets of differential conditioning trials, with an inter-trial interval of 5 min, was retained at least 4 days after conditioning. This conditioning procedure was effective even for cockroaches that had been harnessed in plastic tubes. This study shows, for the first time in hemimetaborous insects, that both freely moving and harnessed insects are capable of forming olfactory memory by classical conditioning procedure. This procedure may be useful for future electrophysiological and pharmacological studies aimed at elucidation of neural mechanisms underlying olfactory learning and memory.     

Immunoreactive material resembling vertebrate neuropeptides and neurophysins in the brain,suboesophageal ganglion,corpus cardiacum and corpus allatum of the dictyopteran Periplaneta americana L.

Summary The peroxidase-antiperoxidase technique (Vandesande and Dierickx 1976) with antibodies raised against several vertebrate neuropeptides and neurophysins, was applied] to 4-m Paraplast-embedded serial sections of in situ fixed brains and adjacent suboesophageal ganglion (SOG), corpora cardiaca (CC) and corpora allata (CA) of the blattarian insect Periplaneta americana L. Substances immunologically related to bovine neurophysin I (NPI) and II (NPII), synthetic arginine vasopressin (AVP) and synthetic oxytocin (OT) were found to be differentially distributed in the central nervous system. The differences among all four antigens demonstrated became clearly evident by immunohistochemical double-staining procedures (Vandesande 1983); no overlapping was observed. The same double-staining technique revealed that these vertebrate-type substances occur in other neurosecretory cells and axons than those containing CRF- and ACTH-like material as reported earlier (Verhaert et al. 1984).     

Synthesis of diacylglycerols by monoacylglycerol acyltransferase from crop,midgut and fat body tissues of the american cockroach,Periplaneta americana L.

Monoacylglycerol acyltransferase (E.C. 2.3.1 type) activity was determined in the crop, midgut and fat body of adult, male cockroaches. Tissue homogenates were capable of acylating [³H]-glycerol-2-monoolein with palmitic acid. The major product was diacylglycerol although some triacylglycerol was also formed. The greatest rate of acylation was observed in the midgut, but the crop and the fat body also displayed a capacity for acylation of monoacylglycerol. The enzyme activity was dependent on time, protein and substrate concentration. The ability of rac-1-monoolein and 2-monooleyl glyceryl ether to substitute for 2-monoolein as an acyl acceptor, was also demonstrated.     

The ocellus of the cockroach,Periplaneta americana (Blattariae)

Summary The ocelli of Periplaneta americana were studied by light and electron microscopy. The view that the ocellus of the cockroach represents a degenerated structure can no longer be supported. All organelles necessary for function are present.The club-shaped retinular cells lie homogeneously distributed in the cupule of the ocellus. Rhabdoms are seen as sickle-, y-, x-or star-shaped structures with up to six cells in formation. Cells were found which had formed two rhabdomeres.The mass of cell organelles lies in the cytoplasm between the cell nucleus and the rhabdom. Smooth endoplasmic reticulum is wound into a spindle formation of considerable size at the origin of the axon in some cells. A cylindrical body in which 10–40 microtubules are packed, as yet unknown in insect retinular cells, is described.The receptory area of the ocellus terminates in a tapetum which contains granules, soluble in alcohol. The axon bundles of the retinular cells run through the tapetum and immediately thereafter make synaptic contact with dendrites of the ocellar nerve cells, while still within the ocellus.The authors are indebted to Mrs. Margaret Weber-Wood for her linguistic assistance